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SULPHUR BACTERIA

BY THE SAME AUTHOR

PRACTICAL BACTERIOLOGY FOR
CHEMICAL STUDENTS

With 55 Illustrations. Crown 8vo. 45. bd. net

BY PROFESSOR D. ELLIS, D.Sc, F.R.S.E., AND
D. CAMPBELL

SCIENCE AND PRACTICE OF
CONFECTIONERY

With 7 Illustrations. Crown 8vo. 5s.

SULPHUR BACTERIA

A MONOGRAPH

BY

DAVID ELLIS

D.Sc. (LOND.), Ph.D. (Marburg), F.R.S.E.

PROFESSOR OF BACTERIOLOGY, THE ROYAL TECHNICAL COLLEGE, GLASGOW

WITH ILL USTRA TIONS

LONGMANS, GREEN AND CO.
LONDON ♦ NEW YORK ♦ TORONTO

1932

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PREFACE.

In the last two decades considerable progress has been made in
every branch of Bacteriology ; and of the different branches,
that dealing with the sulphur bacteria has received much
attention from foreign investigators. An attempt is here made
to present to English readers the present position of our
knowledge of the sulphur bacteria.

The researches on these organisms are spread over a wide
field of investigators, and are described in divers languages.
As for the most part such studies have been confined to partial
aspects of the subject, there should be room for a book such as
the present, which aims at summing up the whole position.
l^J^e only section which is scantily treated is that of Ecology,
and the reason for this is that this subject is very adequately
dealt with in Bavendamm's Die farblosen und roten Schwefel-
bakteriek^

The study of the sulphur bacteria supplies matters of
interest not only to the bacteriologist and the botanist, but
also to the chemist who is interested in the analysis of water,
in the preservation of the purity of water reservoirs, and in the
chemical changes which take place in sewage-laden waters.

These bacteria form a group of peculiar interest to the
botanist because of the possible relationship of many of their
species to the animal kingdom. Their study throws con-
siderable light on the structure, methods of reproduction, and
physiological activities of organisms that stand on the border-
line between plants and animals ; and this work has been

vi PREFACE

primarily written as a contribution to botanical literature.
The author's claim to make the effort rests on a study of the
sulphur bacteria over a period of fourteen years. Some of
the work has already appeared in various scientific periodicals,
but a large portion is here published for the first time.

On the morphological side of the subject all the known forms
are sketched and described with sufiicient fullness to secure
identification. A scheme of classification is drawn up which
avoids the defects of its predecessors. Numerous schemes of
classification of the sulphur bacteria have appeared, but, with
one or two exceptions, these are parts of a general scheme
embracing the whole of the bacteria, and have not followed a
special study of the sulphur bacteria. As a consequence the
later schemes have merely perpetuated the defects of the older
classifications.

There are many gaps in our knowledge of the physiology
of the sulphur bacteria which cannot be adequately filled by
the biologist, and which await the attention of the biochemist.
It is hoped that the present work will form a basis for investiga-
tions on biochemical lines.

I desire to acknowledge my especial indebtedness to my
colleagues, Mr. W. G. Burrell, M.A., Dr. J. A. Cranston, Mr. J.
Muil Leitch, B.Sc, and Dr. Blodwen Lloyd, for their assistance
in the preparation of the manuscript and in the revision of the
proofs ; and to Mr. Leitch for the preparation of the Indices.

DAVID ELLIS.

Department of Bacteriology,

The Royai- Technical College,
Glasgow.

CONTENTS.

CHAPTER I.

PAGE

Introduction .......... i

Connotation of the term " Sulphur Bacteria " ; geographical
distribution ; the sulphur cycle in nature ; methods of investi-
gation ; pleomorphism ; evidence of occurrence of pleomor-
phism in the sulphur bacteria.

CHAPTER II.

The Production of Sulphuretted Hydrogen and its Assimila-
tion BY THE Sulphur Bacteria ...... 20

Natural sources of sulphuretted hydrogen ; the limans ; investiga-
tion of the black sand on the Clyde ; the equilibrium of
hydrogen sulphide in water.

CHAPTER III.
The Metabolism of the Sulphur Bacteria .... 34
Food requirements and sources of energy; metabolism, fermenta-
tion and respiration ; sulphuretted hydrogen ; organic matter ;
carbon supply ; oxygen ; mineral matters ; hydrogen-ion
concentration ; biological explanation of the assimilation of
sulphuretted hydrogen ; the denitrifying sulphur bacteria ;
physico-chemical speculations.

CHAPTER IV.

The Culture of the Sulphur Bacteria . . . . -55

Methods of culture ; methods of Winogradsky, Molisch, Keil,
Jegunow, Skene, Bavendamm, and Ellis.

CHAPTER V.

The Principles of Classification and their Application to the

Sulphur Bacteria . ....... 67

The principles of a natural classification ; review of attributes
used in the subdivision of the sulphur bacteria ; review of
previous classifications : by Winogradsky, by Molisch, bv
Orla Jensen, by Buchanan ; Ellis's classification.

4 2939

viii CONTENTS

CHAPTER VT.

PAGE

The Leuco-thiobacteria (Colourless Sulphur Bacteria) . . 92

BeggiatoacecB : Beggiatoa ; Beggiatoa alba, sheath formation, growth,
autolysis, methods of reproduction ; other species of Beggiatoa ;
pleomorphism of Beggiatoa ; Beggiatoa niirabilis ; Beggiatoa
arachnoidea ; Beggiatoa leptomitifovmis. Thiothrix ; Thiothrix
nivea / Thiothrix tenuis and tenuissima ; Thiothrix annulata ;
Thiothrix marina ; Thiothrix violacea. Thioploca ; Thioploca
Schmidlei ; Thioploca ingrica ; Thioploca minima and mixta.

CHAPTER VH.

The Leuco-thiobacteria (continued) . . . . . .116

Achromatiacecs : Achromatium ; Achromatium oxaliferum [Modderula
Hartwigi and Hillhousia niirabilis), cell contents, growth,
reproduction, and habitat. Achromatium mobile; Microspira
vacillans. Thiophysa ; Thiophysa volutans ; Thiophysa
macrophysa. Thiosphcsrella amylifera. Thiovulum Miilleri.
Thiospirillacecs : Thiospirillum Winogradskii ; Thiospirillum
grannlatum ; Thiospirillum bipnnctatum ; Thiospirillum
agilissimum ; Thiospirillum agilis ; Thiospirillum elongata.
ThiobacillacecD : Thiobacillits Bovistus ; Thiobacillus thiogenus.
Thiopseudomonas ; Pseudomonas bipunctata and hyalina.

CHAPTER VIII.
Rhodo-thiobacteria (Coloured Sulphur Bacteria) . . -134

Lankesteracsce : Lankesteron ; Lankesteron rubescens ; Lankesteron
sulfuratum ; Lankesteron roseo-persicina. ChromatacecB :
Chromatium ; Chromatium Okenii ; variant forms of Chr.
Okenii (Chr. Weissii, Chr. minus, Chr. minutissimum, Chr.
gracile) ; Chromatium Warmingii ; Chromatium Linsbaueri ;
Chromatium violascens ; Chromatium cuculliferum; Rhabdomonas
and Rhabdochromatium ; Rhabdochromatium roseum, minus,
and gracile ; Rhabdochromatium Linsbaueri. Thioporphyra
volutans, reproduction, bud formation, regional rejuvenescence,
ciliation and movement.

CHAPTER IX.
Rhodo-thiobacteria (continued) ...... 160

Rhodothiospirillacece : the terms Thiospirillum, Rhodothiospirillum,
and Ophidomonas ; Rhodothiospirillum jenense. Rhodocapsacea :
Rhodocapsa suspensa ; Rhodothece pendens ; Rhodothiosarcina
rosea. ThiocapsacecB : the terms Micrococcus, Streptococcus,
Sarcina ; Thiocapsa roseo-persicina ; Thiocystis violacea ; Thio-
cystis rufa ; Thiosphcerion violacea ; Thiosphcera gelatinosa.
Amoebobacteriacecs Amasbobacter : roseiis, bacillosus, granula ;
Thiodictyon elegans ; the terms Thiothece and Thiopoly coccus
Thiothece gelatinosa and Thiopoly coccus ruber. Thiopediacecs
Thiopedia rosea. Lamprocystis roseo-persicina, violacea, gela-
tinosa, rubra, and rosea.

CONTENTS IX

CHAPTER X.

PAGE

The Intimate Structure of the Cell . . . . .176

Introduction ; Beggiaioa alba ; Thioporphyra volutans ; Thiophysa
volutans ; Beggiatoa mirabilis ; Chromatium Okenii ; Achroma-
tiuni oxaliferum [Hillhousia mirabilis, Modderida Hartwigi) ;
Achromatium mobile ; Thiovulum Mulleri ; Thiovulum majus ;
Chromatium Linsbaiteri ; summary and conclusions.

CHAPTER XL
Irritability ; Influence of Light ; Chemiotactic Phenomena . 193
Irritability : introduction ; methods of investigation ; influence
of light on the sulphur bacteria ; Engelmann's investigations ;
photokinetic after-effect ; shock movements ; valve action of
shock movements ; Molisch on shock movements. Distribution
of purple bacteria in the various colours of the spectrum ; effect
of colour on the absorption of light ; liberation of oxygen
by the purple bacteria ; effect of light on the growth of the
purple bacteria ; the function of light, photosynthesis ; the
directive effect of light ; summary. Miyoshi's experiments ;
Bengt Lidforss's work ; Molisch's experiments ; irritability and
environment. Buder's researches : ciliary movements, reac-
tion to light, shock movements, localization of area of
sensitiveness, interpretation of results,

CHAPTER XII.

The Mechanics of Ciliary Movement. The Thionic Acid

Bacteria. The Phylogeny of the Sulphur Bacteria . 216
The mechanics of ciliary movement. Thionic acid bacteria ; intro-
duction, methods of culture; Thiobacillus thiopants ; Thio-
bacillus denitrificans : Thiobacillus thiooxidans ; organisms
isolated by Issatchenko and Salismowskaja, Thiobacilhis B.,
Bacillus crystalliferitm. Bacterium retiformans, Pseudomonas
bipunctatum, Pseudomonas hyalina. Nomenclature of the
thionic acid bacteria. The phylogeny of the sulphur bacteria;
factors concerned in the evolution of the group ; summary.

CHAPTER XIII.
The Colouring Matter of the Sulphur Bacteria . . . 232

Introduction ; historical ; investigations of Lankester, Biitschli,
Molisch ; methods ' of investigation ; absorption spectra ;
colouring matter of Thioporphyra volutans; spectroscopic
analysis of colouring matter of Thioporphyra volutans:
spectro-photometric method of examination ; summary

Bibliography ........

Index of Author's Names
Index of Organisms
General Index

243
255
257
259

CHAPTER I.

INTRODUCTION— GEOGRAPHICAL DISTRIBUTION—
THE SULPHUR CYCLE IN NATURE— METHODS OF
INVESTIGATION— PLEOMORPHISAl.

Introduction.

The term Sulphur Bacteria is usually applied to the members
of the group which have sulphur globules in their cells. As a
result of their activities compounds of the highest importance
to the higher green plants are formed ; in fact these organisms
constitute a cog in the machinery of life, a breakdown in which
would sooner or later result in the elimination of mankind
and the higher animals.

It is inevitable that a great diversity of form should
characterize a division of plants the members of which owe
their grouping to a physiological trait that does not neces-
sarily connote the possession of any other character, either mor-
phological or physiological. An examination of the sulphur
bacteria supplies sufficient evidence of the unsuitability of
physiological classifications to express phylogenetic relation-
ships. The sulphur bacteria include almost every variety
of size and form that is to be found among the bacteria and
allied organisms. Thus there are representatives of the
bacillus, the coccus and the spirillum ; and in addition there
are ovoid, filamentous and other shaped forms in the group.
The size varies from one that is barely visible under the
microscope to one that is large enough to permit of micro-
tome sections.

If the bacteria as a group were eliminated from our
classifications and a distribution of the sulphur bacteria among
the other classes became necessary, some would be added to

I

2 SULPHUR BACTERIA

the list of Alga?, others to the lower fungal groups, whilst a
number would find a place among classes of the animal king-
dom. If one compares, for example, the diversity of form
and mode of life of Chromatium, Thiobacillus, Thiospirillum,
and Beggiatoa, the phylogenetic unsoundness of the grouping
becomes manifest. In the present stage of our knowledge,
however, the grouping must remain in spite of this defect
on account of its convenience for the further investigation of
these interesting organisms.

It is doubtful whether any group of plants excels the sulphur
bacteria in the numerous aspects of biological interest that
they show. We have in them the best examples of pleomor-
phism shown by any group of organisms ; some of the mem-
bers hold the first place in their sensitiveness to light. The
sulphur metabolism is a subject of transcending interest.
In addition, the sulphur bacteria show a great variety in form,
in internal structure, and in the different phases of their
development. If we regard these plants from the point of
view of their more immediate utilitarian aspect there is much
that calls for attention. In the first place, as the substance
consumed by them is the foul-smelHng sulphuretted hydrogen,
and as the chief end product in their metabolism is the com-
pound, namely the sulphate, which is absorbed by the higher
green plants, their place in the economy of nature is obvious.
The higher green plants, from which, either directly or in-
directly, all human beings draw their food, are suppHed with
a necessary food material.

Whilst the ultimate result of their activities is productive
of great benefit to mankind their more immediate effects may
be highly inconvenient, for when growth takes place in water
conduits or in stagnant pools, a large volume of sohd organic
matter is formed which chokes up the conduit pipes, and
renders the water unfit for human consumption. Also,
instances are known in which water thus affected has been
rendered unfit for use in various industries.

Indirectly the operations of the sulphur bacteria influence
the play of other forces of great economic importance. The
role of bacteria in the transformation of muds, sands, and other

INTRODUCTION ■ 3

accumulations that contain organic matter, is one that is
important, and is in some instances considerably influenced
by the activities of the sulphur bacteria. The stench of
black muds on the foreshore and the pollution which attends
the heaping together of mud, sand, and organic matter on our
shores cannot be neglected. Among the factors which must
be reckoned with are the activities of the sulphur bacteria.
Again, some of these organisms develop freely in sewage-
contaminated water, and may be utilized in the detection of
sewage contamination in water supposed to be sewage free.
The sulphur bacteria abound in sulphur wells, and although
they do not produce the salts which give such wells their real
or alleged healing qualities, inasmuch as they multiply freely
in such waters, the nature of the products of their metabolism
is of interest to those who consume the water.

Connotation of the Term " Sulphur Bacteria."

The list of bacteria that effect changes in sulphur com-
pounds is a fairly comprehensive one. The changes include
oxidation processes as well as reduction processes. We may
represent the nature of the changes as follows : —

Sulphide ^ Sulphur ^ Sulphate.

Thus complete oxidation as w^ell as complete reduction
of the sulphur element may be effected. The term sulphur
bacteria embraces only those organisms which oxidize the
HgS to S, store the latter temporarily in their bodies, and then
oxidize it to SO4. The term is thus used in a restricted
sense, for there are other bacteria which bring about chemical
changes in sulphur compounds. Thus we have : — •

1. Thionic Acid Bacteria or Thiosiilphate Bacteria, which
oxidize thiosulphates to tetrathiosulphates and sulphates in
accordance with the equations

SNa^SaOa + 50= aNa^SO^ + Na^S^Oe,
NagSgOa + 0 = Na2S04 + S.

2. Denitrifying Thiosiilphate Bacteria, which effect the
oxidation of sulphites, thiosulphates, sulphuretted hydrogen,

4 SULPHUR BACTERIA

or sulphur to sulphates, the necessary oxygen being obtained
by the reduction of nitrates.

3. Siilphale-redncing Bacteria, which effect the reduction
of sulphates, sulphites, and thiosulphates to sulphuretted
hydrogen.

4. Saprophytic Bacteria, which by their activities liberate
sulphuretted hydrogen from the organic molecule.

Although these four classes of bacteria are concerned with
the sulphur metabolism, they are not termed sulphur bacteria.

Subdivision of the Sulphur Bacteria. — The first division is
based on the presence or absence of colour.

1. Colourless sulphur bacteria.

2. Coloured sulphur bacteria.

The colouring matter is composite in structure, and the
sum total of the ingredients imparts to the organism a purple
colour. The purple colouring matter is also found in bacteria
other than the sulphur bacteria. Whilst it has not yet been
proved that the association of this colouring matter with the
sulphur metabolism is other than accidental, it is a note-
worthy fact that all the coloured sulphur bacteria are coloured
with the same group of pigments. Although there are con-
siderable differences in the tints of the various growths of
sulphur bacteria, they differ only in degree, not in kind. The
differences are due to variations in the proportions in which
the constituent colouring substances are present.

Geographical Distribution of the Sulphur Bacteria.

If we consider the conditions which favour the multiplica-
tion of the sulphur bacteria, it is not surprising that their
distribution is universal. They thrive equally well in salt
water and fresh water. They occur wherever saprophytic
decomposition results in the production of sulphuretted hydro-
gen, and wherever, in addition, the water is shallow, the supply
of oxygen not abundant, and the temperature suitable. As
these conditions are obtainable in all parts of the world, with
the exception of certain areas with special conditions (polar
regions, deserts, etc.), the sulphur bacteria are universally

INTRODUCTION 5

distributed. They are noticeable in the pools among the rocks
on the shore where seaweed is undergoing decomposition, in
shallow pools polluted with sewage, at the mouths of sulphur
wells, and in many shallow waters, both marine and fresh, in
which organic matter is present in solution, and in which
the conditions as to oxygen, hydrogen sulphide, etc., are
suitable.

If various putrescent organic masses (remains of animals
and plants) be enclosed in bottles, covered with water, and
stoppered up, and if these be left exposed to light, the
development of purple sulphur bacteria may almost with
certainty be observed in some of them after a few weeks.

Their development on a large scale is seen in shallow land-
locked bays in which seaweeds and other plant debris are in
a state of putrefaction. Near Copenhagen, where such con-
ditions prevail, and where some of the bays on their shore side
are filled with decomposing material, the purple sulphur
bacteria impart their colour to the whole of the water occupied
by the decomposing material.

The same phenomenon has been observed in various bays
in Jamaica, where broad stretches of water off certain of the
shores are tinted purple from the same cause. Isolated
patches of purple arc comparatively common on most shores
on which seaweed is undergoing decomposition. It is not
surprising that observations of the occurrence of the coloured
sulphur bacteria should date from early times, or that they
have been recorded from many sources, for their appearance
in mass is sufficiently distinctive to draw the attention of even
the least observant. The record of the occurrence of the colour-
less sulphur bacteria is not so extensive, owing to the drabness
of the colour of their mass cultures. These usually assume a
grey felted appearance. Thienemann records that Aristotle
remarked on the white patches in certain rivers, and that
Pliny called attention to the blood-red colour of the " Vul-
sinischen " Sea * in the year 208 B.C. It is doubtful, however,
whether the sulphur bacteria were responsible in these cases,

* Now called Lago di Bolsena, in Etruria, Italy.

6 SULPHUR BACTERIA

for the white patches in rivers are almost certainly the result
of the multiplication of those species designated somewhat
loosely as sewage-fungi ; and the blood-red water could not
have been tinted by the sulphur bacteria, for these are found
only in shallow waters.

The observations on coloured sulphur bacteria are numer-
ous. The following recorders among others may be noted : —

{a) Egypt, Arabia, Siberia. — Ehrenberg, Chr. G. (1830) ;
Hirsch, B. (1874).

[b) Germany. — Ehrenberg, Chr. G. (1830) ; Corda, A. J. C.
(1835) ; Cohn, F. (1875) ; Zopf, W. (1882) ; Engler, A. (1882) ;
Winogradsky, S. (1887) ; Zacharias, O. (1903) ; Kolkwitz, R.
(1914) ; Duggeli, M. (1917) ; Buder, J. (1919) ; Gicklhorn, J.
(1921).

(c) Great Britain. — Lankester, Ray (1873) ; Klein, E.
(1875); Ewart, A. J. (1897); Skene, Macgregor (1914) ; Ellis,
D. (1924-30)

(d) France.- — Fontan, A., and Joly, N. (1844) ; Gerardin

(1873).

[e) Russia.- — Weisse, J. F. (1845) ; Winogradsky, S. (1884) ;
Jegunow, M. (1898) ; Nadson, G. A. (1903) ; Omclianski, W.
(1904) ; Arzichowsky, W. (1902) ; Elenkin, A. A. (1914) ;
Issatchenko, B. L. (1913-14).

(/) Italy. — Trevisan, V. (1842) ; Hinze, G. (l 914- 1 5).

(g) Denmark. — Warming, E. (1875).

(h) yapan.—Miyoshi, M. (1897).

[i] Holland. — Beijerinck, M. W. (1904).

(7) Ceylon. — Crow, W. B. (1923).

(k) United States of America. — Waksman, S. A. (1922) ;
Baas-Becking, L. G. M. (1924).

[l] Sweden. — Gertz, 0., and Naumann, E. (191 6).

(m) Poland. — Szafer, W. (1910) ; vStrzeszcwski, B. (191 3).

[n) Pyrenees. — Joly, N. (1882).
It will be evident from this list that the countries represented
in it are those that contain investigators, and that absence
of representation is due to the lack of investigators, not to
the absence of the sulphur bacteria.

Strzeszevvski (i) has made a study of the plant associations

INTRODUCTION 7

in the flora of three sulphur wells in West Galicia. In these
the following grouping prevails : —

1. First Zone.— Rich in HgS ; yellow-green Cyanophycece
and purple bacteria, particularly in the case of the latter, the
more motile forms. Absence of Beggiatoa, Diatomacece, and
Chlorophycece.

2. Second Zone. — Less rich in H2S (0-4 gram per 10
kilogram H2O). Fewer of Cyanophycece, a few Diatomacece,
and no Beggiatoa.

3. Third Zone. — Very small amount of HgS. Rich in
Diatomacecs, BeggiatoacecE, Chlorophycece and non-thermoph-
ilous Cyanophycece. Complete absence of purple bacteria.

The Sulphur Cycle in Nature.

Plant and animal remains are attacked by hordes of
saprophytic bacteria, and, after death, the protoplasmic
molecule undergoes a number of transformations. Such a
molecule may be compared to an accumulator as a storehouse
of energy. The bacteria which nature uses as scavengers
liberate some of the stored up energy in the molecule, and
at the same time obtain material for their sustenance. In
essentials the protoplasmic molecule is composed of a complex
of carbon, hydrogen, oxygen, nitrogen, sulphur, and phos-
phorus atoms. With each transformation the level of energy
is brought lower, and the molecule becomes less complex
in structure. The final stage is reached when the original
component atoms of the protoplasmic molecule have become
changed to carbon dioxide, water, nitrates, sulphates, and
phosphates. After complete oxidation the protoplasmic
molecule has descended to its nadir position as a source of
energy.

Sulphur is liberated as a constituent of the amino-acids
cystine and the associated cysteine, and sulphuretted hydrogen
is formed, either directly or indirectly, from one or other of
these compounds. This sulphide is then absorbed by the
sulphur bacteria, and the sulphur ultimately appears in the
surrounding water in the form of sulphates. The develop-
ment of sulphates is not, however, a simple progression, for

8 SULPHUR BACTERIA

usually there are many kinds of different bacteria in competition
and some of them effect oxidation, whilst others effect the
reduction of the sulphur element. Also the environmental
conditions are continually changing, so that at every change
the organisms which for the moment had gained the upper
hand in the decomposition of the protoplasmic molecule now
find themselves supplanted by other microbes which in their
turn must give way to others with fresh changes in the
environment.

The sulphate may be reduced to elementary sulphur if the
supply of oxygen be scanty, or possibly even to sulphuretted
hydrogen if there be no oxygen present. The elementary
sulphur may unite with iron or other metals to form sulphides.
Again, the sulphide may be replaced by carbonic acid to form
carbonates. Some bacteria oxidize thiosulphates to thio-
persulphates. Ultirfiately, however, all the sulphur is com-
bined in the form of sulphates, in which condition it cannot
be further exploited as a source of energy by bacteria. To
return to our metaphor, in the condition of sulphate the
accumulator is discharged, and must be connected up with
some source of energy if the sulphur is to be made once more
available to build up the protoplasmic molecule. This is
supplied by the sun through the medium of green plants.
The sulphates are absorbed by the roots of green plants,
and enter into combination with the carbohydrates built up
by the carbon-assimilation of the plants. Eventually the
sulphur which has been caught in this stream becomes a part
of the protoplasmic molecule once more, and the cycle is
complete.

As the energy of the universe is derived from green plants
(and a small number of minute animals), and as all animals arc,
directly or indirectly, nourished by green plants, the sulphur
in the protoplasmic molecule of such plants may pass into
and become incorporated with the protoplasmic molecule of
various animals. In such cases the sulphur-containing pro-
teins of the plants enter into the composition of the proto-
plasmic molecule of the animal. When the animal dies, the
fate of the sulphur element, speaking generally, is the same

INTRODUCTION 9

as that of the same element in the protoplasmic molecule of
the vegetable.

The changes to which we have referred are concerned
only with the relationships of bacteria and similar micro-
organisms to the protoplasmic molecule in the economy of
nature. Sulphur may of course be removed from the bowels
of the earth, and may be taken up as a sulphate or a sulphide
into the cycle governed by the activities of microorganisms.
Or again, the sulphur may lie dormant in such substances as
coal, and, after a rest of some millions of years in that form,
be once more taken up into the stream of that cycle. When
coal is burnt sulphur compounds are liberated into the air,
or spread over the surface of the earth. The converse must
also follow^, namely, the removal from the cycle of sulphur
compounds which may lie dormant for seons before being
brought back into circulation. Speaking generally, all
microorganismal and all combustion processes either directly
or indirectly bring about the complete oxidation of the
of the sulphur atom, and the reactions which bring about
this condition are exothermic.

The sulphur bacteria are thus like all plants and animals
in that they require sources of energy to make possible the
various manifestations associated with life. These manifesta-
tions are necessarily in their totality exothermic processes.
As will be shown in the following pages, however, it is probable
that the purple sulphur bacteria are able to do constructive
work in virtue of their ability to absorb energy from the sun's
rays. In this they resemble the plants and animals that pos-
sess chlorophyll, and as in the case of green plants such energy
is directed to the building up from simpler compounds of more
complex substances which thus become storehouses of potential
energy. The conversion of stored energy to what may be named
" vital " energy takes place when the more complex substances
revert to simpler forms, thereby liberating some of their energy.
The same laws regulate the changes of energy in the animate
as in the inanimate world.

SULPHUR BACTERIA

Methods of Investigation.

Our knowledge of the sulphur bacteria has been built up
by the application of three methods of examination, each of
which has its advantages and disadvantages.

1. Direct Observation under Natural Conditions. — This
is the best method of observing the developmental life-his-
tory of these bacteria, provided that the organisms can be
recognized with certainty, and readily distinguished from
all other organisms occupying the same field. The sulphur
bacteria are then observed in the normal course of their
development under natural conditions, and in competition
w^ith other organisms of the same class. The sulphur in-
clusions and the purple colour are noticeable features which
make their identification easy, and facilitate the identification
of the passage of an organism from one phase of development
to another. When observations of the same mass-cultures
are conducted at periodical intervals throughout the whole
year, the course of the life-history of an organism is sometimes
easy to follow. Also, in such cases it is often possible to
observe in organic connection two different forms of growth
which, without the observation of such connection, would be
regarded as specifically distinct. The valuable observations
of Zopf on both the coloured and colourless sulphur 'bacteria
were obtained by this method, and the same applies to the
published observations of the majority of the other mor-
phologists who have studied these bacteria. This method is
particularly valuable when the organism under observation
is present in very large numbers, as is usually the case in mass-
cultures of the sulphur bacteria.

2. Glass Slide and Coverslip Observations. — This method was
favoured by Winogradsky (1-3), and is described in Chap. IV.
In essentials it consists in placing material on a glass slide and
observing it at periodic intervals, so that the same individuals
are viewed after sufficient time has elapsed to enable them^ to
advance in growth. This method is valuable when it supplies
positive results, but no deductions are permissible from the
negative results obtained by its use. If negative results are

INTRODUCTION ii

obtained by this method it does not follow that the results will
be invariably negative under all conditions of growth. The
sulphur bacteria are exceedingly plastic, and it is not to be
expected that all their capabilities will be manifested under
the artificial conditions of the glass slide and coverslip ob-
servations. Under the stress of competition with other organ-
isms in nature they sometimes exhibit a variety of forms
that do not appear when they are cultivated under cloistered
conditions. Hence if a certain phase of development is
observed under natural conditions the value of the discovery
is not nullified if the same development cannot be produced
under certain other artificial conditions. With such plastic
organisms special circumstances bring out special reactions.
Special emphasis is laid on this point because in the observa-
tions of some of the investigators, and notably of Winogradsky,
it seems to be assumed that that which does not appear under
artificial conditions cannot appear under natural conditions
under any circumstances whatsoever.

3. Method of Pure Culture. — Pure cultures of the sulphur
bacteria have been obtained by Keil for the colourless, and by
Bavendamm for the coloured, bacteria. This is the most
valuable method of observation in the investigation of
physiological problems, many of which cannot be determined
in any other way. The same defect is, however, inherent in
this as in the previous method, for the course of the life-
history of bacteria is seldom the same under the artificial
conditions under which the cultivation must necessarily be
conducted, as it is under natural conditions. Hence whilst
the positive results obtained by this method are invaluable,
and particularly in physiological investigations, judgment
must be deferred if the results are negative, especially if such
results are in contradiction to the positive results obtained by
other methods.

All three methods have contributed their share to the
building up of the present body of our knowledge of the
sulphur bacteria.

12 SULPHUR BACTERIA

Pleomorphism.

By pleomorphism is meant the capacity of an organism to
assume diverse forms. A culture of bacteria may contain
several variant forms simultaneously, or these may be formed
in succession. Bacteria in general are noteworthy for the
ease with which changes in form occur in them. The ease
with which certain physiological changes bring about changes
in the external appearance of bacterial cells is a matter of
common knowledge. Probably no other organisms possess
greater plasticity in this respect. Unless, for example, a
wide range is given to the dimensions of a bacillus, a state-
ment of its length or its thickness has little value for
purposes of identification. It is a matter of common ob-
servation, too, that, under certain circumstances the rate of
growth in size and the rate of division of bacteria do not
run parallel. The rate of each is dependent on circumstances
of which at present we have very little knowledge. If the
rate of division is accelerated the individuals of successive
generations become progressively smaller. Under certain cir-
cumstances the rate of division of Crenothrix polyspora, one
of the iron bacteria, may so far exceed the rate of its growth
that the individuals may be so reduced in size that they are
barely visible with the highest powers of the microscope.
Again, the change that takes place in the size of the individuals
of a bacterial culture after frequent subculture is well known.
Further, every living bacterial cell is at all times covered by
a delicate covering of slime formed by transformation of the
outermost layer of the membrane. Given certain conditions,
this slime formation increases in intensity, and the motility of the
cell is retarded, and may even cease altogether. Conversely,
by the elimination of excessive slime formation non-motile
forms may be made to assume motility.* Other instances
could be adduced in illustration of the morphological plas-
ticity of bacteria. Over 2000 species of the genus bacillus

* By subculturing at the very earliest sign of growth on the surface of
agar-slope cultures, the motility of organisms may be considerably en-
hanced, and even forms regarded as non-motile may be made to assume
motility (Ellis (2-3)).

INTRODUCTION 13

alone have been described, and, in all probability, with greater
knowledge of the capacity of bacteria to accomplish morpho-
logical changes it should be possible to reduce this number
considerably. Organisms like Bacillus subiilis, for example,
must have been described under numerous names, for the
aggregate of its characters is never exactly the same from
one generation to another.

The difference between these slight changes in the form
of bacterial organisms and pleomorphism is one of degree
not of kind. The change of form in pleomorphism is more
pronounced and probably more sudden in its appearance.
The sulphur bacteria are at a stage in which, compared with
the lower bacteria, a slight advance in evolutionary progress
has taken place. Developments that are mere tendencies in
the lower bacteria have become more or less crystallized in
them. In some the filamentous condition is normal for the
species, slime formation is a characteristic feature of develop-
ment, and the zoogloea condition with its enclosed mass of
bacteria makes its appearance. Such developments have not,
however, resulted in the stabilization of any one of the forms
of growth, and so in the life-history of some of the species it
is possible to find forms of growth of different morphological
features. The changes in form may take place in successive
generations or the differences may all appear in the same
generation. Or again, the culture may be unimorphic for
several generations and then, under the influence of certain
(at present unknown) changes, become pleomorphic. It is
evident that the proof of the existence of pleomorphism can
be obtained only by chance, or by the long-continued in-
vestigation of an organism, when at length some proof may
be expected. It cannot be demonstrated with regularity,
because the conditions w^hich bring about its manifestation
are at present unknown.

The evidence for the existence of pleomorphism in the
sulphur bacteria may now be set forth.

14 SULPHUR BACTERIA

Evidence of the Occurrence of Pleomorphism in the
Sulphur Bacteria.

Lankester (i) and (2) found a "peach-coloured bac-
terium " on decomposing animal remains (caddis worms) in
an Oxford laboratory. This organism [Bacterium ruhescens)
assumed a bewildering variety of forms, all tinted with a
purple colouring matter which Lankester named Bacterio-
purpurin, and all contained inclusions which we now know
to be sulphur granules. In this case we have either to assume
that a dozen or more different species of the comparatively
rare sulphur-containing purple bacteria had all settled simul-
taneously on the caddis worms, or that all were pleomorphic
variations of one or at most two or three species. Lankester
chose to make the latter assumption, and gave one name to
all the variants, namely, Bacterium rubescens.

Warming examined the purple covering on the surface of
decomposing vegetable remains on the Danish coast, and there
found an organism of a similar protean habit. He regarded
it as another species of the same genus as that described by
Lankester, and named it Bacterium sulfuratiim. At the time
(1876) the number of bacteria of all kinds which had been
described was very small, and both Lankester and Warming
considered that the total number of species was very hmited.
Indeed, Warming deduced from his experience of Bacterium
sulfiiratum that all bacteria had an unlimited capacity for
changes of form, and could all with propriety be brought within
the compass of a single genus. Warming was the first to make
a clear pronouncement on the existence of pleomorphism,
although with the hmited knowledge that was then at his
disposal he overstated the case. The most important part
of Warming's contribution was his discovery of intermediate
forms. He figures two or three hundred varieties all found
in the same medium and linking up by innumerable inter-
mediate forms the most diversely shaped varieties. Recently
Bavendamm has recorded the appearance in his artificial
cultures of an organism which he considers as identical with
Bacterium sulfiiratum. This was a dull red irregular rod-

INTRODUCTION 15

shaped organism of sizes varying from 3/x to I2/^, although
some extended to 58/x. As Bavendanim regards Warming's
Bacterium sulfuratum as a composite species he was not able
to place the organism, and hazarded the conjecture that it
was a Rhahdochromatium.

Zopf, in 1882, made a distinct advance in our knowledge.
He also had found an extremely variable purple-coloured
microbe, which he named Beggiatoa roseo-persicina. Up to
Zopf's time the evidence of pleomorphism had been of a
purely circumstantial nature, as is, indeed, the greater part of
biological evidence. He considerably strengthened the case
for pleomorphism by observing the actual transition of one
type of structure into another. Thus in his Spaltpflanzen,
Tafel 15, Figs. 3a, 3^, and 4 obviously show a stage in the
transition from the filamentous to the coccus form. He
thus observed the development of one fundamental form from
the other. Then again, he traced the passing of coccus forms
into the zoogloea condition. Zopf witnessed the actual
liberation of the end portion of a thread of Beggiatoa alba
and its conversion into a spiral structure, which on liberation
was observed to swim away ; in that form it was indistinguish-
able from a typical spirillum of the lower bacteria. If seen
apart, such an organism would indubitably have been assigned
to the genus spirillum. It is evident from Zopf's work that
the filamentous form, the short rod, the coccus, and the
spirillum may be combined in one species.

In 1888 appeared Winogradsky's Beitrdge ziir Morphologie
iind Physiologie der Bacterien, which has greatly influenced
subsequent investigations. In this work a completely
different view is presented. He starts with de Bary's dictum
that a species is not determinable until its developmental
history is known. The soundness of this axiom cannot of
course be questioned, but there are objections to the
interpretations that are given to it by Winogradsky. The
implication is made in his writings that, if an organism is
put under observation under a given set of circumstances all
the possible, developmental phases of which it is capable
will be exhibited during that period. Winogradsky's method

i6 , SULPHUR BACTERIA

of observation is described on page 53. The organism under
observation is confined between glass slide and coverslip, and
treated with HgS at appropriate intervals (he held that the
introduction of organic matter was not necessary). When
under these artificial conditions the one phase of growth
under observation did not develop into some diflferent develop-
mental phase, he concluded in efi^ect that the transformation
was not possible under any combination of circumstances
under natural conditions. He held that two phases of growth
belonged to one and the same species, provided that it was
possible by a continuous-observation experiment to effect the
transformation of the one into the other. When he failed to
accomplish this, the different phases of growth were to be
adjudged as belonging to different species. On the strength
of the negative results obtained in this way Winogradsky
discounted the positive information obtained by the earlier
investigators ; and he felt justified in consequence in launching
a number of new genera and species, as each phase of growth
observed by him was regarded as a separate organism. Of
the numerous genera and species which are recorded by him
we know practically nothing of the developmental history.
As we know also practically nothing of their internal struc-
ture, or their methods of reproduction, it must be held that
Winogradsky's contributions have added somewhat to the
difficulties of subsequent investigations into the morphology
of the sulphur bacteria.

It was inevitable that the attention given to Winogradsky's
investigations should have obscured the evidence for pleo-
morphism obtained by the earlier writers ; for the greater
part new organisms have been described after observation
under only one set of circumstances, and their pleomorphic
possibilities have seldom been considered. It has inevitably
followed that comparatively large numbers of new organisms
have appeared, and have been labelled without adequate
study of their structure, their developmental history, or their
methods of reproduction. The consequences which have
followed this attitude when applied to the classification of
the sulphur bacteria have been unfortunate, and will be
described in later pages.

INTRODUCTION 17

It is of interest here to note one definite positive result
in favour of pleomorphism which was obtained by Wino-
gradsky himself. He records the growth of cells inside the
slimy covering of an organism which he has named Thiocystis
violacea. The cells which were at first globular were observed
to change shape and become elliptical until the length was
double the thickness. Then he observed the escape of these
altered cells from their slimy covering. The slime lost its firm
consistency, became swollen, and finally disappeared, thus
freeing the enclosed cells, which then disappeared from the
field of view in aggregated masses of varying sizes. The
removal was effected by their own organs of motility which
were probably cilia.

An interesting piece of evidence in favour of pleomorphism
is supplied to us by Bavendamm in his study of Chromatium
Warmingii forma minus. Although this organism has been
studied by previous investigators the variation in question
was not observed until Bavendamm cultivated it under new
conditions, namely, the conditions of pure culture. The
result was the development of buds on the cells, somewhat
similar to the buds which normally appear on yeast cells.
Also the buds of different cells appeared to be able to effect
fusion as though preparatory to sexual reproduction. Al-
though this must not be regarded as a definite example of
pleomorphism, it does show that when the normal conditions
of growth are altered the organisms readily respond by chang-
ing their structure and normal habits.

The Author's Investigations.

The detailed life-history of the organism Thioporphyra
volutans is given in Chap. VIII. We may here, however, call
attention to the evidence in favour of pleomorphism which was
obtained by the investigation of this organism. The species
is normally a large uni- or diplo-coccus of a purple colour,
and with sulphur inclusions (Figs. 33-35). Under certain cir-
cumstances buds are formed on the cocci in great profusion,
and result in the culture fluid assuming a deep purple tint.

2

i8 SULPHUR BACTERIA

The buds, when quite small, are abstricted from the parent
organism, each usually containing a single sulphur granule
(Fig. 35). They are found in the surrounding medium in
aggregated masses, and are devoid of movement. Without
the proof supplied by the organic connection of the buds
with the parent organism they would ordinarily have been
regarded as a species of Lamprocystis.

Although pure cultures of Thioporphyra volutans have not
yet been obtained it is possible to intensify its growth in an
artificial medium (see p. 62), which in consequence of its
growth becomes tinted purple. The individuals, however,
are much smaller in size. They vary from cocci of lO/x in
diameter (the normal size) to small globules that are only
if/i, in diameter. With sufficient assiduity it would have
been possible to arrange the cocci in a descending series in
which any particular form was a fraction of a /a smaller than
the preceding unit, until the minimum of if /a had been reached.

A further proof of pleomorphism was supplied by Chro-
•niatium Linsbaueri, which was found in a small pool of water
in the Epping Forest, near London. The normal form of the
organism is ovoid, and it was this type of structure that was
presented in all but one of the samples that were periodically
sent to the writer. In the exceptional sample, however,
it was found that about 10 per cent, of the individuals were
spiral-shaped. As the general structure and colour of this
organism are of a very distinctive nature, and as the two forms
were absolutely alike in every other respect except that of
shape, there was every reason for the conclusion that both
were variants of the same organism (see Fig. 30 C).

Although the organism Cladothrix dichotoma is not one of
the sulphur bacteria, it is sufiiciently closely related to regard
evidence of pleomorphism in it as contributary evidence
of pleomorphism in the sulphur bacteria. The threads of
Cladothrix dichotoma were observed in a particular solid
medium culture to be violently agitated, and to break up into
spirally wound fragments of short lengths, each of which
developed polar cilia. Intercalary fragments were observed
to " sidestep " from the threads, assume a spiral form, and

INTRODUCTION 19

rapidly separate themselves from the parent threads (see
Ellis (5)).

Another form of pleomorphism is observed in Crenothrix
polyspora, another of the iron bacteria. There are occasions
when this organism departs from its normal structure. The
cells inside the sheath which envelops them break loose and
divide up into minute round fragments, and in that state con-
tinue an existence which is totally different from that normal
to this organism, so much so that seen separately the two
forms would not be assigned to the same species.

The evidence points to the occurrence of pleomorphism,
not as a normal, or perhaps even a frequent phenomenon
in the life-histories of the sulphur bacteria, but rather as one
likely to occur with great frequency if certain conditions pre-
vail, or arise with any degree of frequency. If they do not
arise then it is probable that the organism remains unimorphic
indefinitely. The possibility, however, of its appearance must
be taken into account in framing a system of classification of
the sulphur bacteria.

CHAPTER II.

THE PRODUCTION OF SULPHURETTED HYDROGEN
AND ITS ASSIMILATION BY THE SULPHUR BACTERIA.

Natural Sources of Sulphuretted Hydrogen.

I. Decomposition of Albuminous Substances. — Animal and
plant remains furnish the most important material from which
sulphuretted hydrogen is formed. These are attacked by
saprophytic microorganisms, and in the resulting decompo-
sition sulphuretted hydrogen is liberated. The majority of
the forty or fifty natural proteins which occur in plants and
animals contain sulphur, and probably about 90 per cent,
of the saprophytic organisms in the soil are able to effect
the liberation of sulphuretted hydrogen from albuminous
material (see p. 26).

Emil Fischer and his pupils have shown that the protein
molecule is built up of amino-acids, so that the liberation of
sulphuretted hydrogen will follow from the degradation of
amino-acids which contain sulphur. The sole representatives
of such acids are cystine, the associated cysteine, and glutathione,
which has recently been shown by Nicolet, and Kendall and
his co-workers, to be a tripeptide y-glutamyl-cysteinyl-glycine
bearing the structure
H,N . CH(COOH) . CH, . CH, . CO i NH . CH(CH,SH) . CO ; NH . CH.COOH

[H^N . CH(COOH)CH, . CH,, . COOH] [H,N . CH(CH,SH)COOH] [H^N . CH^ . COOH]
Glutamic acid. Cysteine. Glycine.

Cystine is COOH . CH(NH2)CHo . S . S . CH^ . CHINH^) . COOH.
Cysteine can be converted to cystine by oxidation.

rCHo— S— H CH2— S— S— CH2

I II

2]CH.NH2 +0 >CH.NH2 CH . NH^ + H^O

I II

ICOOH COOH COOH

THE PRODUCTION OF SULPHURETTED HYDROGEN 21

The formation of sulphuretted hydrogen from cysteine is
given in the following equation : —

CH2— S— H CH3

CH . NH2 + H., — > CH . NH2 + H2S

1 I

COOH COOH

or it may be indirectly formed through such intermediate
compounds as

a-Thiolactic acid (CH3 . CH . SH . COOH),
ThioglycoUic acid (CH^ . SH . COOH),
and Methyl mercaptan (CH3 . SH).

The presence of sulphuretted hydrogen in tube cultures is
easily demonstrated by means of filter paper, which has been
previously soaked in lead acetate solution. The paper thus
treated is placed inside a tube containing a growing culture,
and turns black from the formation of lead sulphide if
sulphuretted hydrogen be given off. Or, the gas may be re-
cognized by the formation of the black ferrous sulphide in
the presence of iron salts. The sulphur, when dissolved by
aceto-carmine, crystallizes outside the cells, where it forms
typical fiat octohedra (see Fig. 34^), or the globules may be
treated with hot potassium cyanide and ferric chloride, when
they turn a blood-red colour from the formation of ferric
thiocyanate Fe(CNS)3. The chemical tests, however, are nor-
mally not necessary because the appearance of the sulphur
globules in these cells is so characteristic that they can be
identified from their general appearance.

There are means of effecting the liberation of hydrogen
sulphide other than from an amino-acid split off from a protein
molecule. Thus the decomposition of protein matter by some
of the saprophytic bacteria is attended by the formation of
a scum of sulphur on the surface of the culture fluid. Such
sulphur must have been derived from albuminous matter.
Its union with hydrogen to form sulphuretted hydrogen is
described later.

22 SULPHUR BACTERIA

Selinsky and Brussilowsky succeeded in effecting the
decomposition of a substance in which the S was combined
with two carbon atoms, and such being the case, they con-
cluded that the formation of hydrogen sulphide could have
resulted only after an antecedent liberation of free sulphur ;
and that the hydrogen sulphide resulted from its subsequent
union with hydrogen. It must be noted that this implies the
union of hydrogen with sulphur at ordinary temperature.
As ferments, however, cause reactions within the living body
at ordinary temperatures, which, when carried out by acids,
demand heat, there should be no difficulty in accepting the
possibility of the union of hydrogen and sulphur at the ordinary
temperature when the reaction is controlled by vital forces.

2. Reduction of Inorganic Compounds containing Sulphur.

[a) Reduction of Sulphates. — Beijerinck (2) found that some
bacteria, cultivated under anaerobic conditions, effected the
reduction of sulphates to hydrogen sulphide. Again the pro-
duction of the sulphide is a common occurrence in muds
and other sediments in which the supply of oxygen is small
and the supply of organic matter large. One organism of
this kind which w^as isolated was named Bacterium hydro-
sulfureum. It is pointed out by Nadson that the ability to
reduce sulphate is characteristic of a large number of organisms,
given suitable conditions, and that many of such bacteria,
for example Proteus vulgaris and Bacillus vulgaris, are not
anaerobes, so that the reduction of sulphur may take place in
the presence of an abundant supply of oxygen.

Beijerinck has studied the chemistry of this reduction and
states that in black mud the following changes take place: —

(1) SO4-- > H,S.

(2) H2S + Fe++ > FeS.

(3) H2S + Fe+++ — > FeS + S.

(4) FeS + H2CO3 — ^ FeCOg + H.S.

(5) H^S — ^ S.

(6) S > SO4- -.

(7) S > Hp.

(Quoted from Baas-Becking (i).)

THE PRODUCTION OF SULPHURETTED HYDROGEN 23

Hence the sulphur atom in black mud runs the gamut of
changes from complete oxidation to the form of sulphate to
complete reduction to the form of the sulphide. His conclu-
sions are generally accepted although Baas-Becking points
out that in the case of one of the reactions, namely, No. (4),
the change is carried out in alkaline water with a considerable
buffer value, for the amount of COg must be considerable, which
first lowers the alkalinity and then effects the change indicated
in the reaction. He argues that to effect this reaction in the
mud the concentration of CO2 in the bottom of the sea over-
lying the black mud of the Black Sea must be at least sixty
times greater than the concentration known to exist at the
surface, and concludes that in such places this particular
change is not possible unless there is present another source
of acidity in addition to the carbon dioxide. It is highly
probable that the supply of carbon dioxide is supplemented
under these conditions, for such black sand contains not only
acid-producing organisms but also the organic material which
makes possible their multiplication.

[h) Reduction of Thio sulphates. — Holschewnikoff found
that the two organisms Vibrio hydrosulfureiis and Bacterium
hydrosulfuricum ponticum, which he had isolated from mud
at Wiesbaden, effected the change of sodium thiosulphate
(NagSaOg) to hydrogen sulphide when cultivated in appropriate
media.

(c) Reduction of Sulphites. — Beijerinck showed that the
yeast plant under appropriate conditions reduces oxy-sulphur
compounds to hydrogen sulphide.

Hydrogen sulphide is thus a direct or an indirect deriva-
tive of the decomposition of organic matter under the agency
of various saprophytic microorganisms. Under aerobic con-
ditions it is broken off directly from a constituent of the protein
molecule. Under anaerobic or micro-aerobic conditions its
formation is more indirect and is bound up with a reduction
process.

3. By Direct Union of Hydrogen and Sulphur. — The direct
union of hydrogen and sulphur may be effected by the vital
activity of microorganisms. Some writers have claimed that

24 SULPHUR BACTERIA

a definite principle, a ferment, or something of a similar nature,
is secreted by certain microorganisms, and that this principle
effects the union of these elements. Miquel named it a
ferment sulf-hydrique. The use of the word ' ferment,' to denote
the agent of an endothermic reaction of this nature, is contrary
to the usually accepted meaning of that term. Duclaux
considered that hydrogen was liberated by the organism, and
that the union with sulphur took place outside the cells in
the surrounding medium, not inside them, as claimed by
Miquel. Beijerinck has confirmed this statement, and has
cited the following experiment in justification of his view.
Two flasks are filled, one with fresh water, ferrolactate, flowers
of sulphur, and ditch water, and the other with the first, second,
and fourth only, of these ingredients. Both are allowed to
decompose by the activity of the microorganisms contained
in the ditch water. It is found that ferrous sulphide is formed
in both flasks. As the sulphide is formed in the second flask
which contained no added sulphur, he concludes that in all
probability the sulphide in the first flask was also formed
independently of the sulphur added to it ; and hence that the
union of hydrogen and sulphur is in both cases a secondary
reaction which takes place outside the cell.

It has been claimed by Rey-Pailhade that the synthesis
of hydrogen and sulphur is effected by certain yeasts, and
that a specific substance which he named Philothion is secreted
by the plant to effect the union. No experimental evidence
for the existence of this ferment has been supplied.

The Production of Hydrogen Sulphide under Marine

Conditions.

The deeper layers of marine waters containing hydrogen
sulphide are completely devoid of the fauna and flora which
occupy the upper layers. The Black Sea, which was investi-
gated by the Russian Deep Sea Expedition of 1891, is a case
in point. The water was found to contain the sulphide at all
depths, and below 200 — 400 metres macroscopic organisms were
completely absent. The following table gives the amount of
hydrogen sulphide at the various depths : —

THE PRODUCTION OF SULPHURETTED HYDROGEN 25

Depth in Metres.

Vol. H2S in c.c. per
Litre of Water.

213

427

2026

2528

0-33

2-22

5-55
6-55

The water at the sea bottom contained twenty times as
much hydrogen sulphide as the surface layers.* This seems
to indicate that the sulphide in the lower layers originates
from the sea bottom as a result of the active decomposition
of organic matter under anaerobic or micro-aerobic conditions,
for it would be difiticult otherwise to account for the presence
of the sulphide.

The Limans.

This name is given to the shallow salt marshes at the
mouths of the Dnieper which open into the Black Sea at
Odessa. The bed of these marshes is composed of black
mud which reeks of hydrogen sulphide, and is alkaline in
reaction. On exposure to air it turns a grey colour, but be-
comes black once more when covered with water. Anaerobic
bacteria liberating hydrogen sulphide have been isolated from
this mud, and one has been identified as the Vibrio hydro-
sulfureiis to which reference has already been made. It is
claimed that this organism is responsible for the reduction of
oxy-sulphur compounds to the substance which imparts the
black colour to the mud. This is a hydrate of sulphur and
iron, and forms a black colloidal covering to the other con-
stituents of the marsh bed.

The Author's Investigation of the Black Sand of the
Clyde Estuary.
In various localities on the Clyde, the sand of the shore
is deep black in colour, and its formation is intimately
associated with the multiplication of bacteria which produce
hydrogen sulphide (see Ellis (8)). The blackness disappears

*The adjacent Sea of Marmora and the other neighbouring seas are
normal in respect to their content of hydrogen sulphide.

26

SULPHUR BACTERIA

when the sand is allowed to dry ; it then assumes a grey
or sometimes a golden-brown colour, similar to the normal
sand in the same neighbourhood. When it is again wetted
the black colour returns. The following facts have been
determined : —

(i) Number of Bacteria. — The following figures give the
" total count " of bacteria in sand selected from different
parts of the Clyde : — -
No. of Sample.

I

120,000 per gram^

2

100,000 ,,

3

10,000 ,

4 ■

2,916,000 ,

5

1,000,000 ,

6

3,000,000 ,

7

40,000 ,

8

400,000 ,

9

2,000,000 ,

10

30,000 ,

II

600,000 ,

12

100,000 ,

Calculated on sand with-
>. out freeing it of its water
content.

It was found that at least nine-tenths of these bacteria
liberated hydrogen sulphide under appropriate conditions of
cultivation.

(2) Organic Matter, Total Iron, and Moisture. — The amounts
of these constituents are given as percentages in the following
table : —

No. of Sample.

HoO.

Fe.

Organic Matter.

I

19-0

0-53

2-2

2

25-6

0-56

3-0

3

29-0

0-36

1-2

4

20-4

0-43

07

Two other samples examined independently by workers in
the author's laboratory gave 0-187 pcr cent, and 0*189 per
cent, respectively for the total iron content.

* Subsequent counts on other samples from the same district gave the
figures 600,000, 650,000, 400,000.

THE PRODUCTION OF SULPHURETTED HYDROGEN 27

The sand consists of clay, fine particles of silica, and organic
debris, interspersed with black particles which in most cases
form a covering to the particles of silica.

(3) The Rocks in the Clyde Drainage Area. — To the North
are the Campsie and Kilpatrick Hills, which consist almost
entirely of basaltic lavas rich in magnetite, with an iron con-
tent amounting to lO — 15 per cent, or more. On the South
the drainage area includes the hills that extend through
Renfrewshire and Lanarkshire, which are basaltic lavas.
Along the course of the River Clyde the rocks are mostly
Carboniferous, and include the Carboniferous Limestone
Series containing coal and ironstones, and the Coal Measures
in which these are also abundant. These ironstones contain
FeCOg, but the carboniferous coal and shale beds also contain
large quantities of FeS. Finally, in the upper reaches of the
Clyde, and also on the lower reaches on the north side we
find the Old Red Sandstone Series, the rocks of which are
composed of a sandstone with an iron oxide matrix. It is
therefore natural to find a fair quantity of iron in the com-
position of the sands on the shore of the Clyde.

(4) Formation of Ferrous Sulphide (FeS). — The statements
detailed above show that the conditions are favourable for
the formation of ferrous sulphide. The amount of organic
matter is large enough to provide ample food for the nourish-
ment of saprophytes ; there is ample moisture to permit of
their development ; those organisms are present in abundance
which are capable of bringing about the production of hydrogen
sulphide ; and in addition the sand is rich in iron. It was not
found possible by a simple chemical analysis to make a direct
quantitative estimation of the amount of ferrous sulphide in
the sand. From work done in the author's laboratory it was
concluded that the black colour was entirely due to ferrous
sulphide, because when the iron was extracted, and the residue,
suspended in water, treated with sulphuretted hydrogen,
no black colour was produced. On the other hand, a sample
from which the iron had not been extracted but which had
lost its black colour by drying regained its colour when
sulphuretted hydrogen was passed over it.

28 SULPHUR BACTERIA

These facts are in accord with the conclusions of Doss,
who states that black muds containing abundant organic
material and impregnated with colloidal hydrous ferrous
sulphide occur in many localities, both in enclosed seas and in
gulfs and bays connected with the open ocean. He instances
the eastern Mediterranean Sea, the Black Sea, the Sea of Azov,
the Caspian Sea, and the Dead Sea. Similar muds occur
around Oesel Island in the Gulf of Riga, and around the
mouths of the Elbe and Weser. According to Andrussow (3)
the mud from the bottom of the Black Sea contains an
abundance of ferrous sulphide, and this compound occurs
generally in the black shore muds and sands of the Limans
that border the Black Sea. Omelianski (i) states that this
mud when exposed to air becomes oxidized to a grey colour.
Harder regarded the black mud found on Russian coasts as
ferrous sulphide, and the lighter mud as a mixture of ferrous
and ferric sulphides. He did not, however, advance any proof
in support of this statement. Neither was it possible to con-
firm his statement that the ferrous sulphide existed in a
colloidal state. Even when the sand is wet and black, the
black particles are large enough to be easily distinguished
from the particles of silica.

(5) Cause of Disappearance of Black Colour on Exposure
to the Atmosphere. — It is generally held that the change in
colour is due to the oxidation on exposure to air of black
ferrous sulphide to oxy-sulphur compounds of iron which are
not black in colour. It is also believed that the iron is
dissolved out of iron-bearing minerals, and is removed as
ferrous carbonate, and that it is later deposited and converted
into ferrous sulphide by bacteria. The author considers that
the reaction between FeCOg and HgS is reversible, and that
the light or black condition is determined by whichever
of the two compounds FeCOg or FeS preponderates. The
reaction is as follows : — ■

FeCOg + H2S ^ FeS + HoCOg.

Here we have a delicately balanced state of equilibrium.
With the production of HgS under moist conditions ferrous

THE PRODUCTION OF SULPHURETTED HYDROGEN 29

sulphide is formed, and the sand is black. With dehydration
the reaction is reversed and more FeCOg than FeS is produced ;
and it is this condition that results in a lightened colour.

The Equilibrium of Hydrogen Sulphide in Water.

Sulphur bacteria grow as readily in sea water as in fresh
water, since they are not affected by the degree of salinity
of the water. When, however, they multiply in sea water,
the multiplication takes place in a hard water in which the
concentration of the hydrogen ions is low owing to the high
buffer value of calcium and magnesium salts. The equilibrium
of the sulphide in such a medium has been studied by Baas-
Becking (i).

The H2S will first be dissociated into hydrosulphide ions
and hydrogen ions.

Ki[H3S] = [H^[HS-].

First dissociation constant, Kj = 0-91 X io~' at 18° C.
In the second dissociation we have

K,[HS-] - [H+][S--].

Second dissociation constant, K., = 10 ~^^ at laboratory tem-
perature, also

Ka>= [H^-j[OH-] = 10- 1*.

Hence, as the sum of the positive charges is equal to the
sum of the negative charges,

[H+] = [HS-] + [S--] + [0H-]
_KJH2S] KJHS-] Kg.
[H+] ^ [H^-] ^[H+]
_ K,[H,S] KiK,[H,S] Ko^
^ J [H+] ^ [H+j^ "^ [H-*-]

[U^Y = K/[H-^][H,S] + KiK,[H,S] + Ka>[H+]. . .(i)
[H+]3 - Ka>[H+]

and [H2S] =

K,[H-*-] +K,K2*

* Baas-Becking's paper shows 2KjK, in place of KjKj, but this does
not affect the conclusions.

30

SULPHUR BACTERIA

Noting that the solubiUty of HaS = 0-102 when [H+] is
about I0~^, then, in equation (i),

the first term is of the order I0~' X 10"^ X 10"^ = I0~i*
,, second ,, ,, ,, lO"' X lO'^^ X IQ-^ = lO^^s

,, third „ ,, ,, lO-i* X 10-^ = IO-20

The second and third terms are therefore neghgible in
comparison with the first,

.-. [H^]3=Ki[H,S][H+]
[WY= Ki[H2S]

= 0-93 X io~^
.-. [H+] = 0-96 X lo-^

Baas-Becking obtains the value H"*" = y-d) X lO"^ by using
a method of solution which he names the " Cardanus method "
(unknown to the author) ; and he has drawn up the following
table on the assumption that the metal ions are present in
very low concentration (B"*" = lO"^) : —

H+.

H2S.

HS-.

s--.

10-5
10-^
10-'
10-*
10-9

Z-Z X IO-*
1-2 X IO-*

I-I X 10-5
I X lo-"*

2-2 X IO-*

2 X 10-5
I-I X IO-*

1 X 10-^
g-i X 10-^

2 X IO-*

2 X lO-l^

I-I X lo-i*

1 X IO-"

9-1 X lo-i^

2 X lO-l*

The assumption that all the sulphide ions are in attachment
to the H-ion seems arbitrary, and from observations of natural
waters there appears no warrant for such an assumption.

In order to present these figures graphically he calculates
the logarithm of the concentrations Cg, Ch^s; C^s-, Cg — ,
and, changing the sign throughout, plots the latter three
against the first, which is, of course, the^H of the solution : —

Ch.

Ch2S-

Chs--

Cs".

-5

— 2-66

-4-79

- 14-70

— 6

— 3-92

- 4-96

— 13-96

- 7

- 4-96

— 5-00

— 13-00

- 8

— 6-00

- 5-04

— 12-04

- 9

- 7-66

- 5-70

— 11-70

These results may be presented graphically (Fig. i]

THE PRODUCTION OF SULPHURETTED HYDROGEN 31

With these data it is possible to ascertain the conditions of
equiUbrium in sulphur waters in which, according to Baas-
Becking the^H value varies from 7-6 to 8-6. At^H 7-6 the differ-
ence in the logarithms of the concentrations of HS~ and HgS
is 0-55, as indicated in the graph. Hence the actual ratio of

HS~ n ^^ .

concentration ^^rrr = lo"-*"' =? 3-5.

H,S

TTC-

At pH 8-6 this ratio ^v^ = 35-

Hence in sulphur waters

the HS concentration is 3-5 — 35 times as high as the H2S
concentration.

16

-

14

~ " — ■ — ^-— ->

--^_^£S

12

-

— -^

10

-

8
6

;3HS

p3^^

-—

4

2

-

0

1

\

1

1

7

■pW

Fig. I.

(After Baas-Becking.)

(Baas-Becking appears to obtain 50 — 100 for the same
range of pW values, but this difference does not affect the
main argument.)

Again, at pH ^'6,
and at^H 8-6,

HS-

S
HS

- = 2-5 X 10'

S~

2-35 X 10^

It therefore follows that the concentration of HS" between
pH 7-6 and pH 8-6 is somewhat greater than the concentra-
tion of HgS, and far greater than the concentration of [S ].
From the above facts he makes the assumption that the

32

SULPHUR BACTERIA

sulphur bacteria absorb not the HgS, but the hydrosulphide.
He is supported in this assumption by the fact that the change
from HgS to S is from a lower to a higher level of energy.
Interesting figures bearing on this matter have been published
by Lewis and Randall : —

HS~ = S~~ + H+ — 20,470 calories.
H2S(aq) - HS- + H+ - 9470
H + S(rh) = H2S(aq) + 6490
S(rh) + 2O2 = S04~ + 176,500
Baas-Becking has utilized these or similar figures to indicate
the free energy levels of these compounds in the form of a
scale (see Fig. 2).

It would thus appear that the sulphur
199,950^5 bacteria must expend rather than derive energy

from the conversion of HgS to S. For this
reason it is concluded that the necessary energy
is obtained by the dehydrogenation of the HS~
according to the following equation : — ■
HS- = H+ + S— .
The HS~ is obtained presumably not from
HgS but in other ways, as for example by the
reduction of oxy-sulphur compounds through
the agency of other bacteria.

Following the scheme of oxidation suggested
by Wieland and by Hopkins as taking place in
yeast in the absence of oxygen, Baas-Becking
suggests that the following represents the
reaction by which sulphur develops in the cells
of the sulphur bacteria : —

CH, - S - H

179,480- -HS"
176,500- -S

170,010- -HzS

0-

FlG

so;-
2.

(i)^

CH., - S - S - CH, + 2HS- = 2CH . NH„ + 2SA + 2E

I " I " I "

CH . NH2 CH . NH2 C = O

I I I

c=o c=o

R R

Glutathione
(contains cystine and
glucosamin) . _

E stands for electron

R

Compound

containing
cysteine.

* Refer to p. 20 for the most recent constitution assigned to glutathione.

THE PRODUCTION OF SULPHURETTED HYDROGEN ^^

Thus sulphur is produced and energy hberatcd. The
following reactions then take place : —

(11)

CH„ . S . H CH, - S . S . - CH.,

I " I " I "

2CH . NH, + H2CO3 = CH . NH„ CH . NH., + H . CHO + H„0,

I 1 " I "

c=o c=o c=o

I I I

R R R

(III) H2O, = H20+i02

Thus oxygen is developed inside the cell, which is utilized
to oxidize the sulphur to the sulphate,

S— + 20^= SO4— .
The conclusions of this worker cannot be taken as final, for
all the pieces do not " fit." Thus sulphuretted hydrogen is
more soluble in an alkaline than in an acid medium, and yet
in the table given above an increasing alkalinity (H"^ from
I0~^ to I0~^) is associated with a decrease in the amount
of HgS in solution (from 2-2 X lO"^ to 2-2 X lO"^). Again,
Baas-Becking insists on the absence of organic matter from the
beds in which the sulphur bacteria are developing, and the use-
lessness of such matter in the metabolism of these organisms.
Under natural conditions the reactions which according to
him take place in the sulphur metabolism could not occur
except in the presence of an ample supply of organic matter :
for all of them are the direct results of the decomposition of
organic matter through the agency of saprophytic and other
microorganisms. Finally, the beneficial effect of supplying
the sulphur bacteria with a fluid containing sulphuretted hydro-
gen in solution is well established, and it is proved that the
organisms assimilate this substance directly in their metabol-
ism ; and this in spite of the fact that the energy level of
hydrogen sulphide is below that of sulphur. The sulphur
appears to be excreted and oxidized to sulphate outside the
cell. There is no reason to suppose that the sulphur takes
further part in metabolism any more than it does in the
metabolism of the thionic acid bacteria which deposit the
sulphur outside, instead of inside the cell.

3

CHAPTER III.

THE METABOLISM OF THE SULPHUR BACTERIA.

The Food Requirements and Sources of Energy of the
Sulphur Bacteria.

General Considerations. — The physiological status of any
microbe is determined when its nitrogen and its carbon sources
of supply are known. The protoplasm of the sulphur bacteria,
hke that of all other organisms, is built up of the elements
C, H, O, N, S, P, and of these, definite provision must be made
in all preparations of bacterial cultures for the supply of nitro-
gen and carbon. Of the other elements, hydrogen and oxygen
are present in water as well as in other substances in the
nutrient media, and the remainder are required in such
small quantities that definite provision for their supply is
not necessary. In addition, of course, the supply of mineral
salts must be taken into consideration, but as the differences
among bacteria in this respect are very small they play no
part in making physiological distinctions.

The sources of energy of an organism are also a matter for
consideration. Autotrophic organisms like the green plants
must build up the complex protoplasmic molecule from such
simple substances as carbon dioxide, mineral salts and water ;
and the necessary energy is obtained from the light of the sun.
The presence of chlorophyll in green plants enables them to
" capture " the energy contained in light, and to utilize it
for their metabolic purposes. Thus carbon- assimilation takes
place and carbohydrates are built up from the carbon dioxide
and water of the atmosphere. So far as is known, the energy
of the sun is not directly responsible for the further building
up of carbohydrates into the still more complex proteins, the

34

THE METABOLISM OF THE SULPHUR BACTERIA

i5

formation of which precedes the building up of the most
complex molecule of all, namely, that of living matter. As
however, there is no primary source of energy other than that
of the sun, it must be presumed that more carbohydrate is
formed by the green plant than is required for its immediate
purposes, so that a secondary source of energy is available
to effect the further synthesis of the carbohydrate into pro-
teins by a dissociation of a part of the surplus carbohydrate
into simpler substances. Heterotrophic plants have the ad-
vantage of being able to assimilate substances that already
contain a potential source of energy, which may be utiHzed
for the building up of protoplasm and also for the carrying
out of such vital processes as reproduction, cell division,
motility, etc.

All processes which originate from the vital activity of an
organism are classed as metabolic. If they are such as result
in the production of more complex substances, they are termed
anabolic ; if they result in the breaking down of substances,
they are termed katabolic. All the vital processes in a living
organism are thus to be regarded as consisting of two main
streams, one leading upwards and culminating in the formation
of protoplasm; and the other leading downwards and cul-
minating in the formation of completely oxidized substances,
such as nitrates, sulphates, phosphates, etc.

A closer examination of the various katabolic processes
shows that the relationship of the reaction to the protoplasmic
molecule is not of the same intimate nature in all cases. Three
kinds of katabolic processes may be distinguished :

1. Processes that result from the direct operation of the
protoplasmic molecule itself, that is, those in which there
are no special secretions for the purpose.

2. Processes that are activated by the agency of a special
secretion of the protoplasm ; here the secreted material is in
such intimate relationship to the protoplasm that injury to
the latter results in the destruction of the former.

3. Processes that are activated by a special secretion which,
unlike that of the second class, can be retained in an active
condition after the organism is killed.

3 *

36 SULPHUR BACTERIA

It is not always possible to draw sharp lines of distinction
between these three classes of reactions.

Fermentation.^ The history of the terms Ferment and
Fermentation will give some explanation of the slight confusion
that exists in the use of these words. The word ferment comes
from the Latin fervere, to boil, and referred originally to the
apparent " boihng " that took place when yeast in fermenta-
tion gave off copious bubbles of carbon dioxide. At f^rst the
liquid in which this bubbling took place was named the
ferment, and is so named by bakers to the present day. With
greater knowledge the term was restricted to the organism
that caused this bubbling. With a still further advance in
our knowledge the term fermentation was applied to other
processes in which a medium was chemically affected by
the activities of microorganisms even although there was no
obvious gas production ; such, for example, as the production
of lactic acid in the souring of milk. Then it was ascertained
that in many cases the actual chemical work was done, not
by the organism directly, but by a secretion formed from it,
which in some instances could operate after the organism itself
was dead. Thus arose the distinction between an organized
ferment and an unorganized ferment, the former referring to
the cases in which the protoplasm itself accomplished the
work, and the latter to those cases in which the secretion
was recognizable apart from the protoplasm producing it.
The special secretion then became known as the unorganized
ferment, or more simply as the ferment.

The organized ferments fall into the first of the three classes
of katabolic processes cited above, and the unorganized fer-
ments into the third.

Metabolism and Fermentation.

The view is adopted in this work that all fermentative
processes are of a katabohc nature. Katabolic processes
as a whole all come under one of two categories :—

I. Reactions which convert food substances to a form
more readily assimilable by the organism.

THE METABOLISM OF THE SULPHUR BACTERLi 37

2. Reactions whicli place at the disposal of the plant
(or animal) a supply of energy to carry on various
vital activities.

It is impossible to select a single feature which will enable
us to make a distinction between the fermentative and the
katabolic processes. Indeed all katabolic processes associated
with vital activities may thus w^ith propriety be named fermen-
tative. In actual practice one of two procedures is followed
in attempting a distinction : —

1. The limits are not specifically defined and only those

processes are named fermentative that bear some
resemblance to typical reactions such as, for example,
the fermentation of sugar into alcohol by the yeast
plant.

2, The term fermentation is restricted to the disruption

of easily decomposable carbohydrates and similar
substances where the biological significance of the
process is the gain of energy to the organism.

Metabolism, Fermentation, and Respiration in the
Sulphur Bacteria.

The outstanding feature in the physiology of the sulphur
bacteria is the large absorption of sulphuretted hydrogen ;
and the significance of this absorption is the keynote to an
understanding of their physiology. The gain to the bacteria
must be in terms of energy or of food. The importance of
sulphur as an article of food can be dismissed, for the amount
of sulphur which is absorbed is several hundreds of times
greater than the amount of that substance required for the
building up of the protoplasmic molecule. In this respect the
sulphur bacteria do not require any more sulphur than any
other bacterial organisms. Again, it has been shown that the
energy level of sulphuretted hydrogen in solution is lower
than that of the sulphur into which it is transformed. It is,
therefore, clear that the gain to the organisms cannot be a
direct one, and that the sulphuretted hydrogen must there-
fore participate in some larger operation which is beneficial,

38 SULPHUR BACTERIA

and for the execution of which sulphuretted hydrogen is neces-
sary. The sahent facts may now be considered.

1. Sulphuretted Hydrogen. — The proof of the absorption
of this substance is well established. Provided that it is not
presented in too concentrated a form, large quantities are
absorbed, and the optimum estimated by Bavendamm for
Lamprocystis, namely, a 'concentration equal to 25 mm.
pressure of Hg is probably the optimum also for the majority
of the sulphur bacteria, if not for all of them. It is also
established that these organisms effect within their cells the
transformation of the sulphide into elementary sulphur, for
the latter is found in quantity inside the cells. The agency of
the bacteria in effecting the oxidation of the element to the
sulphate is more open to question. It is true that sulphate
accumulates in the neighbourhood of the cells, but more
definite proof is required of bacterial agency since the oxida-
tion of the sulphur to sulphate in an aqueous medium also
takes place in the complete absence of organisms. As the
sulphate in cultures of the sulphur bacteria is not found within
the cells, its formation may well be due to non-vital agencies.

2. Organic Matter. — Winogradsky (i) and (2) came to the
conclusion that the sulphur bacteria do not assimilate organic
matter, and, further, that their development was hindered,
if not stopped, by the presence of organic matter in more
than the very smallest quantities. The second observation is
obviously incorrect, since under natural conditions these forms
flourish in waters which often contain very large quantities of
organic matter. It is a matter of common observation that
Beggiatoa alba, to take a prominent example, flourishes most
abundantly in water contaminated with sewage matter.
The present writer has found this organism in waters con-
taining sufficient organic matter for the support of hundreds
of thousands of bacteria per cubic centimetre. Again, Thio-
porphyra voliitans flourishes in pools that contain in many
cases saprophytic bacteria to the order of millions per cubic
centimentre. It is very seldom indeed that the sulphur
bacteria, under natural conditions, are found except in the
presence of organic matter in quantity.

THE METABOLISM OF THE SULPHUR BACTERIA 39

The assimilation, as distinct from the toleration of, organic
matter is more of a disputed matter. Nadson inferred the
assimilation of organic matter because he was able to keep
sulphur bacteria in a living condition for a long time in the
absence of sulphuretted hydrogen. It is difficult to see the
validity of the inference. Molisch (4) showed that Chroma-
tium Okenii thrives particularly well in a medium contain-
ing I per cent, peptone and i per cent, dextrin. Skene
emphasizes the unimportance of organic matter, and regards
it as a negligible factor in the metabolism of the sulphur
bacteria. This writer does not do full justice to the facts
which were discovered by him, for whilst the best growth
was obtained when ammonium sulphate was the source of
nitrogen he obtained appreciable growths when such sources
of nitrogen as peptone, asparagin, urea, glycocoll, ammonium
nitrate or calcium nitrate were used. He, however, appears
to have placed a doubtful construction on these facts. Growth
in presence of organic matter was regarded as due to ammon-
ium sulphate, produced by the decomposition of these sub-
stances by saprophytic bacteria. The cultures (which were
raw) were found at the close of the experiment to give
almost as strong a reaction with Nessler's reagent as am-
monium sulphate itself. It must be pointed out, however,
that ammonium sulphate is not invariably produced by the
saprophytic decomposition of these organic substances.

The two investigators who succeeded in obtaining pure
cultures of the sulphur bacteria, namely, Keil (the colourless
bacteria) and Bavendamm (the coloured bacteria), regarded
organic matter as unessential to their development. Both
considered the sulphur bacteria to be obligate autotrophs, that
is, that under no circumstances is organic matter used as a
source of nitrogen.

The following facts, however, show that organic matter
may be occasionally assimilated, that is, that the organisms
Site facultative not obligate autotrophs : —

(i) Skene's results are not consistent with his interpreta-
tion of them, since the organic substances that were found
to favour growth do not all produce ammonium sulphate

40 SULPHUR BACTERIA

when decomposed. This apphes in particular to urea and
glycocoH.

(2) Undoubted increase of growth has been observed after
addition of organic matter (Mohsch, Nadson, Skene).

(3) Undoubted use is made of organic matter by the closely
related non-sulphur purple bacteria (Molisch).

(4) Ammonium sulphate can be replaced by calcium nitrate
(Nadson, Ellis), suggesting the absence of specificity.

(5) In Bavendamm's cultures the individuals did not show
the same healthy growths which characterize the cultures in
nature, suggesting that the growth was weak from the lack of
certain ingredients which are supplied cither directly or in-
directly by organic matter.

(6) The same lack of robustness characterized the present
writer's cultures of Thioporphyra volutans in a medium from
which organic matter was excluded (calcium nitrate being
used as the source of nitrogen).

Whilst individually each of these points is inconclusive,
collectively they appear to justify the conclusion that the
sulphur bacteria satisfy their nitrogen requirements in more
ways than one, and that organic matter plays a subordinate
role as a source of nitrogen. This is rendered all the more
probable because

(1) They grow best in waters containing an abundant

supply of organic matter.

(2) Their artificial cultures from which organic matter was

excluded do not show the same robust growths which
characterize their development under natural con-
ditions.

(3) The maintenance of growth is dependent on substances

that are derived from organic matter.

3. The Source of Carbon. — Particular attention has been
paid to this aspect of nutrition by Skene and by Bavendamm.
Using ammonium sulphate as the source of nitrogen, and
introducing small amounts of various organic compounds,
Skene failed to obtain growth with any of the carbon com-
pounds with which he experimented. He concluded that they

THE METABOLISM OF THE SULPHUR BACTERL4 41

hindered rather than aided growtli. Bavendamm obtained the
same negative results in his experiments with pure cultures,
and considered that the sulphur bacteria derived their carbon
from the carbonic acid of the atmosphere. He observed that
the addition of carbon compounds made no difference to the
growth of Chroniatiiim Wanningii and of Lamprocystis. The
question cannot, however, be held to be completely settled by
the negative results of these two investigators. They have
conclusively proved that the CO2 of the atmosphere com-
pletely suffices for the carbon requirements of the sulphur
bacteria, but it has still to be proved that under other circum-
stances these organisms are not able to make use of carbon
in any other combination.

4. Oxygen. — The influence of oxygen on the sulphur
bacteria may be considered under three heads : —

(1) The directive influence of oxygen as a chemiotactic

agency in effecting the attraction or repulsion of
bacteria.

(2) The possibility of this gas being given off by the purple

sulphur bacteria in the same manner as green plants.

(3) The possible necessity of oxygen to the bacteria during

the process of respiration.

The first and second of these questions are treated in
Chap. XI. The third may now be considered.

Respiration is a function which is common to all organisms.
The majority of organisms require oxygen to effect the de-
composition of highly complex substances in order that the
plant may benefit by the energy which is thereby liberated.
Such plants constitute the majority of the members of the
vegetable world, and there are only a few that respire in a
different way. The two classes of plants are known respec-
tively as aerobes and anaerobes. The anaerobes are confined to
a few organisms among the bacteria and some closely related
organisms. Winogradsky was the first to call attention to
the reaction of the sulphur bacteria to oxygen. He had noted
that in his mixed cultures the bacteria invariably retreated
from the surface of growth if oxygen were present in the same

42 SULPHUR BACTERIA

concentration as in the atmosphere, and that they throve
abundantly in a medium if it was almost devoid of oxygen.
When cultivated in drop cultures the bacteria retreated to
the centre of the drop, and remained there so long as the
oxygen pressure was not changed. Winogradsky ascer-
tained that while oxygen at ordinary pressure was actually
harmful, it was necessary in very small amounts. He did
not experiment with organisms growing in media completely
free from oxygen.

Nadson advanced the opinion that sulphuretted hydrogen
was assimilated for protection against the poisonous effects
of oxygen rather than for purposes of nourishment. He
regarded the sulphur bacteria as obligate anaerobes. Molisch
and Skene, like Winogradsky, considered that a small amount
of oxygen was necessary, and maintained that without it the
bacteria could not oxidize sulphuretted hydrogen first to
sulphur and then to the sulphate. Winogradsky and Skene
thought that they had found sufficient of this gas to satisfy
the needs of the sulphur bacteria in the carbon assimilation
of small green organisms that were always found in natural
waters in the neighbourhood of the sulphur bacteria. This
idea is not convincing, for the sulphur bacteria are fre-
quently found in natural waters in which the supply of green
organisms is either non-existent or is inadequate for such a
purpose.

Bavendamm, working with pure cultures, found that
Lamprocystis and Chromatiiim throve well in a medium com-
pletely devoid of oxygen, but that both these organisms
developed at their best when supplied with oxygen at a pressure
of 5 -2 mm. of mercury.

The conclusions of Bavendamm may be taken as final for
the coloured sulphur bacteria, which resemble nearly all, if
not all, so-called obligate anaerobes in that, whilst they are
able to grow in a medium completely devoid of oxygen, yet
assimilate this gas if it be presented to them in a sufficiently
dilute form.

The above remarks apply only to the relationship of
the coloured forms to oxygen. Although the colourless

THE METABOLISM OF THE SULPHUR BACTERL4 43

organisms Thiothrix and Beggiatoa were isolated by Keil
in 191 2 their oxygen requirements were not ascertained
in pure culture experiments. They are, however, niicro-
aerophilic, as are the coloured forms, so far as may be
judged from the manner of their growth and their physio-
logical requirements.

Observations on Thioporphyra volutans. — No specific ex-
periments have been made to determine the physiological
requirements of this organism, but it was observed that its
growth in various mixed cultures invariably reached its
optimum when the supply of oxygen had been nearlv ex-
hausted by the multiplication of the saprophytic bacteria. It
thus thrives best under micro-aerophilic conditions. On one
occasion, however, it was found to be growing in abundance
on wooden piles only some six inches or so below the sur-
face in the open water of the Clyde. Hence one member of
the coloured sulphur bacteria at any rate is not hindered
in its growth by the presence of an abundant supply of
oxygen.

It may thus be concluded that the majority of the sulphur
bacteria, both coloured and colourless, are micro-aerophilic,
but that one at least [Thioporphyra volutans), and probably
more, thrive in highly oxygenated waters ; and it is highly
improbable that these do not absorb oxygen in quantity.
A diversity in the oxygen requirement is to be expected of
organisms that thrive under such diverse conditions as the
various members of the sulphur bacteria.

5. Mineral Matters. — In all plants the presence of extremely
small quantities of various elements, over and above those
that enter into the constitution of the protoplasmic molecule,
is necessary for healthy growth. Some may be required in
fairly large quantities, as, for example, calcium when used to
combine with the oxalic acid formed in the leaf during meta-
bolism.

In cultivating bacteria it is usually found advantageous
to add to the culture, not pure water, but a solution containing
various inorganic salts in solution. The mixture which has
hitherto been used with the greatest success for the sulphur

1-50

gram.

0-05

) I

0-05

) >

0-05

) ?

o-oi

) )

10-00

grams,

1000

c.c.

44 SULPHUR BACTERIA

bacteria is that used by Licskc in his investigation of the iron-
bacteria. This is made up as follows : —

Ammonium sulphate ((NH4)oS04)

Potassium chloride (KCl)

Magnesium sulphate (MgS04)

Mono-hydric-potassium phosphate (K2HPO4)

Calcium nitrate (Ca(N03)2)

Calcium carbonate (CaCOg)

Distilled water .....

The use of the comparatively large quantities of ammonium
sulphate and particularly of calcium carbonate in this mineral
solution is due to the circumstance that the nitrogen in the
sulphate is required for the building up of the protoplasm ;
whilst the carbonate is required for purposes of neutralization.

These organisms are indifferent to common salt (NaCl).

Issatchenko (4) gives a list of sulphur bacteria that were
found by him in solutions containing either NaCl or Na2S04
in proportions varying from I to 23 per cent.

6. Hydrogeii-ioii Concentration. — The range in the pW
values within which growth takes place must be somewhat
circumscribed, for whilst the medium of growth must be
alkaline the two substances that are required in greatest
amounts in the metabolism of these organisms, namely,
sulphuretted hydrogen and carbonic acid, are of an acid
character. Hence growth is inhibited by the presence of
sulphuretted hydrogen in quantity. Baas-Becking found that
the optimum for the waters containing sulphur bacteria near
Stanford University was 7-6 — 8-6 ; his figures agree with those
determined by Atkins. Under natural conditions when active
growth is in progress the pW is progressively decreased by
the acid substances liberated by the saprophytic bacteria
(CO2, H2S, organic acids, etc.). When a certain degree of
acidity is reached the growth of the sulphur bacteria will
cease altogether, and will recommence only when the surplus
acids have once more been used up by saprophytic and other
organisms.

THE METABOLISM OF THE SULPHUR BACTERIA 45

Baas-Becking makes the erroneous statement that the
sulphur bacteria occur only in hard waters, and that a high
alkalinity is maintained in such waters by the large amount
of calcium and magnesium present. Organisms like Beggiatoa
alba are found in soft and hard, as well as in marine and fresh
waters. Some of these bacteria thrive in waters in which the
alkalinity is not maintained by the presence of carbonates
and bicarbonates. The factors determining the pH of sulphur
waters generally appear to have been determiined by this writer
solely from local conditions. In soft waters, under normal con-
ditions, the pH of the water is kept within a certain range by
the opposing nature of two classes of bacteria. On the one
hand, the saprophytic and other bacteria are constantly pro-
ducing acid, whilst on the other the sulphur bacteria are using
up sulphuretted hydrogen and carbonic acid. Between the
two opposing forces a balance is maintained which, if it is
upset, results in the disappearance of the sulphur bacteria.
The same conditions hold in essentials both in hard and
in soft waters, the differences in respect to the growth of
bacteria being one of degree, not one of kind. In artificial
cultures the maintenance of an alkaline medium is often
guaranteed by the addition of chalk.

Experiment with Thioporphyra volutans.

It was found by chemical tests that the calcium and mag-
nesium content of pools containing a mixture of saprophytic
and sulphur bacteria did not appreciably change during a
period of several weeks in which active growth of both classes
of bacteria was in progress. Hence it was concluded that
any changes that took place in the pH value of the water were
due to changes in the concentration of sulphuretted hydrogen
and carbonic acid. The following results were obtained : —

Time in \\'eeks.

^H.

0

7-2

3

7-3

6

7-1

8

7-6

10

7-4

46 SULPHUR BACTERIA

Although some saprophytes, as, for example, the marine
denitrifying saprophyte examined by Cranston and Lloyd,
maintain an alkaline reaction, the growth of a mixed group of
saprophytes is practically always accompanied by a progressive
development of acid. We may attribute the sharp rise in
the p\l value between the sixth and the eighth week to the
consumption of the sulphuretted hydrogen (liberated by the
saprophytes) by Thioporphyra, which during this period was
observed to grow luxuriantly. The subsequent lowering after
the eighth week was found to be concurrent with a revival
in the growth in numbers of the saprophytes which had
suffered a slight eclipse by the previous growth of the sulphur
bacteria. The most vigorous growth of Thioporphyra occurred
on the eighth week when the pH value was 7-6, a figure which is
within the range set by Baas-Becking for the optimum growth
of the sulphur bacteria.

The Biological Explanation of the Assimilation of
Hydrogen Sulphide.

It has been shown that sulphuretted hydrogen does not
enter the cell in the form of food, and that the change from
the sulphide to sulphur does not result in a liberation of
energy. It has also been shown that an undoubted gain
to the bacteria follows from the assimilation of the sul-
phide. Hence the advantage cannot be a direct one, and
must follow because the hydrogen sulphide is a factor in a
wider scale of operations than its simple decomposition. It
is suggested that this substance enters the cells as an adjunct
in the respiratory process. After absorption the sulphuretted
hydrogen is chemically transformed in such a manner that
hydrosulphide ions are placed at the disposal of the bacteria.
As this is an endothermic process the necessary energy must be
supplied from the general resources of the organism, derived
from other metabolic changes. Then union of the sulphide
with the sulphur amino-acids takes place, resulting in the
production of free sulphur and the liberation of energy. The
probable changes that would then take place have been

THE METABOLISM OF THE SULPHUR BACTERIA 47

indicated by Baas-Becking. This view implies that the hydro-
gen sulphide is a part of the machinery that transfers energy
from what is presumably the less available to the more avail-
able condition. The decomposition of the hydrosulphide
is to be regarded as a respiratory process comparable in its
effect to the changes that follow the absorption of oxygen
in aerobic plants. It is also consistent with the fact that the
absorption of hydrogen sulphide is not absolutely necessary,
since this substance is not the source of either the food or the
energy of the sulphur bacteria. In spite of the statements of
Winogradsky, Skene and Bavendamm, that the addition of
sulphuretted hydrogen is indispensable to the growth of the
sulphur bacteria examined by them, there are facts to show
that growth may take place in the absence of this gas. Thus
Molisch (3) has found that Chromatiwn Okenii may develop
in a raw medium completely devoid of sulphuretted hydrogen,
and Ellis (11) has shown that Chromatiimi Linsbaiieri multi-
plies in a culture containing no trace of this substance. The
sulphur bacteria are therefore able to take advantage of more
than one mode of metabolism. Not only do they change
their form (pleomorphism), they also change their function.
The word pleoenergism may be coined to express this capacity
for adaptation to more than one mode of life.

Pleoenergism of the Sulphur Bacteria.

There are in this group at least four different modes of
obtaining food and energy.

(i) The most important is that which may be regarded as
the' normal mode of existence. The interaction of
sulphuretted hydrogen with the products from the
first stages in the decomposition of organic remains
(sulphur amino-acids) supplies the energy, whilst
the nitrogen and carbon are obtained from non-
organic sources (ammonium compounds and carbon
dioxide, respectively).

(2) Organic remains, or the products of their initial
decomposition (peptones, etc.) in association with

48 SULPHUR BACTERIA

sulphuretted hydrogen appear in certain cases to
be able to supply both food and energy.

(3) Growth and multiplication may take place under the

same conditions as the preceding, but without the
presence of sulphuretted hydrogen.

(4) Under special cases both food and energy may be

obtained without either organic matter or sulphur-
etted hydrogen.

In all these modes, except the fourth, the presence of
organic remains is necessary, and they may be directly utilized,
or they may be used after the saprophytic bacteria have
effected the first stage in their decomposition, with the pro-
duction of peptones, ammonium compounds, etc. In the fourth
mode, the conditions are probably unnatural, and the organism
does not produce normal structures. In nature the sulphur
bacteria are always associated with organic matter, and whilst
normally the energy of the organism is never derived directly
from the organic remains before these have suffered decompo-
sition, they probably draw their nitrogen from the decomposing
remains at several stages in their decomposition of such
remains (peptones, ammonium compounds, nitrates, etc.).
It is also not improbable that in nature the assimilation of
several kinds of nitrogenous food takes place simultaneously.
There is no reason to suppose that the sulphur bacteria are
wanting in adaptability, and certainly the facts do not warrant
this supposition. In this respect the sulphur bacteria are
not different from the majority of other bacteria. Monocnergic
bacteria are probably not common, and the sulphur bacteria
behave as do the majority.

The Denitrifying Sulphur Bacteria. — A study of the small
organism* isolated by Lieske is instructive because the sulphur
metabolism is combined with a process of denitrification. This
organism derives its carbon from CO2 in the form of either
carbonates or bicarbonates. According to Lieske it assimi-

* This organism is not, strictly speaking, one of the sulphur bacteria
because it does not store sulphur in its cells, but the general course of the
metabolism is similar to that of these organisms and may appropriately
be considered in this work.

THE METABOLISM OF THE SULPHUR BACTERL4 49

lates nitrates but not nitrites, and reduces them to free nitro-
gen. In its sulphur metabohsm hydrogen sulphide, sodium
thiosulphate (NagSaOg), and the sodium salt of dithionic acid
(NagSaOg) are utilized as sources of energy and changed into
the sulphate.

The metabolism of this organism is interesting because it
presents certain peculiar features. It is unable to utilize the
energy of the sun as do the chlorophyll plants, and it has no
ready-formed stores of supply as have the parasites and
saprophytes. But energy must be obtained by it to build up
the protoplasmic molecule from simple substances like carbon
dioxide, and to carry out all the processes associated with life,
namely, movement, reproduction, etc. Finally, the organism
is one of the so-called obligate anaerobes, and cannot utilize
oxygen when supplied to it at the normal pressure.

The place of the oxygen of the atmosphere used by aerobic
plants is taken by the oxygen liberated by the reduction of
the nitrate to free nitrogen. We may follow the energy
changes which take place in the metabolism.

The reduction of the nitrate to the nitrite, according to
figures supplied by Waksman, demands an expenditure of
energy equal to ^6-6 calories.

2HNO3 = 2HNO2 + 0., (- 26-6 calories).

But oxygen is produced, and if utilized to break down a
fermentable carbohydrate liberates II2 calories per molecule.
The whole process will result in a gain on balance of

112 — 36-6 = 75-4 calories.

According to the same worker the change from the nitrite
to free nitrogen is an exothermic process and therefore gives a
further gain of energy : —

2HNO2 = N2 + i|0, + H2O (+ 6-8 calories).

Further the i-| molecules of oxygen can be utilized by the
organism with a gain of II2 calories per molecule. Hence

4

50 SULPHUR BACTERIA

in the change from the nitrate to free nitrogen there is a total
gain in energy of

74-6 + (i| X 112) + 6-8 == 249-4 calories.

The sulphur denitrifying bacteria are peculiar in that they
utilize the oxygen derived from the nitrate reduction to oxidize
sulphur compounds in place of the carbohydrate oxidized by
the ordinary denitrifying bacteria. The sulphur compounds
(sulphuretted hydrogen, sulphur, etc.) must be regarded as
fermentable substances and their oxidation as exothermic
processes (or fermentation processes), which have as their
biological purpose the supply of energy to the organisms.
The sulphur compounds must thus be regarded not as them-
selves the suppliers of energy but rather as the vehicles
that arc made use of in the transformation of energy. As in
the true sulphur bacteria they are parts of the respiratory
machinery of these bacteria. It is interesting to note that
Cranston and Lloyd find that the denitrifying organism
examined by them often converted 100 per cent, of the nitrate
supplied to it into free nitrogen, and that if this is universally
true for such organisms the whole of the nitrate is used for
respiratory purposes and none is used to build up the protein
molecule.

Physico-chemical Speculations in regard to Organic
Matter in Sulphur Waters.

There are both biological and chemical methods for the
direct estimation of organic matter in water. Indirect methods
must therefore be regarded as of subsidiary importance.
Consideration is given to the following speculative treatment
of the subject by Baas-Becking, on account of its interesting
physico-chemical deductions. He attempted to prove the
smallness of the organic content of the sulphur waters, rich
in sulphur organisms, which are found in the neighbourhood
of Stanford University, California. He bases his deductions
upon the facts that the CO2 of the atmosphere is in equilibrium
with the CO2 in the water and that the pW value of sulphur
waters varies from pW 7-6 to pH 8-6.

THE METABOLISM OF THE SULPHUR BACTERL4 51

The carbonic acid is dissociated in two stages : —

K,{H,CO,) = [U^][HCO,-], . . (I)

Ki = 3-4 X 10-'^.
K.fHCOa) = (hn(C03--), . . (2)

Ko == 4-6 X 10-11.

Taking into consideration the ionic product of water, we
have

K^=(H-^)(OH-), . . . (3)
K„ = 8 X lo-i^

If we indicate the total base as (B^) and note that the sum
of the positive ions must equal the sum of the negative ions,
we have

(B-^) + (H+) = (0H-) + (HCO3-) + (CO3-).* . (4)

If we assume (B^) = o and consider only the carbonate
relationship, we have

(HCO3-

3-4 X I0-' X (HgCO

(CO3-)

(HCO3-)

(C0.~)

_ 4-6 X io-ii(HC03)

= 2-2 X IQI" X H"^.

Hence in a solution of known pH value we can calculate
the relative proportion of bicarbonate ion and carbonate ion.
Baas-Becking argues that if putrefactive bacteria were present
in sulphur water the HCOs" would increase at the expense of
the CO3 , and so alter the value of H"^. But as the pH
value is always 7-6 — 8-6 this change does not take place ;
hence there are no putrefactive bacteria, and in consequence
there is no organic matter, or not enough to make any material
difference in the equilibrium of the water. It is surprising
that the simple test of ascertaining directly the organic content
of the water by estimating the number of saprophytic bacteria
in it was not resorted to, in order to prove this conclusion.
As to the actual number of bacteria that may be found in such

* Baas-Becking has inserted 2(C03~) here, which is not correct, but this
does not materially affect the equation.

4*

52 SULPHUR BACTERIA

waters, the reader is referred to page 64, in which the numbers
found in waters containing growths of Thioporphyra vohitans
are given. Some sulphur waters contain saprophytic bacteria
to the order of milhons per cubic centimetre, whilst the majority
of such waters contain either tens or hundreds of tliousands
per cubic centimetre. It is exceptional to find that the num-
bers are only in the order of hundreds per cubic centimetre.
Even if it is held that the sulphur bacteria do not directly
assimilate organic matter, it is the case nevertheless, that
they must of necessity find nourishment in the products of
the decomposition of organic matter, and these will be found,
under natural conditions, only in waters containing organic
matter. It is also remarkable that in Baas-Becking's explana-
tion of the metabolism of the sulphur bacteria, an important
role is ascribed to the sulphur amino-acids, and yet it is claimed
that organic matter plays no part in the metabolism of the
sulphur bacteria. '

CHAPTER IV.

THE CULTURE OF THE SULPHUR BACTERIA.

Methods of Culture.

General Considerations. — It is desirable to cultivate the
sulphur bacteria in a medium which does not contain any
other microorganisms : and this is particularly the case in the
investigations of physiological problems. If other organisms
are present it becomes very difficult, and sometimes impossible,
to estimate the exact contribution of the sulphur bacteria
to the production of any particular phenomenon which may
be under investigation. At the same time it is also advan-
tageous to study the sulphur bacteria in a mixed field, for a
truer presentment of their normal state is thereby given :
it is very seldom that bacteria grow and multiply in nature
uninfluenced by other organisms. The best conditions for the
morphological study of these, as well as of other bacteria,
obtain when the organism under investigation is present in
its natural habitat, and is easily recognizable. Under artificial
conditions, as for example in pure cultures, they are apt to
depart from the normal, both in their habits and in their life
histories. We must therefore accept with caution the dis-
counting of certain positive results obtained from the study of
the organisms under one set of conditions, on the strength of
negative results obtained from their investigation under totally
different conditions.

Winogradsky's Methods. — To Winogradsky must be given
the credit for the pioneer work on methods of culture.

{a) Rhizomes of Water-plants {Butomiis, etc.) with adhering
mud were placed in a deep vessel, and water and gypsum added.
The vessel, covered with a glass plate, was then placed in a

53

54

SULPHUR BACTERIA

dark warm room. After some time a smell of HgS was noticed,
and a thick scum collected on the surface. The scum contained
Beggiatoa alba and other colourless bacteria. Gypsum was

added periodically to increase
the growth. This method has
been tried by Molisch, by
Bavendamm, and by the pres-
ent writer, but without suc-
cess.

(b) Macerated Hay and Gyp-
sum supplied another medium.
The hay was placed in water
for ten days and then boiled.
The water was then decanted,
a fresh supply added, and this
again boiled. This process was
repeated several times and the

Fig. 3.

(a) Floor covered with damp paper
on which the glass slide rests
between observations.

{b) Small dish containing water to

which I gram calcium sulphide hay allowed to decompose in

has been added.

water

(c) Wide tube for occasional addi-

tion of a few drops of weak
hydrochloric acid.

(d) Small tube to facilitate the addi-

tion of liquids down tube c.
Winogradsky regarded the sul-
phur bacteria as almost com-
pletely autotrophic* Reused

Beggiatoa alba appeared
early.

(c) Glass Slide Preparations.
— The material was placed
under a coverslip, prevented

Strassburg tap water to which from drying up, and supplied
0005 — o-oio per cent, calcium . .

periodically with Hgo. ihe

butyrate, or sodium acetate,
had been added, or water from
a sulphur well to which small
amounts of HjS had been added.
Since he considered the sulphur
bacteria to be almost completely
autotrophic, he added only min-
ute quantities of organic matter.
He found the organisms to live
up to nineteen days if ad-
equately supplied with sulphur-
etted hydrogen, bnt only eight
or nine days in its absence.

apparatus is shown in the
accompanying diagram (Fig. 3).

(d) Large scale experiments
carried out by method [c] were
also successful.

His conclusions are as fol-
lows : —

1. Sulphuretted hydrogen is absorbed, oxidized first to

sulphur and then to sulphate.

2. The bacteria die if cultivated without sulphuretted

hydrogen.

* An aulotrophic organism is one which is independent of organic matter
in its nutrition.

THE CULTURE OF THE SULPHUR BACTERIA 55

3. The coloured sulphur bacteria require more sulphuretted

hydrogen than the colourless forms.

4. Only a trace of organic matter is tolerated. Relatively

large amounts are toxic, e.g. O-l per cent, peptone
was found to be injurious.

5. The oxygen requirement is very small.

6. Exposure to light is necessary for the growth of the

purple sulphur bacteria.

These conclusions are of special interest because they
have formed the starting-point of later researches. Although
Winogradsky states that only small quantities of organic
matter are tolerated it is noteworthy that in all the experi-
ments given above, with the exception of {c), the fluid in which
the bacteria multiplied must have contained soluble organic
matter in quantity.

The next important investigation is that of Molisch (3),
who cultivated both fresh- water and marine forms. For fresh-
water forms his general procedure was as follows. The material
was covered with water from the river Moldau, at Prague,
and included such substances as hay, boiled egg, bones, earth-
worms, snails, and peptone water. It was left to decompose
and examined periodically for its bacterial content. For
marine species such substances as Zostera (sea-wrack), a
bit of crab, or lish, or some other remains, were placed in sea-
water which had been brought from Trieste. In some of his
cultures a layer of oil was spread over the surface to limit the
supply of oxygen. Molisch obtained an abundant supply of
both colourless and coloured sulphur bacteria, as well as purple
coloured sulphurless bacteria. He was successful in obtaining
pure cultures of the last named,* but not with the sulphur
organisms. Contrary to Winogradsky, he considered that a
larger amount of organic matter was necessary for the growth
of these organisms. He confirmed, however, Winogradsky's
conclusions that oxygen can be utilized only at diminished
pressure, and that exposure to light is necessary for the coloured

* The cultivation of the sulphurless purple bacteria was first ac-
complished by Esmarch when he isolated Spirillum rubvum.

56 SULPHUR BACTERIA

forms. For pure cultures obtained from the raw cultures
mentioned above, he used the following medium : —

River water or sea water . . looo c.c.

Peptone . . . . .5 grams.

Dextrin or glycerine . . • 5 ,,

Agar 18 grams or gelatine lOO grams.

Colonies were obtained by plating from this medium. He
also obtained colonies by plating a drop of the inoculated
melted nutrient medium under a coverslip on a glass slide and
ringing the coverslip to exclude atmospheric oxygen.

Molisch's results are noteworthy in that they showed that the
purple sulphurless bacteria, which are genetically allied to the
purple sulphur bacteria, grow abundantly when supplied with
organic matter, and in that they showed that a greater intensity
could be secured in the growth of the coloured sulphur organism
Chromaiium in a medium containing organic matter but no H2S.

A very important advance was made by Keil when he
succeeded in preparing pure cultures of the colourless sulphur
bacteria, Beggiatoa and Thiothrix. This investigator assumed
that the following factors were necessary : sulphuretted
hydrogen, oxygen, carbon dioxide, and some source of nitro-
gen supply, and then proceeded to show that the influences
which favourably affected growth were favourable only
within certain limits. The upper limit of oxygen pressure
was 20 mm. Hg, the lower limit 10 mm. Hg, and the
optimum 15 mm. Hg. Using a fixed oxygen supply of 15
mm. Hg pressure he determined experimentally that the
optimum concentration of sulphuretted hydrogen was exerted
at a pressure of 0-8 mm. Hg (about 5 c.c. HoS in a vessel of
4500 c.c. capacity). The lower limit of carbon dioxide was
0-5 mm. Hg, approximately the amount normally present in
the atmosphere. The upper limit was 360 mm. Hg, and the
optimum 25 mm. Hg.

Keil then made use of these three substances in appro-
priate amounts to obtain pure cultures.

Pure Cultures. — [a] Thiothrix. — A clump of material made
up almost wholly of Thiothrix threads was washed and

THE CULTURE OF THE SULPHUR BACTERIA 57

placed in sterilized fresh water, or a solution made up as
follows : — •

Percentage.

Percentage.

CaH,(C03)2

• 0-34

Ca3(P04)2

0-02

MgH^iCOs)^ .

0-27

KCl

o-oi

CaSO^

• 0-31

K2S

O-OI

MgSO^ .

• 0-51

FeS

O-OI

Na2S04 .

0-2I

CaS

O-OI

A small quantity of ammonium sulphate was added, and
the three gases O2, H2S, and CO2 introduced.

In the second week, the threads of Thiothrix had begun
to liberate segments from their free ends. The culture was
periodically washed with sterile water. It was maintained
by Keil that by this method his cultures were free from im-
purities because the subsequent addition to them of O-l — 0-2
per cent, of sterile peptone did not result in a development
of saprophytic bacteria.

[h) Beggiatoa. — Pure cultures of Beggiatoa were obtained,
but with greater difficulty, due to the threads being free.
The purity of the cultures was tested in the same way. Keil's
conclusions were : —

1. The best source of nitrogen is ammonium sulphate.

2. Organic matter, although not harmful, cannot be used

as a source of N. (Contrast the conclusions of Wino-
gradsky and Molisch.)

3. Nitrate cannot be used as a source of nitrogen.

4. Oxygen, carbon dioxide, and sulphuretted hydrogen

are necessary for development, but their amounts
must be within the limits given above.

5. A carbonate of one of the alkaline earths is necessary.

Either the calcium or magnesium salt may be used.

6. The organism is indifferent to the salts of the alkalis.

7. A chloride furthers growth, and the culture fluid must

contain phosphorus.

8. Growth increases with rise of temperatures up to 30° C.

The threads rapidly degenerate above 35° C. Beggi-
atoa is somewhat more resistant than Thiothrix.
Thermal death point : Thiothrix, 37° — 38° C.
Beggiatoa, 45° C.

9. The colourless sulphur bacteria are indifferent to light.

58

SULPHUR BACTERIA

Jegunow (i — 5) cultivated sulphur bacteria in a flat glass
vessel about the length and width of an ordinary glass slide,
and of a depth about a sixth of the width. Some slimy m.ud
from the bed of one of the limans near Odessa was placed in
the vessel, together with water from the same source. The
vessel was exposed to air in an upright position. As oxygen
was supplied from above, and hydrogen sulphide from below,
: the organisms took up some intermediate position, where
they could obtain access to both. At the selected level they
formed what Jegunow names a Bacterial Plate, the shape of

Fig. 4.

which is shown in Fig. 4 (I) at a and on a larger scale at Fig.
4 (II). By altering the content of hydrogen sulphide he was
able at will to raise or lower the level of the bacterial plate.
The chief peculiarity of this plate is the series of downward
directed projections which are formed by it. The movement
of the bacteria composing each projection resembles that of
water in a spring. They travel down the projection, and on
reaching the bottom turn round and travel up again until
the top of the plate is reached. When viewed through a low-
powered microscope, when the bacteria are viewed in the mass,
the general appearance of a fountain is presented, which made

THE CULTURE OF THE SULPHUR BACTERIA

59

Jegunow bestow a second name to the aggregated bacteria,
namely Fountain Plate.

By letting down into the fluid a very line and weighted
thread which had first been treated with very dilute ferric
chloride, and then with ammonia, so that tlic thread was
coloured a faint bluish-yellow, he was able to show some inter-
esting relationships between the organism and the medium.
Below the plate the thread turned black, following the for-
mation of ferrous sulphide. This demonstrated the presence
of hydrogen sulphide below the plate : —

FeClg + 3NH4OH = Fe(0H)3 + 3NH4CI,
Fe(0H)3 + H2S = Fe^Ss + 6H,0.

In the plate itself the bacteria were filled with globules
of sulpfhur, whilst above the plate the colour was taken out
of the thread by the action of the sulphate formed by the
bacteria.

It must be stated that the phenomena described above
are not of frequent occurrence, and their incidence is probably
due to physical causes, in this case probably the presence of
slime.

Macgregor Skene, working with raw cultures of purple
sulphur bacteria, concluded that when more favourable results
were obtained by the addition of certain organic compounds,
this was to be attributed not directly to the organic compounds
but to the ammonium sulphate liberated from these com-
pounds by saprophytic bacteria. The most vigorous develop-

t tooK place m the lollc

)wmg medmm : —

Ammonium sulphate

0-75 gm.

Magnesium sulphate

0-05 „

Potassium dihydric phosphate .

0-05 „

Potassium chloride

0-05 „

Calcium nitrate

o-oi ,,

Sodium chloride

27-00 gms

Calcium carbonate .

10-00 ,,

Distilled water

1000 c.c.

6o « SULPHUR BACTERIA

A small conical flask (50—100 c.c.) containing a depth of
I — 1-| cms. of this solution was infected with sulphur bacteria,
and exposed to an atmosphere of sulphuretted hydrogen.
In 10 — 30 days a vigorous development of bacteria took
place, and a rich red-purple zoogloea covered the chalk
sediment.

Source of Nitrogen. — The most satisfactory of the nitro-
genous compounds experimentally added was found to be
ammonium sulphate, only slightly less satisfactory album.en,
peptone, and asparagin. As already stated these organic
compounds are not regarded by Skene as being directly assimi-
lated. The most suitable concentration of ammonium sulphate
was found to be O-i per cent.

Sources of Carbon. — Using ammonium sulphate as the
source of nitrogen he next determined the source of carbon. A
large number of organic substances were put under requisition,
but Macgregor Skene came to the conclusion that not one of
them was assimilated by the sulphur bacteria. Since vigorous
growth occurred in the second and third generations in the
mineral solution given above, he concluded that the carbon
constituents necessary for the building up of bacterial cells
could not have been contained in the original infecting material,
and such being the case the carbon could have no origin other
than the carbon dioxide of the atmosphere. Without pure
cultures he was not able to confirm this statement. A series
of experiments is recorded by this investigator to determine
w^hether the sulphur bacteria obtained COg from the atmosphere
directly or through the medium of an autotrophic bacillus
which constantly appeared in his cultures and which was easily
isolated.

Eight flasks were infected with a pure culture of the bacillus
and set aside for twelve days. Four were then sterilized, and
to the collection four uninoculated flasks were added. Of
the twelve flasks three from each series were infected with
purple sulphur bacteria, so that the following experimental
conditions could be obtained : —

THE CULTURE OF THE SULPHUR BACTERLi 51

3 flasks containing live autotrophic bacillus + CO^ from atmosphere

+ sulphur bacteria.
3 flasks containing dead autotrophic bacillus + CO, from atmosphere

+ sulphur bacteria.
3 flasks containing 0 autotrophic bacillus — CO, from atmosphere

+ sulphur bacteria.
I flask containing live autotrophic bacillus + CO, from atmosphere

— sulphur bacteria.

I flask containing dead autotrophic bacillus -)- CO, from atmosphere

— sulphur bacteria.

1 flask containing 0 autotrophic bacillus — COj from atmosphere

— sulphur bacteria.

The results were inconclusive, the first nine flasks showing
equal growth, and the last three, of course, none.

I'ests to ascertain if H.jS is necessary to purple sulphur
bacteria. — Flasks infected with sulphur bacteria were treated
as follows : —

Results.

1. Placed in air ........ 0

2. Placed in HjS gas ....... Strong growth

3. Usual salts added, 0-5 per cent, sodium thiosulphate + air

+ light 0

4. Usual salts added, 0-5 per cent, sodium thiosulphate + air

-light .0

5. Usual salts added, 0-5 per cent, sodium thiosulphate — air

+ hght 0

6. Usual salts added, 0-5 per cent, sodium thiosulphate — air

- light 0

7 . Sodium bisulphate, sodium sulphide, sodium thiosulphate,

potassium sulphide, calcium sulphide, iron sulphide,

each used in turn in place of HgS gas . . . all 0

Hence he concluded that HgS gas is a necessary addition.
Tests to ascertain the necessity of light to the purple sulphur
bacteria : —

1. Lieske's solution (see p. 44) ....

2. ,, ,, + dextrose, 0-15 per cent. .

3. ,, ,, + calcium lactate, 0-15 per cent. .

4. ,, ,, + pot. formate, o' 1 5 per cent.

5. Ammonium sulphate, 0'075 per cent. ;

+ dextrose, o'lj percent. .

6. ,, ,, 00-75 per cent. ;

+ calc. lactate, o"i5 per cent.

7. ,, ,, 0-075 per cent. ;

+ pot. formate, o"i5 per cent.

From which he concludes that hght is necessary

Results

Light.

Dark

+

0

recorded

0

,,

0

"

0

..

0

.'

0

iry.

0

62 SULPHUR BACTERIA

He found further that

[a) The purple bacteria grow better in dayhght than in

red light.

[b) The same bacteria grow better in red than in blue light.

Bavendamm repeated the cultural methods of Skene for.
the coloured, and of Keil for the colourless, sulphur bacteria.
He also used successfully Lieske's mineral solution for the
cultivation of the iron bacteria. All the culture fluids received
an abundant supply of pure "calcium prascipitatum." In
one instance he found that the growth of a raw culture of
Lamprocystis obtained from a mass of decomposing Chara
was so abundant that colonies of the organism rose to the
surface in large numbers. By an ingenious arrangement, for
details of which the reader is referred to his book, he was able
to regulate the supply of oxygen and of sulphuretted hydrogen.
He obtained the following results : —

1. HgS optimum supply 25 m.m. Hg pressure.

2. O2 „ ,, 26 „

Hydrogen was added to raise the pressure to that of the
normal atmosphere. His raw cultures were disturbed by the
appearance on the surface of a thin whitish-yellow layer of
sulphur in which were embedded numbers of a small colourless
bacillus. By subculturing he finally obtained a growth of
Lamprocystis which did not form a surface layer of sulphur.
He satisfied himself that the culture was now pure by trans-
ferring some of it to sterile bouillon, a procedure which did
not result in bacterial growth taking place in this fluid.

Experiments with Thioporphyra volutans. — A few tentative
experiments have been made with this organism by the author.
In nature it flourishes only in sea water containing organic
matter resulting from the decomposition of seaweed, or from
the presence of sewage. Both in nature and in artificial
cultures active growth results only if the supplv of oxygen
is limited. In nature this is secured by the rapid con-
sumption of the dissolved oxygen by the numerous aerobic
saprophytic bacteria that are also present. In the laboratory

THE CULTURE OF THE SULPHUR BACTERIA

63

the vessels containing decomposing seaweed show a much
stronger growth of the purple organism when stoppered.
The growth of the organism is hkewise m.uch better in pools
or in cultures in w hich the sulphuretted hydrogen is enough to
cause a perceptible smell. Finally, active growth follows ex-
posure to subdued light, but does not take place in the dark or
when exposed to bright light. Dr. Blodwen Lloyd, in the
author's laboratory, has succeeded in cultivating Thioporphyra
in a medium made up of potassium nitrate 0-5 gram, sodium
dihydric phosphate 0-25 gram, calcium malate 5 grams, and
sea water lOOO cc. The nitrogen in this culture was thus
obtained from the nitrate, and the carbon almost certainly
from the CO2 of the atmosphere. On further cultivation,
however, the size of the organism rapidly diminished in this
artificial medium, probably because the nitrate was not the
best source of nitrogen. So far as the investigation has pro-
ceeded, the results confirm those obtained by Keil, Skene, and
Bavendamm, but it is not yet known whether under natural
conditions nitrogen is derived, as Molisch believes, from organic
matter, or, as Skene thinks, from products resulting from the
decomposition of organic matter.

Changes Effected in the Constitution of the Water
BY Thioporphyra volutans

The chemical changes which take place in a marine pool
during the growth of Thioporphyra volutans have been investi-
gated (Ellis and Stoddart). An analysis of the water of a
neighbouring marine pool in which there was no decomposing
matter is given for comparison in the following table : —

H2S.

Oxygen.

Ca.

Mg.

so.,".

PH.

Grams
per Litre.

cc.
per Litre.

Grams
per Litre.

CO.

per Litre.

Grams
per Litre.

Grams
per Litre.

Grams
per Litre.

0-00032

0-2I

0-013

S-89

0-20

0-315

0-408

8-1

A pool containing decomposing matter was kept under
observation for ten weeks during the summer, and samples of

64

SULPHUR BACTERIA

water from it were periodically analysed. During this period
the pool, which also contained putrescent seaweed, became
coloured a deep purple owing to the growth of Thioporphyra.
Whilst the chemical changes cannot all be ascribed to Thio-
porphyra volutans, the fact that this organism during the period
of investigation was far more numerous than any other indi-
cates that it was responsible for a very large percentage of
the chemical changes that took place. This applies particularly
to hydrogen sulphide and the sulphates, which are respectively
assimilated and secreted by this organism.

The following table gives the results of the chemical
analyses : —

HoS.

Oxygen.

Ca.

Mg.

SO4".

pn.

Grams

c.c.

Grams

c.c.

Grams

Grams

Grams

per

per

per

per

per

per

per

Litre.

Litre.

Litre.

Litre.

Litre.

Litre.

Litre.

At commencement

of investigation

•00022

•15

•015

IO-5

■20

•315

•690

7-2

After 3 weeks

•00071

•465

•016

II-4

•25

•317

•672

7-3

,. 6 ,,

•03

19-71

•003

2-1

•126

•16

•368

7-1

,, 8 ,,

•0072

47

•0028

1-96

•33t>

•49

I-I9

7-6

,. lo

•0045

3-0

•001 s

1-26

•18

•235

•588

7-4

The results are represented graphically in Fig. 5.
In addition, a total count was made of the saprophytic
bacteria, with the followingf results : —

Time in Weeks.

No. of Saprophytes
per c.c. (approx.).

Sulphur Bacteria.

0

3

6

8

10

1,000,000
1,000,000
2,000,000
5,000,000
5,000,000

Few : objects in water not purple.

Objects in water slightly purple,
deep

These tables show that for six weeks the content of hydro-
gen sulphide and the number of saprophytic bacteria had
steadily increased, the increase in the former obviously result-
ing from the increase in the latter. During this period the

THE CULTURE OF THE SULPHUR BACTERL4 65

sulphur bacteria had also increased, and at its close had im-
parted a tinge of their colour to the various objects in the pool.

H2S in soluh'c
Oxygen >• »

A

\

16
12
8

/

/

\

r—

- —

/

/

s

V

!)

Y

N»-.

— <

*—— .

"^1

i 6

Time in weeks

4 6

Time in weeks.

76

1 74

X
0- 7-2

7n

/

^

^

^

^■'''><~.

/

/

^-H

/

4 6

Time in weeks.

Fig. s.

10

The rise in the .hydrogen sulphide content is an indication
that the supply by the saprophytes was greater than the

5

66 SULPHUR BACTERIA

demand for it by the sulphur bacteria. After the sixth week,
although there was a marked increase in the number of
saprophytes, the sulphur bacteria had increased at a far
greater rate, until the demand for hydrogen sulphide was
greater than the supply. The gradient downwards in the
sulphide graph is very steep between the sixth and the tenth
weeks.

On the other hand, the downward decline of the oxygen
curve is steady during the whole of the period of investi-
gation, an indication that both the saprophytes and the
sulphur bacteria were using up this element in respiration.

Whilst the graphs for hydrogen sulphide and for oxygen can
be interpreted in terms of the vital activity of the sulphur
bacteria, the same cannot be said of the SO4" graph. The in-
determinate character, of this graph indicates that, like the Ca
and the Mg, which have similar graphs, its rise or fall does not
coincide with the rise or fall in the numbers of the sulphur
bacteria. This fact is consistent with the supposition that the
change from S to the sulphate is not directly dependent on the
vital activities of the sulphur bacteria.

The smiall fluctuations in the pW values reflect the small
changes that follow the absorption of hydrogen sulphide by
the sulphur bacteria, the production of other acids by the
saprophytic bacteria, the partial neutralization of these acids
by the calcium and other compounds, and the changes caused
by evaporation or by rainfall. The results in general confirm
those obtained by other investigators for other sulphur bac-
teria, but cannot be regarded as conclusive, because the con-
tribution of other organisms which were necessarily present in
the pool under observation could not be exactly estimated.

The growth of Thioporphyra reached its zenith in a medium
containing enough organic matter to support 5,000,000 bacteria
per cubic centimetre. Winogradsky's conclusion that the sul-
phur bacteria tolerate only minute quantities of organic matter
is not justified. Without pure cultures it was not possible to
ascertain if Thioporphyra assimilates organic matter directly.

CHAPTER V.

THE PRINCIPLES OF CLASSIFICATION AND THEIR
APPLICATION TO 1TIE SULPHUR BACTERIA.

The Principles of a Natural Classification, and their
Application to the Grouping of the Sulphur Bacteria.

All bacteria are usually assigned to the plant kingdom,
mainly because the genus Bacillus, which includes most
bacteria, is, in its structure and life-history, of a distinctive
plant nature. This cannot, however, be said of some of the
other genera in this group. Some, as for example. Spirillum,
are more allied to animals that to plants. In the classification
of more highly evolved organisms the ditihculty would be a
serious one, but bacteria are at a stage of development where
the distinction between a plant and an animal is not so well
defined as in higher organisms. They are in fact not sufiiciently
far removed from ultramicroscopic organisms that cannot be
assigned exclusively to either the plant or to the animal
kingdom.

All those bacteria which contain sulphur inclusions are
grouped under the name sulphur bacteria. These organisms
are morphologically very varied but physiologically similar.
Physiological similarity does not, however, necessarily imply
genetic affinity, and the systematist is therefore confronted
with a difficulty. Fie must effect the grouping of a large
number of species on the basis of a common physiological
attribute, when some of the constituent units can be assigned
to the plant kingdom, whilst others readily find a place in
the animal kingdom. A grouping on physiological lines is
unsatisfactory, for, with increasing knowledge, it is becoming
more evident that the mode of metabolism of an individual
bacterium is not fixed and unalterable. As a class bacteria

67 5*

68 SULPHUR BACTERIA

are very adaptable. For example, certain bacteria lead both
a saprophytic and a parasitic existence according to the nature
of the habitat ; and there are denitrifying bacteria that undergo
the sulphur metabolism. The insufiticiency of a physiological
grouping is apparent in the sulphur bacteria, for whilst the
group includes almost every known variety of form and
structure, the possession of sulphur inclusions does not connote
any other distinction common to the whole group ; and this
is the surest indication that the classification on the basis of
a common physiological trait is not genetically sound. But
it is nevertheless adopted in this book on account of its con-
venience in limiting the area of investigation. The further
subdivision of the group is followed on morphological lines.
All existent classifications have the defects of the classifi-
cations of higher plants in the days of Linnaeus. These were
based on artificial distinctions rather than on natural afiinities,
and to a certain extent the difficulty will always remain in the
classification of lowly forms with the high degree of plasticity
possessed by bacteria. Of the earlier attempts the best is that
by Migula (i — 3), which is based on morphological characters.
This classification would have been completely successful were
it not for the fact that the distinguishing features selected
by him were in several instances transient and not permanent
attributes.

In the classifications of Engler (1912) and of Meyer (1912),
the sulphur bacteria are incorporated in a general scheme in
which a few changes are made in the division of the sulphur
bacteria to bring them into conformity with the general scheme
adopted by the authors. There had not been during the
period (1909-12) any investigations which threw any further
light on the afitinitics of the sulphur bacteria. One addition
had been made to their number, namely, Hillhousia, dis-
covered by West and Griffiths in 1909.

Of the recent classifications those of the American Society
of Bacteriologists are not completely satisfactory. They
violate the first essential of any logical system of grouping
which demands that it should not be possible to fit any
organism into more than one group. Inclusion in one group

THE PRINCIPLES OF CLASSIFICATION 69

should necessarily spell exclusion from any other group, and
this is not the case in the American classifications.

In the lengthy and elaborate schemes that have appeared
from America there has been no attempt to question the
validity of the characteristics used for the groupings. All the
attributes used in earlier groupings have been taken at their
face value. Those parts of Buchanan's scheme that deal
with the sulphur bacteria will be here noted.

Review of the Aitributes used in the Subdivision of
THE Sulphur Bacteria.

The chief difficulty in grouping these forms arises from the
fact that hitherto so little has been done to distinguish transient
from permanent characters. If the former arc used for the
diagnosis of bacteria, confusion inevitably follows the effort
to establish the genus and species of any particular unknown
organism on the basis of its morphological distinctions. The
range of constant attributes among bacteria is very limited.
The author has recorded (Ellis (i — 3)) the results of the ex-
amination of the more important morphological characters
generally used in the classification of bacteria. These are
given below : —

(i) Division in one, two, or three planes. — It was found
experimentally that a Sarcina which normally divides in three
dimensions of space, could be made to break up into smaller
units of one, two, three, or four cells. An observer remarking
the cells in this last condition, would not be able to allocate
them to the genus Sarcina, for they would not show the
characteristic cell-arrangement in three dimensions of space.

In the same way species of the genus Micrococcus, which
is distinguished by the divisions of its cells in two dimensions,
were induced to break up into smaller groups varying from
one to four or a small number of cells.

On the other hand, the Streptococci, which divide in only
one dimension, do not, in the author's experience, depart from
that method of division. Hence it follows that an organism
showing globular cells in a culture may be a Sarcina, or Micro-
coccus, or a Streptococcus. It is possible only by an extended

70 SULPHUR BACTERIA

investigation to ascertain to which of these genera any particular
organism belongs. The author is of the opinion that no
difference exists between the genera Sarcina and Micro-
coccus. An organism is generally ascribed to Sarcina if
observed dividing in three dimensions, and to Micrococcus
if in two dimensions, but it has been shown that the same
organism may use both methods of division. It is an easy
matter, by subculture, to effect the transformation of a
Sarcina into a typical Micrococcus. Hence the number of
planes of division is not constant, and is therefore not a suitable
criterion for the classification of bacteria.

(2) Motility. — In Migula's system a distinction is made
between a Sarcina and a Planosarcina, the former being non-
motile, and the latter motile. A similar distinction is drawn
between Micrococcus and Planococcus. Twenty-four species
of Sarcina, Micrococcus and Streptococcus were cultivated by
the author in such a way that the cells were as short a time
as possible in the presence of their excretions. The result
of such cultivation was invariably the same. The aggregated
cells were found to break up into uni-, diplo-, tri-, and tetra-
cocci, and at the same time motility was developed. Con-
versely, the aggregated stage was found to be a non-motile
one ; this loss of motility was correlated with an increased
slime-production. It was found possible by frequent sub-
culturing to reduce the slime formation and so to induce
motility in sixteen species of Sarcina, five species of Micro-
coccus, three species of Streptococcus, and five species of
Bacterium, all usually described as non-motile. The bacteria
investigated were the following : — ■

Sarcina piilmo)ium, auresce^is, flavescens, rosea, flava, mo-
hilis, fimentaria, gasoformans, striata, vermiformis ,
oleus, ventriculi, fuscescens, marginata, and two
unidentified species.

Micrococcus helvolus, citreiis, grossus, and two unidentified
species.

Streptococcus tyrogenus, pallidus, and pyogenes.

Bacterium hirtum, tomentosum, filamentosum, rugosum.
and cerinum (Ellis (i — 3)).

THE PRINCIPLES OF CLASSIFICATION 71

It is therefore highly probable that the capacity of
movement is a distinctive feature of all bacteria under certain,
at present incompletely known, conditions. It is well to bear
in mind that motility is one of the fundamental properties of
protoplasm, and is more readily manifested in unicellular
aqueous organisms because of the obvious advantage which
they obtain by its occurrence. At any rate it is certain that
the fact of motility in an organism can only have a restricted
use in a natural system of classification.

(3) The Size of the Cells. — This is an unreliable criterion for
classification, as may be demonstrated by the cultivation of
any one species of bacteria for an extended period, and under
difi^erent conditions of growth. The production of long or
short rods is often dependent upon variable external conditions,
whose effect upon the organism cannot be predicted before-
hand. The tendency of a normal bacillus to form long filaments
under certain conditions is well known. Even the thickness
of any particular species of bacteria varies. At the same time
a large organism, e.g. Bacillus megatherium, is always large
when cultivated under normal circumstances ; and others, as
for example some of the nitrate bacteria, are always very small.
It is therefore possible to assert that an organism is large or
that it is small, but unless the given range of its measurements
is a wide one, any statement of the size of an organism cannot
be relied upon as a help in its identification.

Some of the sulphur bacteria show an extraordinary
variation in size. This applies not only to differences in
different generations, but frequently to differences in the same
generation. Bacterium sulfuratum may show hundreds of
different sizes in the same field. It is a sound rule, and one
followed in this book, to ascribe to the same species all those
organisms that are similar except as to size, and in which the
sizes are so graded that the individuals form a series separated
only by minute dimensional differences. This attitude is
justified by the frequent occurrence of pleomorphism (see
pp. 14 et seq.) in the sulphur bacteria. In the cultivation of
some of the sulphur bacteria a very small change in the environ-
mental conditions sometimes results in the production of indivi-

72 SULPHUR BACTERIA

duals of varied size, a fact which makes it practically impossible
to fix upon a certain defmite size as being an attribute of any
particular organism. The method adopted by Winogradsky
in his study of Beggiatoa alba is not one which is justified in
the subdivision of bacteria. When lie found various sizes
of that organism in the same field connected together by
numerous gradations, he arbitrarily selected a particular
range, and bestowed upon the individuals within that range
a specific name. In this way several " species " of the genus
Beggiatoa were established by him. Such " species " are not
of the nature of " microspecies " or " elementary species " or
" pure lines." The variations in size appear to be the
ordinary manifestation of every culture of Beggiatoa; it is an
aspect in the pleomorphic tendency of this species, and no
more significance is to be attached to tlie variation than
to the variation in the number of sulphur granules in the
different individuals. W inogradsky established the following
" species " : —

Threads up to i/x thick, Beggiatoa minima.
1— 2-5/A ,, ,, media.

2-5— 4/x ,, ,, alba.

4—5-5/^ :, ,, major.

The figures were arbitrarily chosen by him and he admits
that any others would have done " weil sie nur auf Convention
beruht." In other words, they are intended merely as catalogue
names, and in that case it is not correct to label them as
"species." Suppose a culture of this genus in, say America,
shows sizes of filaments varying in thickness from, let us
suppose, i/Lt to 6jLt, and a culture of the same genus, in say
Germany, gives a crop of threads of thicknesses varying from
■|/i, to 5/x. According to this " Convention " all the threads in
both cultures between ijx and 2-5jLt must be labelled Beggiatoa
media. Whilst there is still some ambiguity in the meaning
of the term species all must agree that it connotes a unit that
is sharply separated from other units, whether the bond of
union is known or unknown. It is more reasonable to fix
the name Beggiatoa alba (chosen for priority) for all thicknesses

THE PRINCIPLES OE CLASSIFICATION 73

of this organism, until there is evidence that there are other
distinctive differences between the different classes of threads.

(4) Cilia Insertion : Presence or Absence of Cilia. — An
investigation was instituted (Ellis (2 — 3)) on the mode of inser-
tion of the cilia to ascertain its constancy in the life-history
of a species. A culture of a species of Bacillus was kept
for several months, under different conditions. At frequent
intervals cilia preparations were made, and it was found
that under all conditions the peritrich ciliation was main-
tained. l"he same procedure was followed with a culture of
Pseiidomonas, with the same result, namely, that the polar
ciliation characteristic of this genus was also a constant feature.
So far as it was possible to conclude from the limited scope
of these experiments the mode of ciliation may be used for
purposes of classification.

A distinction must also be made between organisms pro-
pelled by cilia, and others, like Beggiatoa alba, that are motile,
but which are apparently without cilia. Even if later re-
searches show that Beggiatoa and similar organisms do possess
cilia, these are almost certain to be of a different character from
the cilia of the bacterial organisms known at present to possess
them. Hence it may be possible with further knowledge
to make use of this fundamental difference in the mode of
ciliation.

Migula in his classification made use of the number of cilia.
In the author's experience the number of cilia has been found
to vary in the same species. The cilia of Spirillum voliitans
were found to vary from one or two up to thirty and even a
greater number. The number of cilia seems to have some
relationship to the amount of slime formation, and this in its
turn depends on the nature of the external cultural conditions,
and the other factors which promote or retard growth. Hence
the mode of insertion of cilia may be used for classification,
but not tlieir number.

(5) Pleomorphism. — In Chapter I. it has been shown that
some of the sulphur bacteria are pleomorphic; for example,
that the organism known as Lam.procystis is a form that occurs
in the life-history of at least three organisms, namely Bacterium

74 SULPHUR BACTERIA

siilfiiratiim, Beggiatoa roseo-persicina, and Thioporphyra volu-
tans ; and probabh^ of other sulphur bacteria not yet fully in-
vestigated. It has also been shown that the zoogloea condition
is frequently found in some of the sulphur bacteria ; that in
some species almost every shape known in the sulphur bacteria
may be found ; that in some cases the actual transition from
one form to another has been directly observed ; and that
two organisms, apparently specifically distinct, have been
observed in organic connection. Hence pleomorphism may
be a useful aid in the separation of species, if it can be shown
that in certain species its appearance is a regular occurrence.

(6) Mode of Geyniination of the Spore. — The germination of
the spore in the genus Bacillus is not uniform in all species
of that genus. As, however, spore formation, with one or
two exceptions, is confined to that genus, this feature cannot
be used in the classification of the sulphur bacteria.

ij) Method of Cell Division. — With greater knowledge it
may be possible to utilize the undoubted differences that exist
in the method of division of the cells. In the genus Bacillus
cell division is preceded by the formation of a plasma-derived
wall cutting across the cell. This wall then undergoes
longitudinal division, the process resembling in all essentials
the method of cell division which is followed in the higher
plants. On the other hand, in the genus Spirillum, the cell
contents withdraw from a certain area, separating the two
daughter cells which even at this stage are organically separated;
and are kept in place only by a connecting bridge of slime.
Complete separation follows the disappearance of the slime (see
Ellis (i)). lliis method occurs where a definite cell membrane
is not formed, and it is general among animal cells. With one
or two exceptions, e.g. Beggiatoa mirabilis, the cells of the
sulphur bacteria divide by the second method. This difference
in the mode of division could be used with advantage in the
classification of the sulphur bacteria were it not that the number
in which the first method prevails is so small that its use would
not have much value, and that there are too many organisms
among the sulphur bacteria of which the exact method of cell
division is unknown.

THE PRTXCIPLES OF CLASSIFICATION 75

(8) Intimate Structure of the Cell. — The structure of the cell
is simple and no nucleus is differentiated. In addition, the
membrane in the majority of the sulphur bacteria is not well
marked (see Chap. X.). So far as is known at present, the
presence of sulphur inclusions in the cell does not imply the
constant presence of any other solid substance in the cell.
The intimate structure of the cell is too uniform in the sulphur
bacteria to permit of use being made of it in classification.

(9) Colour. — The primary division in all classifications of the
sulphur bacteria is based on the presence or absence of colour.
The distinction of colour is most useful from the practical point
of view, and it is probable also that the differences between
the coloured and the uncoloured sulphur bacteria are deep
seated. The coloured forms probably utilize the energy of
light, as is done by green plants ; and if this be the case
their metabolism is essentially different from that of the
uncoloured forms. To some extent the distinctive features
of the coloured and the uncoloured forms are different, but
they are not sufficiently well marked to justify their use as
definite connotative marks to distinguish one kind from the
other.

The terms Leuco and Rhodo are already in use as prefixes
for the colourless and the coloured forms respectively, and it
is proposed to retain them, so that the first division in the
author's revised system will be

1. Leuco-thiohacteria.

2. Rhodo-thiohacteria.

A difficulty has arisen in taking this step. The name
Thiospirilhmi was originally used by Omelianski for all sul-
phur spirilla, both coloured and uncoloured. If this primary
division on the basis of colour is to be retained, it will be
necessary to make a distinction between the coloured and the
uncoloured spirilla. To preserve uniformity of nomenclature
it is proposed to make the following distinction : —

Thiospirilhmi— \Jnco\oured sulphur spirilla.
Rhodothiospir ilium — Coloured ,, ,,

76 SULPHUR BACTERIA

Another difficulty arose in the nomenclature of Thiothrix.
The author has found a coloured Thiothrix, which appears to
differ in no single particular from the normal uncoloured species,
except in colour. Its genetic connection with Thiothrix is so
patent that it must be retained in that genus in spite of its
possession of colour.

(lO) Diversity of Habit. — Several attempts have been
made, notably by Bcijerinck and by Orla-Jensen, to frame
classifications based on differences in the mode of life of
different bacteria. Such attempts would have much to
recommend them, were it not that the majority of bacterial
organisms could be included not in one but in several
groups. It has already been pointed out that many para-
sites, like Bacillus cholercE for instance, lead a saprophytic life
when living cells are not available. Many other instances
may be cited in support, and it may be stated of bacteria in
general that the more that is known of their metabolism the
greater the conviction of the diversity of their habits. All
physiological systems fail to satisfy the first requisite of a
logical classification, namely, that it should not be possible
to place an organism in more than one group. 7he sulphur
bacteria are not cultivable on ordinary media, so it is not
possible to use such cultural characteristics as gelatine lique-
faction, or sugar reactions, for the diagnosis of species. The
reaction of the sulphur bacteria to the Gram stain has not
yet been investigated, so that its use is problematical.

Summary.

The foregoing remarks may now be summarized as follows :
(i) Spatial Division of the Cells. — Of limited application.
(2) Motility. — A positive result is valuable, but a negative
one is not. Absence of movement may be due to an abnormal
development of slime, with a consequent restriction of cilium
development. Caution must therefore be exercised. The
sulphur bacteria may be divided into three groups on the basis
of motility : (i) Motile with cilia ; (ii) Motile without cilia ;
(iii) Motility unknown.

THE PRINCIPLES OF CLASSIFICATION 77

(3) Size of Cells. — Of limited application, on account of the
variations both in length and in thickness. Certain ranges
of dimensions may be given, if they are not regarded as having
an absolute value.

(4) Ciliation. — 1 he mode of distribution of cilia is constant.
Bacteria may be divided into three classes on this basis :
(i) With peritrich ciliation ; (ii) with polar ciliation ; (iii) with
no cilia. The number of cilia is not constant, except where an
organism has not more than one cilium.

(5) Pleomorphism constitutes a factor which may be used in
the grouping of the sulphur bacteria. Our knowledge of this
phenomenon in the sulphur bacteria is at present too scanty
to make much use of it.

(6) Mode of Germination. — Not applicable.

(7) Method of Cell Division. — Two methods are known : (i)
with formation of transverse wall in the plasma ; (ii) without
such a formation. The value of this difference is considerably
lessened because practically the whole of the sulphur bacteria
belong to the second class.

(8) Intimate Structure of the Cell. — The differences are not
sufficiently marked to give them value for grouping purposes.

(9) Colour. — The presence or absence of colour is the most
important distinction that is found in the sulphur bacteria.

(10) Diversity of Habit. — As many, perhaps all, bacteria do
not follow only one mode of life, a grouping based on the mode
of life of the sulphur bacteria can have very little value for
the major partitions, although there is a possibility of its use
in the minor divisions.

There are thus many difficulties to encounter in the
classification of the sulphur bacteria. The confused state of
the classifications of the present day arises from the bestow-
ing of names on what appear to be new species, after only
an inadequate investigation. Genera have been founded on
characters whose constancy is open to question ; and very
little pains have been taken to ascertain whether the life-
history of the organism under investigation is limited to the
one phase that is presented to the observer.

Probably more classifications of bacteria have been issued

78 SULPHUR BACTERIA

within the last thirty years than of any other class of plants ;
and yet there has been practically no critical examination of
the soundness of the characters that have been used to effect
the division of these organisms. The classification sponsored
by the American Society of Bacteriologists is no exception, at
any rate in that part of it which relates to the sulphur bacteria.
It is proposed in the present work to attempt a remedy in the
limited field of the sulphur bacteria, and to modify Buchanan's
classification after elimination from it of what is regarded as
of dubious value.

Review of Previous Classifications.
Winogradsky's Classification of the Sulphur Bacteria.

The predecessors of this grouping may be left out of account,
as they were based on very scanty and inaccurate knowledge.

Winogradsky, 1888, distinguished fourteen genera, in-
cluding the genera of previous investigators : —

I. Colourless Bacteria.

(i) Beggiatoa. — Threads equally thick, freely motile, and
not forming gonidia.

(2) Thiothrix. — Threads unequally thick, attached, and

forming motile gonidia.

II. Coloured Bacteria.

(A) Cells united in families.*

a. Division in three directions of space.

(3) Thiocystis. — Single or several families of small cells,

surrounded by a slime cyst, and capable of movement.

(4) Thiocapsa. — Several families of round cells, loosely

embedded in a common envelope of slime. Not
motile.

(5) Thiosarcina. — Families in packets.

b. Division at first in three, later in two directions of

space.

* The term family is used here in the sense of a cluster or an aggregate of
similar cells.

THE PRINCIPLES OF CLASSIFICATION 79

(6) Lamprocystis.— -Fami\\es at first forming a solid mass,

later forming a reticulated hollow sphere.

c. Division in two directions of space.

(7) Thiopedia. — Families in plates, cells arranged sym-

metrically in fours, and capable of motion.

d. Division of cells in one direction of space.

(8) Amoshobacter. — Families amoeboid and motile. Cells

connected by plasma threads.

(9) Thiothece. — Families with thick slime cysts. Cells

motile and loosely enclosed in a common envelope
of slime.

(10) Thiodictyon. — Families made up of rods arranged net-

wise.

(11) Thiopolycoccus. — Families compact. Cells non-motile,

small and closely pressed together.

(B) Cells free, and capable of movement.

(12) Chromatinm. — Cells cylindrical-elliptical.

(13) Rhabdochromatium. — Cells rod- or spindle-shaped.

(14) Thiospirillum. — Cells spirally twisted.

Remarks on Winogradsky's Classification.

The attributes selected for the grouping are simple, and for
the most part easily ascertainable. The most serious fault
of this system lies in the inconstancy of the characters used
in the grouping, and this applies in particular to the partition
of these organisms according to the number of dimensions in
space in which they divide. Motility or its absence is also
an uncertain factor.

The following features in the species are constant : — •

[a) The division into coloured and uncoloured forms.

[b) The division into free cells and aggregates of cells

(families).

[c) The shape of the cell (rod, globular, etc.)

As Winogradsky discounted the facts of pleomorphism, and
in consequence magnified the importance of variants into
species and even into genera, the value of his system is

8o SULPHUR BACTERIA

considerably impaired. Of the fourteen genera the generic
names Lamprocystis, Thio splicer ion, and Thiopolycoccus have
been discontinued for reasons given later. The remaining
eleven genera are retained with modifications. Since the
publication of Winogradsky's system a comparatively large
number of new genera have been added.

Both the defects of Winogradsky's scheme and the discovery
of new genera which do not readily fit into his scheme, make it
necessary to modify the bases of grouping used by him.

This classification is, however, one of great importance
since it has formed the pivot about which have turned the
majority of subsequent systems. The genera established by
Winogradsky have been incorporated into the general schemes
of classifications of bacteria. They appear with a few modifica-
tions in the elaborate classifications of De Toni and Trevisan
(1889), Hansgirg (1888), Cornil-Babes (1890), Migula (1890
and 1894), Ludwig (1892), Sternberg (1892), Fischer (1895),
Lehmann and Neumann (1896), Chester (1897), Kendall
(1902), and Molisch (1907). During the period 1888-1907
there were no investigations to determine the validity of
the features selected by Winogradsky for the grouping of
the sulphur bacteria. Their diagnostic value had not been
challenged, and in all, the facts of pleomorphism were either
unknown, or if known, disregarded. The most noteworthy
contribution to our knowledge of the sulphur bacteria dur-
ing this period was Molisch's Die Purpurhakterien, and in
the system established by him notable changes were intro-
duced.

During the period 1888- 1907 a new genus of colourless
sulphur bacteria was discovered by Hinze (1903), and
named Thiophysa voliitans. No consideration, however, was
given to this organism by Molisch because this writer dealt
only with the coloured sulphur bacteria.

Molisch's Classification of the Sulphur Bacteria.
Order RJiodohacteria.

Bacteria coloured red, rose, violet, or carmine, by the
presence of bacteriopurpurin.

THE PRINCIPLES OF CLASSIFICATION 8i

1. Cells contain free sulphur.
Family (i) Thiorhodacece.

[a) Cells united into families.

Sub-family a. Thiocapsacece. — Division of cells in

three directions of space.
Sub-family h. LamprocystacecE. — Division of cells

first in three, then in two directions of space.
Sub-family c. Thiopediacece. — Division of cells in two

directions of space.
Sub-family d. Amcehobacteriacece. — Division of cells in

one direction of space.
ib) Cells always swarming.

Sub-family^. ChromatiacecE. — Not capsulated.
Sub-family /. Rhodocapsacece. — Capsulated.

2. Cells do not contain free sulphur.
Family (2) Athiorhodacecs.

[a) Cells united into families.

Genus a. Rhodocystis. — Cells rod-shaped, many being
embedded together in a common capsule.

Genus b. Rhodonostoc. — Cells spherical or short rods
in chains, each chain enclosed in a capsule.

[b] Cells free.

Genus a. Rhodococcus. — Cells spherical, non-motile.
Genus b. Rhodobacterium. — Cells straight rods and

non-motile.
Genus c. Rhodobacillus. — Cells, motile rods.
Genus d. Rhodovibrio. — Cells, short, bean, or comma

shaped ; monotrichous, actively motile.
Genus e. Rho do spirillum. —Cells, spiral ; actively

motile ; polar ciliation.

Remarks on Molisch's Classification.

Molisch did not investigate the colourless sulphur bac-
teria, and in the present work we are not dealing specifically

with the sulphurless purple bacteria. His classification has

6

82 SULPHUR BACTERIA

therefore only a partial application. He divides the coloured
sulphur bacteria into six groups, the first four of which are
divided according to the number of directions in space in which
the cell divides, and the last two are separated from the rest by
being free instead of forming families. I'his classification, in
one respect, is not an advance on that of Winogradsky, for
in it also the number of dimensions in space in which an
organism divides is made the basis of the primary grouping.

Molisch reduced Winogradsky's fourteen groups to six, as
he omitted the following : Thiocystis, Thiosarcina, Thiothece,
Thiodictyon, Thiopolycoccus, Rhabdochromatium, and Thio-
spirillmn. No reasons are given except that the classification
is made " auf Grund meiner Erfahrung." As Molisch's re-
searches did not deal, except incidentally, with the grouping
of the sulphur bacteria, his classification of these forms is
necessarily incomplete. He, however, added two genera to
the sulphur bacteria, namely Rhodocapsa and Rhodothece.
Both are sharply marked off from all the genera hitherto
included in the sulphur bacteria. In his own classification
they are included presumably in Rhodocapsaccce, although
he does not specify the genera of this sub-family in his
scheduled classification.

A notable departure in the method of classification of
bacteria was made by Orla Jensen (1909). In the interval
the list of sulphur bacteria had been supplemented by the
discovery of the genus Thioploca by Lauterborn (1907).
Orla Jensen introduced entirely new factors in his classifica-
tion. Whilst using morphological characters for the main
groupings, the families and genera are separated on the basis
of physiological difterences.

Orla Jensen's Classification.

Two Orders are distinguished.

I. Cephalotrichince. — Cells spherical, rod-shaped, or spiral.
Endospores formed only in a few sulphur-free spirilla. When
motile, mono- or lopho-trichous. Typically water bacteria.
Energy almost exclusively by oxidation processes.

THE PRINCIPLES OF CLASSIFICATION 83

2. Peritrichime. — ^Cells spherical, or rod-shaped, never
spiral. Either peritrichous or non-motile. Not typically
water bacteria. Energy not by oxidation processes.

Seven families are included in the first order : —

Oxydobacteriacece.

Luminibacteriacece.

Reducibacteriacece .

* Thiobacteriacece. — Containing sulphur but no bacterio-

purpurin.

* Rhodobacteriacec?. — Containing sulphur and bacteriopur-

purin.
Actino)}Lyces.

* Triihobacteriacere. — Water forms. Absence of mycelium.

Filamentous.

Four families are included in the second order : —

Acidobacteriacece.

Alkalibacteriacece.

BntyribacteriacecE.

Putribacteriacecs.

All the sulphur bacteria then known were placed in one or
other of the three families marked with an asterisk.

Family Thiobacteriacece :

1. Cells not spiral.

A. Cells rod-shaped or not spherical.

* Snlphomonas. — Autotrophic, rod shaped.
Thionionas. — heterotrophic, oval.

B. Cells spherical.
Thiococcus. — Cells spherical.

2. Cells spiral.

Thiospirillum.

Family Rhodobacteriacece :

Rhodoinoiias [Chromatium).
Rhabdotnonas [Rhabdochromatium) .

* Sulphomonas is not rightly included in ThiobacteriacecB as the cells do
not store sulphur.

6*

84 SULPHUR BACTERIA

Rhododiclyon ( Th iodictyoii).
Aynoebomonas [Amcebobacter).
Rhodothece [Thiothece).
Rhodopoly coccus {Thiopoly coccus) .
Rhodococcns ( Thiopedia).
Lamprocyslis.
Rhodocystis (Thiocystis).
Rhodocapsa ( Th iocapsa ) .
Rhodosarciva {Thiosarcina).
Rhodospirilhun [Thiospirillum).

Family Trichobacteriacece :

1. Not containing sulphur granules.

2. Containing sulphur granules.

a. Beggiaioa. Threads not attached. Motile.

b. Thiothrix. Threads attached.

Remarks on Orla Jensen's Classification.

All the fourteen genera introduced by Winogradsky were
accepted, but their names were changed. In addition four
new genera were added. The first of these calls for particular
attention, because it is a genus composed of cells in which
sulphur is not stored. This is Siilphomonas , the name in this
system for the thionic acid bacteria, which by this time had
been discovered by Nathansohn. Thiophysa volutans appears
under the name Thiomonas. The third and fourth genera,
namely Thiococciis and Thiospirillum, were added as logical
sequences to the insertion of, rod-shaped genera in the family
Thiobacteriacecp. The teriri Thiospirillum was not well chosen,
as it had already been used by Winogradsky with a different
meaning.

It has already been affirmed that all classifications with
pliysiological attributes as the main basis of the grouping
cannot be regarded as satisfactory, because bacteria are not
confined to only one mode of life.

THE PRINCIPLES OF CLASSIFICATION 83

Buchanan's Classification (1917).

He distinguishes six orders of the class Schizomycetes.

Order i. — Enhacteriales.
Order 2. — Chlamydohacteriales .
Order 3. — Actinomycetales.
Order 4. — Thiobacteriales.
Order 5. — Myxobacteriales.
Order 6. — Spirochcetales.

He gives the following key to the families of the order
Thiobacteriales, which includes all the sulphur bacteria.

Family (i) Achromatiacece. — With sulphur, but no pur-
purin.
Unicellular : not motile, and not filamentous.
Genus i. Achromatiiim. — Cells ellipsoidal, containing

calcium oxalate, and perhaps sulphur.
Genus 2. Thiophysa. — Spherical cells, with sulphur

granules in a central vacuole.
Genus 3. Hillhoiisia. — Cells longer, and very large
(42 — 86/a), with peritrichous cilia.

Family (2) Beggiatoacece. — Like family (i), but filamentous.

Genus I. Thiothrix. — Non-motile filaments, thread un-
equally thick ; attached.

Genus 2. 5^^^w/oa.— Filaments, motile, not attached,
thread cylindrical ; filaments not in bundles, nor
surrounded by a gelatinous sheath.

Genus 3. Thioploca. — As Genus 2, but filaments in
bundles surrounded by a gelatinous sheath.

Family (3) RJwdobacteriacece. — Containing bacteriopur-
purin, and with or without sulphur granules. These he divides
into two sub-families : —

Chromatioidece. — Containing sulphur granules.
Rhodobacterioidece. — Without sulphur granules.

Chromatioidece. — Divided into five tribes as follows : —

ThiocapsecE. — Cells in families : division in three
directions of space.

86 SULPHUR BACTERIA

Lamprocystecc. — Cells in families : division first in three,

then in two directions of space.
ThiopediecE. — Cells in families : division in two planes,

forming plates of cells.
Amoehohacteriacecc. — Cells in families : division in one

plane.
ChromatiecE. — Cells free, capable of swarming.

(It is not necessary to consider the Rhodobaclerioidece in
this work, as they are not sulphur bacteria.)
Key to the Thiocapsecc :

Genus i. Thiocysiis. — -Motile : families small, compact,
. enclosed singly or several, in a cyst.
Genus 2. Thiosphcera. — Motile : cells large (7 — 8/x),

loosely bound by gelatine into loose families.
Genus 3. Thiosphcerion. — Motile : cells small, united

to form solid, spherical families.
Genus 4. Thiocapsa. — Non-motile : cells spread out in
flat families, loosely enveloped in a common gelatine.
Genus 5. Thiosarcina. — Non-motile : cells in regular
packets.
Key to the Lamprocystecc :

One genus Lamprocyslis. As for family.
Key to the ThiopediecB :

Genus i. Lampropedia. — Cells arranged regularly in

fours.
Genus 2. Thioderma. — Cells in a film, or membrane, not
regularly disposed in tetrads.

Key to the Amoehobacteriece :

Genus i. Aniabohacter. — Cells connected by plasma

threads : families amoeboid, and motile.
Genus 2. Thiodictyon. — Cells arranged in a net by their

ends.
Genus 3. Thiothece. — Cells not arranged in a net, and

loosely aggregated in gelatine : motile.
Genus 4. Thiopolycoccus. — Same as the preceding,

only cells are not motile, and closely appressed into

a colony.

THE PRINCIPLES OF CLASSIFICATION 87

Key to the Chroniatiecs :

Genus i. Chroniatium. — Cells motile, with polar cilia

and cylindrical.
Genus 2. Rhabdomonas. — Same as preceding, only cells

have a tendency to a spindle shape.
Genus 3. Thiospirillum. — Same as above, only cells are

spiral.
Genus 4. Rhodocapsa. — Cells spherical, or nearly so,

and non-motile, not capsulatcd.
Genus 5. Rhodothece. — Same as preceding, only cells

capsulated, and in pairs.

Remarks on Buchanan's Scheme of Classification.

In the above scheme only that part which deals with the
sulphur bacteria is here considered. All the niemxbers of this
group are included in the order Thiohacteriales, which is one
of the six into which the Schizomycetes are divided. There are
twenty-two genera in this order, but no new species of sulphur
bacteria.

The different genera of the sulphur bacteria arc all included
in the one order. It is evident that the classification of the
sulphur bacteria has been drawn up without specific investiga-
tion of these organisms, and without discrimination between
the values of various attributes. The characters chosen for
effecting the grouping are merely mixtures of the characters
used in previous classifications. The result is naturally the
perpetuation of all the errors that are found in the previous
schemes. Whilst Buchanan's scheme appears to be more
complete than older classifications, it is nof, so far as the
sulphur bacteria are concerned, and in essentials is no advance
on Winogradsky's plan.

In the scheme drawn up under the aegis of the American
Society of Bacteriologists, the grouping of the sulphur bacteria
follows very closely the plan laid down by Buchanan. New
generic and specific names appear, but as these did not appear
as the result of independent investigations, the scheme is
cumbersome without compensating advantages.

88 SULPHUR BACTERIA

A committee of the Society of American Bacteriologists
(Bergey, Breed, Hammer, Harrison, and Hunton) prepared
a Manual of Determinative Bacteriology in 1923, and in the
classification of the sulphur bacteria they have adopted
Buchanan's scheme without change. The same applies to
Pribram's classification which appeared in 1929.

Ellis's Classification of the Sulphur Bacteria.

Buchanan's classification has the merit that it takes
cognizance of all the sulphur bacteria that have been dis-
covered in recent years. The present writer's scheme has
been drawn up after an inspection of the stability of the
attributes used in the classification of bacteria in general, and
of the sulphur bacteria in particular. Also the life-histories of
these bacteria have been studied, and observations have been
made on the majority of the organisms included in this group.
In this way the defects of the earlier systems have been made
manifest, and so far as has been possible, they have been avoided.
As a quarter of a century has elapsed since a scheme of classi-
fication of the sulphur bacteria has been drawn up after a
special investigation of the group, there is much to rectify.
The acceptance of pleomorphism is a fact of great importance
in framing a scheme of division. The instability of dimensional
division in space is also a fact that strikes at the roots of former
classifications, because of the im.portance attached to it. The
genus Lankesteron has been created because it was felt that
the organisms examined respectively by Lankester, Warming
and Zopf were different species of a highly pleomorphic genus
of the sulphur bacteria. Within the last twenty-five years,
since the last classification was made after a detailed study
of the group, several additions have been made to the group,
and the author has added still another genus, namely, Thio-
porphyra. In addition a slight change has been made for the
ake of preserving uniformity of nomenclature. The Spirilla
genera of the sulphur bacteria have been divided into two,
namely, Thiospirilliim (colourless), and Rhodo-thiospirillum
(coloured). The term Thio-pseudomonas was also introduced

THE PRINCIPLES OF CLASSIFICATION 89

for purposes of uniformity. It has been found necessary to
discard some hitherto famihar names. The best known of
these is Lamprocystis. It has been shown already (p. 18)
that the organisms known by this name are forms of growth
of one or other of several organisms, and not independent
species. It is considered that the attributes by which this
genus is known, namely, that division is at first in " three
directions of space " and subsequently in " two directions of
space," is a fugitive character, and due in all probability to
some change in the constitution of the sHme which envelops
the cells. The validity of this alleged distinctive feature of
Lamprocystis has never been confirmed by any subsequent
writer.

The following genera are discarded for reasons given be-
low : — Thiosphcera, Thiosphcerion, Thiothece, Thiopoly coccus,
and Hillhousia. Of these the first four are discarded because
it is considered that they represent merely phases in the life-
histories of various pleomorphic bacteria. Zoogloea struc-
tures, when they occur in organisms other than the sulphur
bacteria, have not been regarded as other than forms of growth,
not as separate organisms. There is still some doubt whether
the genus Hillhousia is specifically distinct from Achromatium.
The balance of evidence supports the view that they are
identical.

Ellis's Classification.
The sulphur bacteria arc divided into two groups : —

A. Leuco-Thiobacteria. — Colourless sulphur bacteria.

B. Rhodo-Thiohacteria. — Coloured sulphur bacteria.
A. Leuco-Thiobacteria. — Four families.

Family i. Beggiatoacece. — Normally straight filaments, but
may show spiral or globular forms ; normally motile ;
occasionally enters zoogloea condition.

Genus i. Beggiatoa. — Normally straight, motile,

filaments.
Genus 2. Thiothrix. — Filaments attached, and often

enclosed in sheath of hardened slime.
Genus 3. Thioploca. — Aggregates of threads in har-
dened slime.

go SULPHUR BACTERIA

Family 2. AchromatiacecE. — Free, motile, cells normally
spherical or spheroidal in form : reproduction by
fission and by zoospores.

Genus i , Achromatium. — Cylindrical, ellipsoidal, cells ;
reproduction by fission and by zoospores ; move-
ment chiefly of translation and slow.

Genus 2. Thiophysa. Cells globular, or ovoid, uni-
and diplo-cocci common ; movements by trans-
lation and rotation ; reproduction by fission.

Genus 3. Thiosphccrella. — Ellipsoidal cells ; thick
membrane surrounded by slime ; movement slow ;
reproduction by fission.

Genus 4. Thioviduin. — Oblate spheroid or this figure
with one end slightly elongated ; reproduction
by fission and division at right angles to long axis.

Family 3. Thiospirillacece. — Free, spiral, cells ; polar cilia.

Genus i. Thiospirillum.—Free, motile, spiral, cells;
division by fission. .

Family 4. Thiohacillacecc. — Short rods ; division by fission.

Genus l. Thiobacillus. — Peritrich ciliation.
Genus 2. Thiopseudomonas. — Polar ciliation.

B. Rhodo-Thiobacteria. — Seven families.

Family i. LankesteracecB. — Normally filamentous ; highly
pleomorphic, with rod, spiral, and coccal, forms ;
occasionally forms zoogloea?.

Genus i. Laiikesieron. — As for family.

Family 2. Chromaiacece. — Spherical, or ellipsoidal, free cells ;
one polar (probably compound) cilium.

Genus I. Chromatimn. — Rose-pink colour; uni-cocci ;
reproduction by fission, and probably also by sexual
cells.
Genus 2. Thioporphyra. — Purple colour ; uni-, diplo-,
and tri-cocci ; reproduction by fission, by budding,
and probably by endospores.

THE PRINCIPLES OF CLASSIFICATION gi

Family 3. Rhodothiospirillacece. — Free, spiral, cells ; polar
ciliation.
Genus i. Rhodothiospirilhim, as for family.

Family 4. Rhodocapsacece. — Free cells each surrounded by
slime ; motile.

Genus l. Rhodocapsa. — Uni- or diplo-cocci, or short

chains.
Genus 2. Rhodothece. — Rod cells ; contain aerosomes.
Genus 3. Rhodosarcina. — Cocci arranged in packet form.

Family 5. Thiocapsacece. — Globular cells, forming clusters
enclosed in slime.

Genus I. Thiocapsa. — Cells non-motile on escape from

slime.
Genus 2. Thiocystis. — Cells motile on escape from slime.
Genus 3. Thiospharion. — Violet colour ; slime colonies
of globular form ; cells motile on escape from slime.

Family 6. Amahobacteriacece. — Rods or spherical cells en-
closed in slime ; cells connected by plasma threads.

Genus i. Amcehobacter. — Sphaeroidal cells in slime
which move in unison when they separate or close in.

Genus 2. Thiodictyon. — Rod cells united like a Hydro-
dictyon net.

Family 7. ThiopediacecE. — Globular cells in slime arranged
in regular formation.

Genus i. Thiopedia. — As for family.

CHAPTER VI.

LEUCO-THIOBACTERIA (COLOURLESS SULPHUR
BACTERIA).

Family i. Beggiatoacecs. Genus i. Beggiatoa ; Genus 2. Thiothrix ;
Genus 3. Thioploca.

The Leuco-Thiohacteria. — This group includes all the
colourless sulphur bacteria.

Family i. Beggiatoace^.

Straight filaments sometimes spiral or rounded. The fila-
ments are unicellular, and either homogeneous or divided into
sections by the formation of thin transverse bands of hyaline
slime at more or less regular intervals. Slime is formed outside
the membrane. In Beggiatoa slime formation is scanty ; in
Thiothrix the slime hardens and forms a cylindrical sheath
with transverse bands ; in Thioploca numerous filaments are
enclosed in a well-developed mass of slime.

The filaments are motile, the degree of motility being
determined chiefly by the amount of slime formation.

Reproduction is by fission, and in one genus (Beggiatoa)
also probably by asexual spores.

Genera —

1. Beggiatoa.

2. Thiothrix.

3. Thioploca.

Genus I. Beggiatoa (Trevisan), 1842.

Colourless threads, non-septate, and typically motile.
One species {B. alba) shows high degree of pleomorphism.
The varieties of form include filaments, large and small cocci,
short rods, and ovoid structures. These may be motile or

92

THE LEUCO-THIOBACTERIA 93

non-motile, and may be either free or enclosed in slime
(zoogloea condition).

The developmental life-history and methods of reproduction
are fully known only in B. alba. In this species reproduction
is by fission, and probably also by formation of endospores.
Raw cultures in nature form a white slimy or felted cover on
the surface of various objects undergoing decomposition ; or
if the organism is growing in sewage-laden water, on the bed
of the stream.

Beggiatoa alba (Voucher), Trevisan (i), 1842.
Synonyms. — Oscillaria alba (Vaucher) ; Hygrocrocis Van-
dellii (Meneghini) ; Beggiatoa punctata (Trevisan)
Beggiatoa media, and Beggiatoa minima (Winogradsky)
Literature. — Vaucher, 1803; Meneghini (1833-36); Tre
visan (1842) ; Cohn (10), 1875 ; Warming, 1875
Zopf (i), 1882; Winogradsky (2), 1888; Butschli (i)
1890; Fischer (i), 1891 ; (3), 1897; Mitrophanow
1893; Corsini, 1905; Keil, 1912 ; Ellis (1924, 1927)
Bergey, 1924 ; Bavendamm, 1924.

Description. — In mass cultures Beggiatoa alba typically
forms a dark grey, loosely textured, felted shroud, covering
the surface of its host : or if the water is sewage-contaminated
a covering on the bed of the stagnant or slow-moving water.
Beggiatoa is typically made up of long threads of cylindrical
form with rounded ends, and unless very long these are of uni-
form thickness, 2 — 6/x. The movement is normally slow and
without undulation. Lengths of even a centimetre and more
have been observed.* Long threads do not move forward but
exhibit a slow swinging jerky movement at the ends, resembling
that of Oscillatoria. The average length is 50 — lOOju.. The
cells contain, under favourable conditions, an abundance of
sulphur globules.

Motile, colourless threads, with oil-like contents, in a water
containing organic matter in sohitioii, may with certainty be
referred to Beggiatoa alba.

* A filament of this length is on the same scale as an inch-thick rope
about 140 yards long ; a remarkable instance of cohesion in a substance of
such soft matter.

94 SULPHUR BACTERIA

Sheath Formation,

The existence of a sheath-covering in Beggiaioa was re-
corded as early as 1884 by Winter, in Rabenhorst's Krypto-
gamen Flora; and in more recent work it is mentioned by
Selk, 1907, and by Keil, 191 2. Further, Koppe, 1923, has
recently shown that Beggiatoa arachnoidea occasionally be-
comes attached to objects in the water by the development
of cushions of slime at one end of the thread. The occurrence
of slime formation, however, as a normal feature in the life-
history of Beggiatoa alba has not hitherto been recognized.

Sheath formation is characteristic of all conditions of
the Beggiatoa cell, but in motile threads its amount is too
slight to be perceptible without special treatment. A similar
formation is found in the iron bacteria, e.g. Crenothrix poly-
spora, but whereas in these the sheath formation is a normal
occurrence of healthy cells, in Beggiatoa alba, as in yeast,
active slime formation is abnormal. The autolysis of the cells
is always accompanied by excessive slime formation (see below).
The thread appears to be divided transversely by walls such as
are normally formed in the division of cells of the higher plants.
In this case, however, they are merely transverse bands of
hardened slime. Their mode of formation is described below.

Growth.

Under favourable conditions a thread grows to double its
length in twenty-four hours, and during this time undergoes
one division. According to Winogradsky, this rate of growth
continues as long as the thread receives an adequate supply
of sulphuretted hydrogen. The amount of this substance
daily absorbed by a normal thread is substantially greater
than the amount of protoplast in that thread. Growth is
intercalary.

Autolysis.*

Mass cultures of Beggiatoa alba in nature usually disappear
completely with the advent of unfavourable circumstances.

* Autolysis refers to a process initiated inside the cell which results in
the disappearance of the organism by self-liqueiaction.

THE LEUCO-THIOBACTERIA 95

Tlie filaments frequently suffer dissolution with dramatic
suddenness, for the whole procedure which is detailed below
may occupy not more than a few minutes. The procedure
in autolysis follows one of two courses.

First Method. — After coming to rest the filament breaks into
fragments, as shown in Fig. 6c. The sheath at this stage can
be made visible by treatment with iodine. Between the short
lengths into which the thread has divided (Fig. 6d) transverse
sheaths of slime are formed. Then, beginning with one of the
cells at or near the middle, each swells in turn (Fig. 6e). The
order of swelling seems to be strictly maintained. By the
time the fourth or fifth has begun to swell, the starting cell
has completely disappeared. The process continues until the
last cell has dissolved.

Second Method. — When autolysis takes place in a filament
in which a well-developed and hardened sheath has already
been formed, a change in some of the details of the process is
observed. As before, the thread breaks up into a number of
short lengths, but the fragmentation is accomplished inside
the hardened sheath (Fig. 6h) ; and it is within the sheath that
the dissolution of the cells is effected. In Fig. 6h is shown an
example in which all except three cells have disappeared.
In this figure a septum of slime is also shown.

Cause of Autolysis. — Winogradsky (2) states that autolysis
takes place as a result of the depri\-ation of sulphuretted hydro-
gen, but he did not advance any proof in support of this state-
ment. Actually, it takes place when inimical conditions of
growth reach a certain stage of intensity, and this may result
not only from the dearth of sulphuretted hydrogen but also
from an insufficiency or an excess of oxygen, or from other
causes. When the conditions of growth are not satisfied the
filaments disappear, so that in most cases not one but several
factors are concerned. It has been suggested that the process
is furthered by the secretion of a specific ferment, but no proof
of the existence of such a ferment has been advanced. It
is very unlikely that a process which is accompanied by a
series of remarkable morphological changes originates or is
furthered by a ferment. It must be borne in mind that the

96

SULPHUR BACTERIA

Fig. 6.

THE LEUCO-THIOBACTERIA 97

protoplast segments into a number of equal or approximately
equal lengths, and that the morphological changes outlined
above take place in a regular sequence. Autolysis must be
regarded as a vital phenomenon, as much so as the capacity
of a plant to respond to such outside influences as light,
gravity, etc. In the course of this response ferments may or

Fig. 6. — Beggiatoa alba (Vaucher). Trevisan (i), 1842.
a. — Photomicrograph (shghtly touched up) of a short filament containing

a few granules of sulphur. x 1200.
b.- — Fragment of thread undergomg autolysis. Transverse walls are clearly
visible. At one point the protoplast has receded from both sides of the
transverse band of sheath (at b) leaving a clear space. This manner of
separation is characteristic of the process. X 1500.
c. — Shows the first stage in autolysis. The thread has broken up into

fragments of approximately equal lengths. x 1500.
d. — Second stage in autolysis. Transverse bands of hardened slime are

seen between the segments. x 2000.
e. — Third and final stage in autolysis. Nine segments are shown, of which
three are in process of swelling, preparatory to dissolution, x 1500.
(For further explanation see text.)
/. — Short filament with four endospores. x 1500.
e — Endospore.

s — Sulphur globule, somewhat contracted, lying in its vacuole.
g. — Semi-diagrammatic representation of a portion of thread to show
alveolar arrangement of the plasma. x 4500.
V — Vacuole,
s — Sulphur globules.
g — Minute granules in plasma.
h. — Filament in last stage of autolysis. Whilst the sheath has remained
comparatively unchanged, its contents have almost disappeared.
X 1500. (For further information see text.)
c — Rerhains of the disappearing segments.
sh — Sheath.

b — Transverse band of sheath-material.

d — Debris left after disappearance of another portion of the
filament.

may not be developed, but if formed they are to be regarded
as concurrent, not as causative agents in the production of the
phenomenon, and their presence as an incidental, not as a
necessary feature.

The mechanism of the process is a physical one. All
living cells contain osmotically active substances, and normally,
equilibrium is maintained because the outward pressure is

7

98 SULPHUR BACTERIA

balanced by the resistance of the somewhat hardened and
chemically changed outermost layers of the protoplast. The
balance is evidently a very delicate one, and one which is
easily destroyed by a further change in the constitution of
the resisting outer layers when these are transformed into
slime. This causes the outwardly directed pressure to over-
come the resistance of the outer layers, with the results described
above. It is as though the leather casing of an inflated foot-
ball were gradually changed into a softer material. Expansion
would occur and ultim.ately there would be a complete dis-
appearance of the ball. A hypothetical ferment may be pre-
dicated to bring about the transformation of the outermost
layers, but there is no necessity to assume its existence to
explain what in the last resource is a vital phenomenon, which
does not necessarily demand the existence of a ferment for
its manifestation.

Methods of Reproduction.

(A) Fission.~T\\e normal method of reproduction is by
fission. At first the two daughter cells are attached by a
band of slime continuous with the surface slime. This is made
clearer on staining the slime with iodine or carbol-fuchsin.
Later the daughter cells gradually draw apart, but the contin-
uity of the slime is maintained until the cells are about their
own length apart.

Zopf (2) states that the separation is facilitated in station-
ary threads by the swinging movements of the free end, and
Winogradsky (2) affirms that a break usually occurs at a place
on the thread where a large vacuole is found.

(B) Endospores. — Structures similar to endospores were
observed on one occasion inside the threads of a Beggiatoa
growing in a pool on the rocks near the Millport Marine Bio-
logical Station, Scotland.

The following reasons suggest that these structures were
spores : —

[a) Constancy of size and shape (4/Lt long and 3-5/x broad).
[h) Difiiculty of staining.

THE LEUCO-THIOBACTERIA 99

(c) The approximation of the size to that of the normal
bacterial endospore in the genus bacillus.

Their germination has not been observed.
Sexual reproduction apparently does not occur.

Species of the Genus Beggiatoa.

When many individuals of Beggiatoa are observed in the
same microscope-field, threads varying in thickness from i^
and less to approxim.ately 3/x are found, and a similar variation
is to be found in the lengths of the cells in the threads.

Winogradsky (2) has arbitrarily fixed on certain ranges of
size in such a mixture and given a specific name to each selected
range. He distinguishes different species in accordance with
the dimensions griven in the following: table :• — -

Thickness in /u..

Length of Cells in /ix.

Beggiatoa alba
media
„ minima*

4—5-5
i-o — 2-5
Up to i/x

2-9— 5-8
4-0-S-5

A criticism of this procedure is given on page 72.

Pleomorphism of Beggiatoa alba.

Zopf (l) has found this organism to be highly pleomorphic:
it can exist in a motile free state, or in a non-motile attached
form. The long threads show a differentiation of base and
apex.

The filament breaks up under certain conditions and forms
a mass of cocci. f In Fig. ja is shown a filament which has

* The name Beggiatoa minima has been apphed to two different organisms,
this name being given by Winogradsky to one organism, and hy Warming
to another. That described by Warming has since been taken away from
the Bacteria by Lauterborn, and placed in another group under the name
Spirophis minima.

t Zopf uses the term coccus, not to designate an individual belonging
to the group CoccacecB, but to indicate a small rounded mass of plasma.
In the accepted use of this term nowadays, a coccus is a spherical mass of
plasma surrounded by a definite cell membrane.

7*

TOO SULPHUR BACTERIA

already assumed an irregular form preparatory to breaking
up, and which is already showing at its basal end small
independent masses of plasma, each containing a few grains

/

Fig. 7. — Pleomorphism of Beggialoa alba.
At a is shown a filament breaking up into separate clumps of plasma, each

including a number of sulphur granules. In the lower part of the

figure, four of these clumps are shown.
In b the formation of these clumps is shown completed, although the

alignment of the filament is still maintained.
In c, d, e are shown the different forms into which the members of the

clumps develop, namely, free cocci, short rods, or zoogloea masses.

Either cocci or short rods may be found in the zoogloea masses.
At / is shown a rigid spiral cell formed by separation from the filament.

of sulphur. In the next state (Fig. yb) the filament has given
place to a number of separate clumps of plasma. Next, the
clumps break up into micrococci which may become motile in.

THE LEUCO-THIOBACTERIA loi

that form, or after they have developed further to the rod form
(Fig. Jc). Or the cocci may multiply and enter into the zoo-
gloea condition (Fig. Je). The last-named condition may be
brought about by inoculating material rich in Beggiatoa into
a fluid made up of a mass of Spirogyra and Cladophora in a
partial state of putrefaction in water. Also Zopf observed a
fragment of a filament being cut off from the end, when it
assumed a twisted form and swam away (Fig. 7/). He
states that he made a direct observation of this occurrence :
" Ich beobachtete diesen Faden zufallig einige Zeit, und sah
nun wie das spiralige Stiick in ein Hin- und Her-schwanken-
gerieth um schliesslich abzubrechen und als starre Spiralige
hinweg zu schwimmen." This fact has been doubted, but
the author has observed (Ellis (5)) an intercalary cell being
detached from a thread of Cladothrix dichotoma, when it
swam away in precisely the same manner as the detached
fragment of Beggiatoa^ alba observed by Zopf. Finally, Zopf
relates that the elongation of cocci and short rods into short
filaments is sometimes followed by the assumption of the
spiral shape.

Beggiatoa alba may therefore exist in the coccus, the bacillus,
and the spirillum shapes ; and each one of these may enter the
zoogloea condition. Further, each kind may be motile or
non-motile. The later development of these different forms
is unknown, but there is no reason to doubt that, given the
proper conditions, they develop once more into the filamentous
condition.

Other Species of Beggiatoa.

Our knowledge of other species of this genus is somewhat
scanty, and even their title to be included in the genus is
doubtful.

Beggiatoa mirabilis (Cohn), 1865.

Literature. — Cohn (5), 1865 ; Warming, 1875 ; Engler,
1883; Butschli (i), 1890 ; Kolkwitz (i), 1897; Flinze
(i), 1901, and (2), 1902 ; Kolkwitz (10) 1918 ; Zuelzer,
1924 ; Bavendamm, 1924.

SULPHUR BACTERIA

5Jr-> . '^ .• .- • ■ '; I v"

-■n'» - ■}'gTO"'"»''...'T.i:?3grr>

Fig. 8.

THE LEUCO-THIOBACTERIA 103

Description. — This remarkable organism is composed of
threads varying in thickness from 15 to 45/.t. In spite of its
large size it has the same simple structure of cell as Beggiatoa
alba. It differs, however, from that species in possessing a
sharplv defined external membrane and transverse walls similar
to those of the genus Bacillus. Cohn depicts transverse walls
and their existence has been confirmed by Hinze, who has de-
scribed their structure in detail. Warming, however, depicts a

Fig. S. — Beggiatoa mirahilis (Cohn), 1865, and Beggiatoa mirabilis (Warming).

a and b show parts of filaments of the organism which Warming described
under this name. Transverse walls are not formed, x 500.

c-f show parts of filaments of the Beggiatoa mirahilis as figured by Cohn
and Hinze.

c. — Shows the outer walls, the transverse membranes, the vacuoles and the
sulphur globules, x 1200.

d (after Hinze) . — Two cells separated by a delicate transverse wall : sulphur
globules and bodies alleged by Hinze to be chromatin (see p. 182^.
X 1200.

e (after Hinze) . — The first phase in the development of the transverse wall :
formation of an annular ring. Seen in section at a^. X 1200.

/. — Diagrammatic representation of the annular ring, shown in section in
the preceding diagram.

g.- — Photomicrograph of Beggiatoa mirahilis as described by Warming. From
material from the South of England. X 500.

h. — Beggiatoa mirahilis of Warming.— K diagrammatic representation of
the internal structure of a thread preserved in formalin. The plasma
appears as an irregularly reticulate knotted mass. The nucleus is
absent, and there are no transverse walls. At the time of writing
living threads have not been obtainable, but there seems little doubt
that in the living condition the structure of the plasma conforms to
that shown in Fig. 6b. x 1200.

species by the name of Beggiatoa mirahilis (Fig. 8 a and h) which
is essentially different from the organism figured by Cohn, in
the complete absence of transverse w'alls. The thread appears
to be roughly segmented, but this is not really the case, the
fictitious appearance being caused by the occurrence of trans-
verse bands of plasma at more or less regular intervals. In
Fig. 8g is shown a photomicrograph of an organism found in
the South of England (in material kindly sent to the author
by Miss Nellie Carter) which corresponds in all essentials to
the organism described by Warming. Viewed with a low

I04 SULPHUR BACTERIA

magnification the thread appears to be segmented, but with
a higher magnification no signs of segmentation are seen.
The disposition of the plasma is seen in Fig. 8A. Small pro-
jections of plasmatic material appear to jut out from the sides.
The projections take the form of very irregular-shaped rods
and point in all directions. There thus appears to be a single
continuous vacuole along the whole length of the thread,
occupying the space not filled up with the plasma. It is
possible that transverse walls may be a feature in the further
development of the threads, but such w^ere not observed.
Opportunities for studying the life-history of this organism
have not yet occurred, but it is unlikely that it is identical
with the Beggiatoa mirabilis of Cohn and of Hinze.

In Hinze's account of Beggiatoa mirabilis the appearance
and development of the transverse walls are given. Its
development begins with the formation of a ring-shaped
flat projection round the inside of the cell (Fig. 8^). This is
shown diagrammatically in Fig. 8/. This annular plate is
pushed during development farther and farther inwards until
the growing wall stretches across the whole cell. By inter-
calary growth the dividing cells attain the size of the mature
cells. According to Hinze some of the cells die oft and sepa-
ration takes place at these points. Each of the separated
threads then elongates afresh. They are naturally straight
and of a somewhat stift' consistency, but become somewhat
wavy from the unequal distribution of the numerous particles
of dirt and other debris w^hich settle on their surface.

Beggiatoa arachnoidea (Agardh), Rabenhorst (2), 1865.

Literature. — Agardh (2), 1827; Corda (l), 1835, (2), 1836;
Kutzing (2), 1843 ; Cohn (5), 1865 ; Warming,
1875 and 1876; Engler, 1883; Koppe, 1923;
Bavendamm, 1924.

Description. — This organism was first mentioned by Agardh
under the name Oscillatoria arachnoidea. Since that time
several changes in nomenclature have occurred, and in 1865
Cohn transferred it to the genus in which it is now placed.

THE LEUCO-THIOBACTERIA 105

The threads are 5/x — 6/x thick, and are divided into cellular
segments by transverse bands of clear protoplasm placed at
regular intervals. They form in the mass, thin, chalky-white
slimy layers, of the texture of a spider's web. The segments
are either as broad as they are long, or half as long as they
are broad.

Habitat. — Found originally in the sulphur springs at
Karlsbad. It occurs in waters, both fresh and salt, containing
mud rich in HgS.

Beggiatoa leptomitiformis (Meneghini), Trevisan (i),
1842.

Literature. — Meneghini (2), 1844 ; Massart, 1902.

Description. — This species is found in great numbers in
various sulphur springs. The threads are not segmented.

They measure i-8 — 2-5/n in thickness and form a chalky-
white covering to the objects on which they settle.

Habitat. — Sulphur springs.

Genus 2. — Thiothrix (Winogradsky), 188S.
Founded by Winogradsky to designate a group of organisms
very similar to, and frequently in association with, Beggiatoa.
The genus differs from Beggiatoa in the following respects : —

1. Absence of free miovement.

2. Presence of an anchoring slimy organ of attachment.

3. Development of a slime sheath.

4. Formation of " conidia " at the free end of the threads.

These are thrust out of the sheath, and after a slow,
creeping, independent movement, attach themselves
to objects in the water, w^here they form new threads.

It is not always possible to distinguish these two general
for Beggiatoa alba sometimes develops excessive slime, when it
becomes stationary, and resembles Thiothrix. Again, another
species, Beggiatoa arachnoidea, possesses an anchoring organ
of attachment, and thus also resembles Thiothrix. But slime
development resulting in an enveloping hollow sheath is a
normal feature in the life-history of Thiothrix. This sheath

io6

SULPHUR BACTERIA

hardens and becomes detached from the remainder of the
organism, forming a covering which encloses the Hving part of
the cell. Finally, although short lengths of Beggiatoa do not
show any diflerence in thickness at different parts of the thread,
longer filaments show differences which may be pronounced.
In Thiothrix the presence of a fixed hardened slime sheath
has some influence in determining the thickness, since normally
the apex, where the slime is softer, is thicker than the base,
where the slime is firmer.

'X

M

H

a t> c

Fig. g. — Thiothvix Rod-Gonidia X looo.

a. — Young thread before active slime formation.

b. — Slightly older thread, in which slime formation has commenced. Slime

shown at x.
c. — Later stage. Slime has hardened into a sheath.
Rod-Gonidia (d) are in process of expiilsion from open apex of sheath.

Description. — Colourless sulphur-containing threads, non-
motile in mature form, and surrounded by an enveloping
sheath of hardened slime. Usually attached by a slimy
anchoring organ to various objects in the water. Distinction
between base and apex much more marked than in Beggiatoa,
and the middle portion may occasionally be thicker than

THE LEUCO-THIOBACTERIA 107

either the base or the apex. The thread readily breaks up
into more or less equal segments which for a time are held
together by connecting bands of slime. The whole surface
of the cells is covered by a layer of slime which subsequently
hardens, and forms round the cells an enveloping hollow
tubular sheath. With further growth the apex of the line of
cells inside the tubular sheath is pushed out. Then each cell
as it wins clear of the top of the sheath becomes separated and
moves away. There is thus a steady emergence of cells from
the sheath, pushed from behind by the pressure of growth.
In some cases the liberated cells develop motility and swim
slowly away. The name rod-gonidlwn is usually given to a
cell of this kind. After swimming for a short time it comes
to rest on some object and forms a new thread (Fig. 9).

Note on the Terms "Conidium" and " Rod-Gonidium."
The use of these terms is incorrect in the sense in which they are
employed above, as it implies that they are special organs of asexual re-
production. The method of reproduction in Thiothvix is one of simple
fission, but it is complicated by the circumstance of the fission taking
place inside a sheath of slime. In essentials it consists merely in fragments
of a thread being successively detached from the apex.

Thiothrix nivea (Rabenhorst), Winogradsky, 1888.

Literature. — Pollini, 1817; Oerstedt (2), 1844; Kiitzing (2),
1843; Rabenhorst (2), 1865; Corsini, 1905; Swellen-
grebel, 1909; Keil, 1912; Bavendamm, 1924.

Synonyms. — Conferva alba (Pollini) ; Leptomitus (x^Vgardh) ;
Leucothrix Mucor (Oerstedt) ; Hygrocrocis nivea
(Kiitzing) ; Beggiatoa nivea (Rabenhorst) ; Thiothrix
tenuis and Thiothrix tenuissima (Winogradsky,
Migula, Buchanan, and Bavendamm).

Description. — Bavendamm suggests that this species was
described as far back as 1817 by Pollini under the name Con-
ferva alba, and at various times, as is shown by the literature
quoted, attention has been called to it by various investigators.
Rabenhorst in 1865 gave to it the name Beggiatoa nivea, which
w^as subsequently (1S89) changed by Winogradsky to Thiothrix

io8

SULPHUR BACTERIA

nivea. The last-named investigator divided one species into
three on the sole basis of differences in the thickness of the
threads. As all these different forms were found in the same
medium, and as numerous intermediates were also present,
the subdivision of the species is not warranted.

Fig. io. — Thiothrix nivea. X looo.
a, b, c- — Thiothrix nivea. Shows young filaments with slime cushions at

the base of each.
d. — Thiothrix tenuis. Filament from the free end of which " conidia" are

in process of liberation. Four of these are shown at point of separation

from the parent filament.

Note on the Variant forms of Thiothrix nivea.

The following figures are supplied by Winogradsky as
the distinguishing marks of the three " species " mentioned
by him : —

THE LEUCO-THIOBACTERIA

109

Thiothrix nivea
„ tenuis

tenuissima

Apex.

2— 2-5/x I-7M 1-4— I-5M

All threads of uniform thickness and — i — i-i/x
All threads of uniform thickness and = 0-3 — 0-5^

As all these forms may be found in the same microscope-
field, the inference that may be drawn is that threads of Thio-
thrix are of uniform thickness
when very young, and that they
show inequality of thickness
when they further develop. It
appears as though Winogradsky
had assigned different specific
names to different stages in the
development of one organism.

Habitat of Thiothrix nivea. —
Found in sulphur springs and in
waters, both fresh and salt, which
contain sulphuretted hydrogen
in solution.

Thiothrix ANNULATA (Molisch).

Literature. — (Molisch (5).)

Description. — The threads are
attached by a well-developed
slime anchor (about 3*5/x in
thickness). The young colonies
are composed of short, sulphur-
filled threads, all in attachment
to the same slime anchor (Fig. \ia). After further growth
a long thread is formed which is at first unicellular, but
later is segmented by transverse septa. In Fig. 11& is shown
a still older thread in which the upper part is septate, but free
from sulphur. Very old threads may attain a thickness of
7/A, and exhibit knotty thickenings separated by ring-like,
constricted, sulphur-free areas (Fig. lie). Fragments break
off at the constricted parts and a portion of such a fragment

Fig. II. — Thiothrix marina
(Molisch, 1907).

no SULPHUR BACTERIA

is shown in Fig. lid. The threads, except in very young stages,
are not uniformly thick. The following measurements were
obtained from a typical example : base 2/x, middle 3 — 4/x, and
apex i-8/i. The septa were I/a apart.

Habitat.— Found by Molisch in sea-water from Trieste
containing decomposing Algse ; and by Bavendamm in similar
material from the neighbourhood of Berlin, and from the
briny wells of Sperenberg (Brandenburg, Prussia).

Thiothrix marina (Molisch), 1907.

Literature. — Molisch (5), 1907 ; Bavendamm, 1924.

Description .^This species forms a white pellicle on the same
material as that on which 77?. aniiuJata thrives. Bavendamm
considers it to be identical with Th. tenuis^ that is, Th. nivea.

The threads are relatively long and thin, being 0-8 — i'3ju,
thick and 130 — 300/x, sometimes up to 500/i., long.

Thiothrix violacea (Ellis).

In all systems of classification of the sulphur bacteria, the
first division is based on the presence or absence of colour.
This procedure is satisfactory in the majority of cases, as the
distinction is felt to be a natural one, and its utility is obvious.
The author has, however, found in Fossil Marsh, near Glasgow,
a coloured Thiothrix. A difficulty in the allocation of this
species thus arises. In its structure and life-history it is a
typical Thiothrix, and yet being coloured, it should find a
place in the Rhodo-thiohacteria. It is proposed to place this
organism provisionally in the genus Thiothrix, on account of
its obvious relationship.

Description. — ^Violet coloured, uniformly thick filaments,
showing sheath-formation. Reproduction by fission, and
possibly by cndospores. The threads are approximately 3^
thick and are found attached basally to various objects in the
bed of a stream containing domestic sewage. There is no
special anchoring cushion of slime. Transverse walls (of
slime) are not visible in young threads, but they appear
in old threads, and also in threads that have lost their

THE LEUCO-THIOBACTERIA iii

sulphur contents (Fig. I2a). In older threads a sheath of
slime is formed from the outermost layers of the cells, which

n\

9^

o

abed

Fig. 12. — Thiothrix violacea (Ellis). X 1200.
a. — Fragment of old thread made up of the hardened slime sheath, the

transverse bands of slime, and unidentified particles.
b. — Fragment of old thread containing seven round bodies, possibly endo-

spores. Previously formed transverse bands of slime have been swept

away by growth in the cells within the hardened sheath.
c. — Fragment of a thread with an open end and transverse bands of slime.
d. — Thread showing so-called " conidia " formation. The " conidium "

is attached to the parent thread by a band of slime, which can be seen

by treatment with carbol-fuchsin.

subsequently hardens and forms a hollow tube containing
the cells in single row formation. In Fig. 12 h and c, are shown

112 SULPHUR BACTERIA

two old threads, of which & is a hollow sheath of hardened
slime and containing seven of the objects which may be
endospores ; whilst c is a similar thread, but in this the trans-
verse bands of hardened slime are also present. It also con-
tains one of the objects that may be endospores.

The plasma is homogeneous, with the sulphur globules
and pigment uniformly distributed. In older threads from
which the sulphur globules have disappeared a large number of
sm^all granules are seen. The nature of these has not been
determined.

Methods of Reproduction.

(A) Reproduction by fission is the common method. It
may take place terminally, or it may be intercalary. Inter-
calary grov/th is found only in young threads. In older
threads only the tip goes on dividing, and short lengths of the
filament arc successively broken off. These are often named
" conidia " or " rod-gonidia," which are misnomers, for the
difference between such reproduction and fission is one of
degree not of kind (see also note on p. 107). In Fig. I2d
is shown a young thread in process of liberating a short length
from its tip. Connection with the parent thread is still
maintained by a band of slime. The same process is in force
even when the filament is enveloped by a sheath, for the top
of the sheath is open, and the cells are pushed out of the
opening by pressure from behind.

(B) Endospores. — In older filaments a number of bodies
are occasionally seen which may be endospores (Fig. 12 h and c).
They are roughly cylindrical-elliptical, and approximately
of equal size (about 3/^). They were found in old threads
of which nothing remained except the sheath. For lack
of opportunity, the germination of these structures was not
followed so that their spore nature has still to be proved.

Habitat. — Found in a running stream containing domestic
sewage, near Glasgow.

Note. — The investigation of this organism was unfor-
tunately cut short by its disappearance following the intro-
duction of sanitary measures, which diverted the course of
the sewage.

THE LEUCO-THIOBACTERIA

113

Genus 3. — Thioploca (Lauterborn), 1907.

Literature. — Lauterborn (5), 1907; Wislouch (i), 1912;
Koppe, 1923 ; Bavendanim, 1924.

A well-defined genus living in the calcareous mud of
shallow marine waters.

a - KM- b

Fig. 13. — Thioploca Schmidlei (Lauterborn). x 5000.

Description of genus. — Colonial motile threads of the
Igiatoa type bound together in a com.mon envelope of
slimy matter. Each colony has the appearance of a rope of
slime, but the individual movements of the contained threads
do not result in their separation from the colony. The outer
surface of the slime rope is covered with particles of mud and
gritty matter, and is constricted at regular intervals (Fig. 13&),

8

114 SULPHUR BACTERIA

each constriction being ring-shaped and transverse. The
threads are septate, and their ends often pointed.

Wislouch states that the members of this genus are
shghtly blue in colour, due to the presence of phycocyan and
chlorophyll. It is probably physiologically intermediate
between the Cyanophycece and the Leuco-thiohacteria, but
it is included in this group for the sake of completeness.

Thioploca Schmidlei (Lauterborn), 1907.

Literature. — Lauterborn (5), 1907 ; Koppe, 1923.

Description. — Each "rope" may extend to 3 — 4 centi-
metres, and may itself be one of a number of strands forming
a thicker rope. The thickness of such a " rope " is 50 — 60)u-,
and within it the closely adpressed threads at its centre exhibit
gentle gliding movements passing over one another, but they do
not leave the " rope " in their movements. Also there appears
to be some directive force in the community as a whole, for
the movements of two neighbourhood threads are always in
opposite directions. Sculpturing effects are produced on the
surface of the " ropes " owing to the incrustation of mineral
matter from the surrounding water. At certain periods, or
under certain conditions, the slime opens out and from it
emerge threads, which swim away and doubtless form the
nucleus of new colonies, although their development has not
been investigated. Reproduction is by fission. Lauterborn
also observed the intertwining, after separation, of the two
sections of a dividing thread.

The threads are septate, the length of each segment being
i-| times its breadth. The threads in a young colony, accord-
ing to Lauterborn's figures, do not show septation. The
sulphur granules are so numerous and small that the threads
appear black under the microscope.

Habitat. — Found in calcareous mud below 15 — 20 metres
depth either in marine, or brackish, or fresh water. Recorded
from Ermatingen ; from the neighbourhood of Solingen, in
a bay of the Rhine ; from near Strassburg; from the Neva;
and from the neighbourhood of Konigsberg.

THE LEUCO-THIOBACTERIA

115

Thioploca ingrica (Wislouch), 1912.

Literature. — Wislouch (i), 191 2 ; Kolkwitz (7), 1912 ;
Koppe, 1923 ; Bavendanim, 1924.

Description. — The rope-Uke masses which constitute this
species are white in colour, are about i centimetre long, and
are composed of threads rich in sulphur. The threads creep
along both directions of the longitudinal axis. Each rope
contains from ten to twenty threads, some with blunt, others
with pointed ends.

Habitat. — Same as preceding species. Has been found in
Leningrad, in a harbour in the Gulf of Dantzig (Frisches Haff),
and in the Boden See (Switzerland).

The differences between the two species of this genus may
be tabulated as follows : —

Th. Schmidlei.

Th. ingrica.

Lengths of slimy masses
Thickness of sHme envelope
Thickness of thread
Length of cells

A few cms.

50— 60/X

5— 9M
5—14/^

Up to I cm.
Up to 80^
2—4-5/^
1-3— 8m

Recently two species of this genus have been described
by Koppe, and named respectively Thioploca minima and
Thioploca mixta. As the distinction between them and the
two preceding species is confined to a difference in dimensions
only, and as they are found mixed with Thioploca ingrica,
and as in Thioploca mixta different thicknesses of thread are
found in the same colonies, we are entitled to conclude that
these latest additions are merely variant forms of Thioploca
ingrica.

8*

CHAPTER VII.

LEUCO-THIOBACTERIA (COLOURLESS
SULPHUR BACTERL\).

Family 2. AchromatiacecB. Genus i. Achromatium ; Genus 2. Thiophysa :

Genus 3. ThiosphcBrella ; Genus 4. Thiovulum.
Family 3. Thiospirillacecs. Genus i. Thiospirillum.
Family 4. Thiobacillacecs. Genus i. Thiobacillus ; Genus 2. Thio-

pseudonionas.

Family 2. — Achromatiacem.

Free, motile, and usually spherical or globoid. In one
genus [Thiovuhmi) cilia have been demonstrated. Reproduc-
tion by fission ; and in one genus by zoospores.

Genus i. — Achromatium (Schewiakoff), 1893.

Literature. — Warming, 1876; Schewiakoff, 1893; Frenzel,
1897; Lauterborn (i), 1898, and (6), 1913; Massart,
1902 ; Zacharias (2), 1903 ; "West and Griffiths
(i), 1912, and (2), 1913 ; Kolkwitz (3), 1909 ; Virieux
(i), 1912, and (2), 1913.

Free, motile, and spherical, or spheroidal or cylindrical-
elliptical. Reproduction by fission and by zoospores.

Achromatium oxaliferum (Schewiakoff), 1893.
Literature. — See above.

Habitat. — Widely distributed in most European countries,
and probably outside Europe.

Description. — The cell is ellipsoidal and normally appears
full of dark spherical bodies, as shown in Fig. 14A. The
movement is very slow, and consists, usually, of a forward or

116

THE LEUCO^THIOBACTERIA 117

backward motion with an occasional rotation on its longi-
tudinal axis. No organs of movement have been found,
although Zacharias states that young individuals possess a
clearly visible cilium as long as the organism itself. This has
not been confirmed.

Although probably first mentioned by Warming in 1875,
the fi.rst descriptive account of the organism was given by
Schewiakoff in 1893. He described and named Achromatiiim
oxaliferum, which is considered to be identical with the
organism subsequently described by Frenzel (1897) under the
name of Modderula Hartwigi, and by West and Griffiths (1909)
under the name of Hillhoiisia mirahilis. In the works of the
earlier writers (Schewiakoff, Frenzel, Lauterborn) an attempt
is made to distinguish between the peripheral layer (Rinden-
schicht) and the central body (Centralkorper), obviously under
the influence of Biitschli's now discredited view of the structure
of the bacterial cell, and so the plasma is described as being com-
posed of two layers, a central with larger, and a peripheral with
smaller, meshes. This marked distinction between the size of
the meshes has not been observed by later writers as Virieux,
and West and Griffiths, who have examined the structure of
the cells w4th greater accuracy. Dr. B. M. Griffiths regards
Hillhousia as specifically distinct from Schewiakoff's Achroma-
tiiim oxaliferum and Frenzel's Modderula Hartwigi, because in
it there is no distinction between a peripheral and a central
plasma. This distinction, however, does not occur in any mem-
ber of the sulphur bacteria nor in any one of their immediate
allies. The distinction apparently did not exist in the organism
examined by Schewiakoff and by Frenzel. It is noteworthy
that, although the organism named Modderula was stated
to be quite common, no organism of the bacterial group
containing a differentiated plasma has since been found.
If, therefore, we take the view that Modderula (including
Achromatium) does not possess a differentiated plasma, the
sole distinction between Hillhousia and this organism vanishes,
anrl on the groimd of priority the name Achromatium oxa-
liferum must be held to cover all three generic namics (see
Fig. 51 a and b, and pp. 182-4).

ii8

SULPHUR BACTERIA

THE LEUCO^THIOBACTERIA 119

Marked differences in size are noted in this organism : —

Schewiakoff . . . I5/^ X 9 — •22/x.

Frenzel .... 9 — ■I5ju, X 6 — -33ju..

West and Griffiths . . 40 — 60ju X 20 — ^},yL..

Nadson .... Up to I02/Lt in length.

The smallest appears to have been that recorded by Baven-
damm, which was only 3/x in length.

Achronialiuni supplies another illustration of the fact
that all widely distributed bacteria exhibit a wide range of
dimensions, and that within the extremes of this ran^e are

Fig. 14. — HiUhousia. (West and Griffiths.)
A. — Normal aspect of living specimen of HiUhousia niirahilis. X 500.

The cell is filled with globules of calcium carbonate.
B. — H. mirabilis after removal of calcium carbonate by dilute acetic

acid. The conspicuous granules are grains of sulphur, x 850.
c. — Rhombic crystals of calcite, obtained by allowing the organism to dry,

and then irrigating with distilled water. X 850.
D. — After treatment for 15 minutes with 5 per cent, phenol. Shows lamel-

lose cell wall.
E. — Diagrammatic representation of two stages in the division of H.

mirabilis.
F. — Cell after removal of cell contents.
G. — Photomicrograph of H. mirabilis from material kindly sent to the

author by Dr. B. M. Griffiths. X 450.
a-f are taken from West and Griffiths' paper (see Bibliography).

a multitude of intermediate forms. Hence one specific name
should cover all that fall within the range. A new specific
name is called for on the score of differences in size only when
a distinct gap occurs in the grading. In plastic organisms like
the sulphur bacteria, a variation in the size of the different
individuals in a culture has no more genetic significance than
the variation of the number of sulphur globules in the cells.

Methods of Reproduction.

(A) Fission. — Two stages in division arc shown diagram-
matically in Fig. 14E. This process has not been followed
in detail.

I20 SULPHUR BACTERIA

Virieux claims that nuclear material is present as chromatin
granules. If so, their disposition during cell division should
be a matter of interest. The method of division is that which
is common to the great majority of the sulphur bacteria. The
plasma separates without the previous formation in it of a
definite cell wall.

(B) Zoospores. — The cell gradually expands, finally bursting
its membrane, and liberating a number of zoospores that,
during this period of expansion, have formed inside. Each
zoospore resembles the adult in structure, and differs only in

Fig. 15. — Achromatium oxalijermn. (After Virieux.)
a. — Zoospores. They show the diversity of form and characteristic

alveolar structure of this organism, x 1500.
h. — Liberation of zoospores from the nivother cell. X 1000. (For further

explanation see text.)

showing some diversity in shape (Fig. 15). It measures 4 — 6/x.
in length, although some reach 10 — liju. It exhibits the
alveolar structure characteristic of the adult, and in each
are 2 — 3 granules and a few corpuscles (see p. 184). Cilia
are not developed. The zoospores on liberation increase
without any further change to the size of the adult cell.

Habitat. — Present in water, both fresh and salt, in which
H2S is present as the result of vegetable decomposition, and
in which also calcium salts are abundant. The organism has
been found in various places in Germany (Lauterborn, Frenzel,
Bavendamm, etc.) ; in Belgium (Massart) ; in England (West

THE LEUCO-THIOBACTERIA 121

and Griffiths) ; Austria and Czecho-Slovakia (Molisch). It
has also been recorded from Ireland, Denmark, Russia, and
South Africa. Its presence has been frequently noted in lake
deposits of vegetable debris and diatomaceous muds ; and in
vegetable debris in marshes. Distribution is probably world-
wide.

AcHROMATiUM MOBILE (Lauterborn), 1915.

Syn. Microspira vacillans (Gicklhorn).

Literature. — Lauterborn (7), 1915 ; Gicklhorn (i), 1920 ;
Bersa (i), 1920 ; Utermohl, 1923 ; Koppe, 1923.

Description. — The general appearance of this organism is
Lauterborn placed it in the genus Achro-
later writers

shown in Fig. 16
matium, but
have consideredits differences
sufficiently marked to merit
the formation of a new genus,
Utermohl, and Koppc
gave it the generic name of
Macromonas, and what is
apparently the same organ-
ism is described by Gickl-
horn (i) under the name of
Microspira vacillans. Pend-
ing a more comprehensive

Fig. 16. — Microspira vacillans.
X 700.

investigation the organism is best retained in the genus in
which it was placed by Lauterborn.

Microspira vacillans {as described by Gicklhorn).*

The organism is a single ellipsoidal cell, som^ewhat bent in
the middle (Fig. 16), measuring 12 — 30^^ X 8 — 14/x. It con-
tains numerous globules of sulphur, and in addition two or
three large bodies showing a bluish tinge. These, according

* This writer's work was not available, and the statement about the
composition of these bodies is made on the strength of the title of Bersa's
work Uber das Vorkommen von kohlensaurem Kalk in einer Gnippe von
Schwefelbakterien.

122 SULPHUR BACTERIA

to Bersa, are composed of calcium carbonate. There is a
single large polar cilium, which is plainly visible without special
preparation, and which is as long as, or even longer, than
the cell. Cell-division takes place by simple fission following
upon a slight elongation of the cell, which during the process
does not come to rest. The stages in division are the same
as in Achromatium oxaliferum. After fission has taken place
the two daughter cells are connected for a period by a trans-
verse band of liyalinc material. The cells gradually draw
apart, and complete separation is efl'ected. The number of
sm.all globules of sulphur is increased when sulphuretted hydro-
gen is added to the medium.

Habitat. — Found in foul mud samples from the University
garden in Graz (Austria).

Genus 2. — Thiophysa (Hinze).

Literature. — Hinze (3), 1903 ; Nadson (5), 1914.

A genus of two species. The cells are globular or ovoid,
colourless, and motile. The movement, which is slow, is one
of translation and of rotation. Cilia have not been found.
The cells contain a peripherally placed plasma, and a large
central vacuole. Division takes place l)y fission, following
a slight preparatory elongation. Both uni- and diplo-cocci
are conmion.

Thiophysa volutans (Hinze).

Literature. — Hinze (3), 1903 ; Nadson (5), 1914 ; Kolkwitz
(10). 1918.

Description. — This species was found in a sulphur spring
in the Gulf of Naples, near Castellamare. The floor containing
the organism was made up of fine calcareous sand, and smelt
strongly of H2S. The calcium does not appear to penetrate
the cell membrane. Diameter of cells, 7 — i8/a; maximum =
28-9)U, long, and i7-9/i, broad (Fig. 17).

It has also been found in the brine ditches at Artern
(Saxony), and at the sea-bottom near Hapsalu in Esthonia.

THE LEUCO-THIOBACTERIA

123

Thiophysa macrophysa (Nadson).

Literature. — Nadson (5) 1914.

Description. — This species is distinguished from the
preceding by its larger average size. The diameter of cell is
21 — 40jU.. It is, therefore, a much larger organism than the
preceding, and, in addition, there is a physiological distinction,
for whilst the Th. voliitans is confined to marine waters, this
species flourishes only in a medium containing much less
sodium chloride, namely \ per cent.

b c

Fig. 17. — Thiophysa voliitans.

X 500.

Genus 3. — Thiosph^rella (Nadson).
Literature. — Nadson (5), 1914 ; Bavendamm, 1924.
One species, Thiospha;rella amylifera. The following de-
scription is translated from Bavendamm's work, as Nadson's
work was not available. Cells round, often ellipsoidal, 6^ long,
and 4-8)U, broad. The membrane is relatively thick, dense,
and highly refractive, and surrounding it is a colourless
slime layer. The protoplasm is normally not quite colour-
less, but has a steel-grey tint. In the plasma are sulphur
globules of various sizes, and, in addition, a substance of a
starchy nature, which colours a dark red or violet when treated
with iodine. The cells possess a slow movement, similar to
that of Achromatium. Reproduction is by fission (Fig. 17
a and b).

Genus 4. — Thiovulum (Hinze).
Literature. — Warming, 1876 ; Migula (3), 1900 ; Molisch
(5), 1912 ; Hinze (6), Lautcrborn (7), 191 5 ; Baven-
damm, 1924.

124 SULPHUR BACTERIA

Description. — The cells are ellipsoidal, sometimes pointed,
or considerably flattened. The membrane is sharply defined,
and the plasma is made up of delicate strings which traverse
the whole cell. In some the plasma may be massed all at one
end, leaving a large vacuole in the remainder of the cell. The
sulphur globules are soluble in 90 per cent, alcohol, and
insoluble in hydrochloric, nitric, and acetic acids. Also, the
peripheral plasma contains dull green, round, or oval, or some-
times angular, plates of varying sizes. The poorer the cells
become in their content of sulphur, the more numerous are
these plates. They are regarded as some form of reserve
material. The cilia are numerous, and peritrich, each about
a third of the length of the cell, and o-Jix in thickness. Re-
production is by transverse fission.

Hinze distinguishes two species : —

Thiovuhtm majus, between 1 1 X 9fx and 18 X iJix.
,, minus ,, g-6 x 7-2/x and 11 x gfji.

Thiovulum Mulleri (Warming).

Literature. — Warming, 1876 ; Migula (3), 1900 ; Molisch
(5), 1912 ; Hinze (6), 1913 ; Bavendamm, 1924.

Description. — In 1876 Warming described and depicted an
organism, which he named Monas MiiUeri, that belongs
indubitably to the same group as Thiovulum. Thus the
generic name Monas * has prior claim, but as this name is
already applied to a genus of one of the Flagellate groups, it
may conveniently be ignored.

The cells are ellipsoidal or globular (Fig. 18). Tire size
varies from 6'3ju. to i2-8ju. in length, and from 5-6/x to 15/x in
breadth. Hinze has recommended that the species Thiovulum
MiiUeri be limited to such members as are within the dimensions
4*9 — I2ju,in length. The suggestion is not to be recommended,
for there is obviously no specific distinction between the micm-
bers of these dimensions and those immediately shorter or

* Monas. — The term is applied to a division of Flagellates, a group which
is divided according to the number and position of the flagella or cilia.
The genus Monas is distinguished by the possession of one flagellum.

THE LEUCO-THIOBACTERIA

125

longer, and there is more to be lost than gained by unnecessarily-
adding to the number of species.

The cells show quick, agile movements and appear to be
devoid of cilia. Reproduction is by longitudinal fission. The
intimate structure of the cell is given on page 185. Hinze

Fig. 18. — Thioviilum Miilleri. X 2000.

claims that a certain organ in the cell which is colourless, but
which possesses a centrum that can be stained with Delafield's
hiematoxylin, is a nucleus, and that cell division is accompanied
by the division of this organ (see Fig. 19). He also states
that when the HgS supply is scarce, starch or an amyloid
compound is stored in the cell, probably as a food reserve.
An interesting feature in
Hinze's work is his observa-
tion that the cells are still in
possession of a large number
of sulphur granules when, after
an active life, they have come
to rest and apparently died.
Thus there are other determin-
ants besides sulphur in the
metabolism of this organism.

Habitat. — Found on the
Danish coast, in various parts
of Germany, in Austria, and in the Gulf of Naples. It is
probably an organism of universal distribution. It lives in
water above mud rich in sulphuretted hydrogen, and thrives
particularly well in salt water, although it is also found in
fresh waters.

Fig. 19.

-Thiovulum Miilleri.
X 1500.

126 SULPHUR BACTERIA

Note oil Thiovuhmi.— -The discussion of this organism should
more appropriately appear in a work on the Flagellates, but
place is given to it in this work because the limits of the
group are defined by a physiological trait, namely the sulphur
metabolism. It is obvious, however, that the sulphur-organ-
isms are polyphyletic and that whilst the majority belong to
the Schizophytes, some, like Thiovulum, cannot be classed as
Bacteria.

Family 3. — Thiospirilla cem.

Free motile, colourless cells, containing sulphur. Division
by transverse fission.

As the spiral sulphur bacteria differ from the Spirillacece
of the Eiibacteria only in the inclusion of sulphur granules,
the term Thiospirillum used by Omelianski is preferable to
the term Thiospira used by Wislouch. The former has also a
claim on the ground of priority. Following the nomenclature
adopted in this book the following generic terms may be
distinguished : —

Spirillum. — Colourless spiral bacteria : sulphur absent.

Thiospirillum. — Colourless spiral bacteria, containing
sulphur.

Rhodothiospirillum. — Coloured spiral bacteria, containing
sulphur.

Rhode spirillum. — Coloured spiral bacteria ; sulphur ab-
sent.

The prefix " Rhodo " was first used by Molisch in his
Purpurbakterien to designate purple coloured bacteria, and
included the sulphur-containing and the sulphur-free forms.

Genus i. — Thiospirillum (Omelianski).

Literature. — Omelianski (2), 1905 ; Molisch (2), 1912 ;
Wislouch (2), 1914; Perliljeff, 1923; Bavendamm,
1924.

Colourless, spirally wound, with polar cilia. Division by
transverse fission, as in all Spirilla.

THE LEUCO-THIOBACTERIA

127

Thiospirillum Winogradskii (Omelianski).

Literature. — Omelianski (2), 1905 ; Molisch (5), 1912.

Ihis species was found in 1903 at the bottom of a high
glass vessel which had been filled with mud and water, from
the limans near Odessa, and to which some lime had been
added. It is a large, motile, colourless, or almost colourless,
spirillum, about 40/x long and 3-5/z thick,
as sketched by Omelianski (Fig. 20). The
normal form has one spiral turn but some
show two such turns. The single cilium, /<0-
which is probably compound, is polar. A^^'Qi

Habitat.— Yownd in Russia (Omelianski) ;
Austria (Molisch) ; and Germany (Kolkwitz).
It is common in water, both fresh and salt,
containing hydrogen sulphide.

Thiospirillum granulatum (Molisch).

Literature. — Molisch (2), 191 2.

An organism found in an artificial culture
containing fresh w-ater, mud, gypsum, and
roots of Elodea. This is a doubtful species,
as it was found in the same medium which
contained Beggiatoa alba and Cladothrix, both
of which are pleomorphic, and in some stages
have a spiral form. It is provisionally in-
cluded here. The length is given as 21 — 40/x
and its thickness 2 — 3"5/i..

^•

^m

Fig. 20. —

Thiospirillum
Wiyiogradskii
(Omelianski) .
X 2500.

Thiospirillum bipunctatum (Molisch).

Literature. — Molisch (2), 1912 ; Wislouch (2), 1914.

A marine organism showing a half, or a complete turn, with
a clear zone in the middle. The sulphur granules are located
in this middle portion. There is one cilium at each pole.
Volutin is present. Length of thread, 6-6 — lO/x, and thickness
1-9— 2-4/x (Fig. 21).

128

SULPHUR BACTERIA

Habitat. — Found in mud from Trieste, in which hydrogen
sulphide was present. Also in mud from the harbour of Reval
(Esthonia).

Thiospirillum agilissimum (Gicklhorn), Ellis.

Literature. — Gicklhorn (i), 1920 ; Bavendamm, 1924.

The generic name Spirillum was given to this organism by
Gicklhorn, and, following Wislouch, changed by Bavendamm
to Thiospira.

A small S-shaped, or comma-shaped, spirillum, found in
mud samples from a pond at Graz (Poland). It measures
lOjx in length and about i-S — 2-C)/,i in thickness. Gicklhorn

Fig. 21. — Thiospirillum
hipunctatum (Molisch).

Fig. 22. — Thiospirillum agilissimum
(Gicklhorn), Ellis. X 3000.

describes it as one of the smallest forms of sulphur-spirilla.
Its motility is very great. The sulphur droplets are pressed
close together, and number 4 to 1 2 in each cell. The membrane
is very delicate (Fig. 22).

The introduction of hydrogen sulphide into the medium
containing the organism liad no apparent effect in promoting
growth.

Thiospirillum agilis (Kolkwitz), Ellis.

Literature. — Kolkwitz (3), 1909 ; Lauterborn (7), 191 5 ;

Koppe, 1923 ; Bavendapim, 1924.
This organism was found in mud rich in hydrogen sulphide.
Its characteristics are given as follows : Thickness i/i, one

THE LEUCO-THIOBACTERIA 129

or two windings, widtli of winding 6/i., sulphur globules
disposed in a single line.

Habitat. — Found in various parts of Germany.

Note. — It is doubtful whether this organism is sufficiently
defined from^ the above description to justify giving it a specific
name,

Thiospirillum ELONGAiA. — Perfiljeff, 1923.

Literature. — Perfiljeff, 1923 ; Bavendamm, 1924.

Bavendamm gives the following description. Slender
cells with steep windings, and pointed ends. Dimensions
1-2 — I-5/X thick (in the middle) and 12 — 28ju long. The ends
are filled with volutin granules. Two polar cilia. Membrane
with spiral thickening.

Habitat. — Found in fresh water overlying mud rich in
hydrogen sulphide, near Leningrad.

Note on the Sulphur spirilla. — It is unfortunate that
practically nothing is known of the life-histories of these
interesting forms, and specific namics have been given almost
entirely from the appearance presented by the adult organisms.

Family 4. — Thiob.a cilla ce.e.

Free, colourless, motile or non-motile cells. Division by
transverse fission. The term Bacillus in Migula's classification
is used to designate motile, rod-shaped cells, and the term
Bacterium is used for similar cells that are non-motile. It is
doubtful, however, whether the distinction can be maintained.
Five species of the genus Bacterium were chosen at random,
and in all it was found possible to induce movement (see Ellis
(3)). It is, therefore, extremely probable that the motility
or non-motility of these organisms is conditioned by the nature
of the environment, and that the two terms apply to the same
organisms. In this work the term Bacterium is not used with
generic significance, and the term Bacillus is used for both the
motile and the non-motile rod-shaped organisms. There is
another reason for discarding the word Bacterium. Its proper
use in the English language is obviously as the singular of the
word Bacteria^ and it would therefore be better to restrict its

9

i3o SULPHUR BACTERIA

use to designate a single member of the group of Bacteria.
The word Bacterie which often appears in German hterature
as the singular of Bacterien (or Bakterien) is not so suitable.
The following terms are used in this work :- —

Thiobacilhis. Short rods, colourless, and with sulphur contents.
Rhodobacillns. ,, coloured, and no ,, ,,

Rhodothiohacillus. ,, coloured, and with ,, ,,

If the ciliation is polar instead of peritrich the term
Pseudomonas miust replace th.at of Bacillus, with the same
meaning as in Migula's classification. Thus there are theo-
retically six genera, four coloured and two colourless. In
this section only the genera Thiobacilhis and Thiopseudo-
monas are considered. Both Thiobacillus and Rhodobacilhis are
already in current use, with the same meaning as is given to
them in this work. The former word, however, is frequently
used loosely to indicate certain thionic acid bacteria, and in
particular Thiobacillus thiooxidans which in the strict sense
is not one of the sulphur bacteria. Up to the present the term
Thiobacillus has been used in the publications of American
writers as a convenient catalogue name, devoid of any phyletic
significance.

Genus i. — Thiobacillus (Ellis).*

Colourless, motile, or non-niotile, rod-shaped bacteria.
Cilia presumed peritrich.

Thiobacillus Bovistus (Molisch), Ellis.

Syn. Bacterium Bovisia (Molisch).

Literature. — Molisch (5).

Description. — A marine organism living a colonial life en-
closed in bladder-like slimy colonies measuring up to 4 mm.
in thickness. The outer surface of the bladder is made up of

*It has been shown (Ellis (i)) that the mode of insertion of the ciha
in bacteria is a constant feature. An organism with peritrich cilia is always
peritrich, and in the same way an organism with polar ciliation is constant
to that mode of insertion. This is important for systematic purposes.
As the majority of the rod bacteria have peritrich cilia it is advisable to
assume that an organism of this class has peritrich cilia when the ciliation
is unknown and, pending further investigation, to place it in the genus
Thiobacillus.

THE LEUCO-THIOBACTERIA

131

a denser material which functions as a protective covering,
and in its substance are short uncoloured rods, containing
sulphur globules each about i|/x in thickness and about 2-5jLt
in length. The bacteria are confined to the surface layers
and in the centre the material is more fluid (Fig. 23 a and b).

&\ .

•^.

a

V.

Fig. 23.

a. Thiobacillus Bovistus. x 5.

c. Pseitdomonas bipunctatus. X 1500.

b. Thiobacillus Bovistus.

d. Pseudoinonas retijornians.

The slimy globules are white in reflected, and dark or blue-
black in refracted light. Under certain conditions the slime
disappears and the bacteria are set free.

We must regard this organism as one in which the zoogloea
condition is permanent, that condition being probably best
adapted to its mode of life. The slight differentiation in the

9*

132 SULPHUR BACTERIA

composition of the slime seems to indicate the first step in the
evolution of the group towards a miore complicated organization.
Habitat. — Marine.

Thiobacillus thiogenus (Molisch), Ellis.
Syn. Bacillus thiogenus (Molisch).

A marine bacillus. It is described as being 0"9 — i-3/x
thick and 2-6/x long.

Habitat. — Marine.

Description. — The two organisms Bacterium crystalliferimi
(Gicklhorn) and Bacterium reliformans (Gicklhorn) do not store
sulphur ill the cells but deposit it outside. Accordingly they
cannot be included in the sulphur bacteria. Bavendamm
suggests that their place is probably in the group of thionic
acid bacteria.

Genus 2. — Thiopseudomonas (Ellis).

Description.- — Colourless, rod-shaped, motile or non-motile
cells. Cilia polar.

The introduction of this genus is necessary although so
far no definite species can be assigned to it. The two terms
Thiobacillus and Thiopseudomonas must necessarily go together,
for in most cases all motile rod-shaped forms are assigned to
the genus Bacillus, before it is known whether the cilia are
peritrich or polar. In most cases the difficulty of staining
the cilia accounts for the want of knowledge as to the dis-
position of the cilia.

The two organisms Pseudomonas bipnnctatiis (Gicklhorn)
and Pseudomonas hyalina (Gicklhorn) have been erroneously
jjlaced in the genus Pseudomonas by their discoverer. In
their general character they have no resemblance to the
organisms placed in this genus. They are described here
because they have been labelled as sulphur bacteria on the
strength of their habitat, namely, water containing hydrogen
sulphide in solution. Also, they do not store sulphur in their
cells.

Pseudomonas bipuncta tus. Colourless transparent cylin-
drical cells, 8 — I2/X long and 3 — 5/a broad. The single polar

THE LEUCO-THIOBACTERIA 133

ciliuni is invariably behind, and about I2ya in length. The cell
moves very slowly, covering about 6oo/x per minute, and the
movement consists of a slow forward rotation about the
longitudinal axis. The cell contains two, sometimes three,
highly refractive drops of calcium carbonate. Multiplication
is by simple fission, the cell becoming more and more con-
stricted in the middle until separation takes place. The
daughter cells remain pear-shaped for some time after separa-
tion (Fig 236^).

PsEUDOMONAS HYALINA . This species differs from the pre-
ceding ill being only 4 — 6/x long and 2 — 2'5/x broad. Gicklhorn
considered them to be distinct species from his failure to find
intermediate forms. The organisms P. hipunctatus and
P. hyalina, members of the thionic acid bacteria, are further
treated in Chap. XII.

CHAPTER VIII.

RHODO-THIOBACTERIA (COLOURED SULPHUR
BACTERIA).

Family i. Lankesteracecs. Genus i. Lankesteron.

Family 2. Chromatiacece. Genus i. Chromalium; Genus 2. Thioporphyra.

Family i. — Lankesterace^e.

Literature. — Lankcster (i), 1873, and (2) 1876; Warming
1876 ; Zopf (l), 1882 ; Ellis (ll), 1929.

A family of highly pleomorphic species which may be
filamentous, globular, ellipsoidal-cylindrical, or spiral. The
organisms enter the zoogloea condition in any of these phases.
May be motile or non-motile. One genus, Lankesteron.

Proof of the occurrence of pleomorphism in the sulphur
bacteria was submitted in Chap. L If the facts there given
are accepted, the three organisms, Bacterium ruhescens de-
scribed by Ray Lankester, Bacterium sidfuratiim described by
Warming, and Beggiatoa roseo-persicina described by Zopf, must
be accepted as highly pleomorphic species. Hitherto each has
been regarded as a mixture of species, and so has not found a
place in existing classifications. The three organisms show a
sufficient resemblance to justify their inclusion in the same
genus.

The names Bacterium and Beggiatoa are obviously un-
suitable. The former has already two meanings in bacterio-
logical literature, and the term Beggiatoa is already in use to
indicate a genus in which Beggiatoa roseo-persicina would be
unsuitably placed. The term Lankesteron is suggested as a
generic name for the inclusion of these species, and from it we
derive the family name of Lankester acece.

134

RHODO-THIOBACTERIA 135

Genus i. — Lankesteron (Ellis).

Literature. — Lankester (i), 1873, and (2), 1876 ; Warming,
1876; Zopf (i), 1882, (2), 1885, and (3), 1895;
Winogradsky (i), 1887, and (2), 1888 ; Migula (i),
1887, and (5), 1904 ; Bergey, 1923 ; Bavendamm,
1924; Buchanan, 1925, and Ellis (11), 1929.

A widely distributed genus, found in marine and fresh
waters containing plant and animal remains. Characteristics
as for the family Lankesteracece.

Lankesteron rubescens (Lankester), Ellis.

Syn. Bacterium rubescens (Lankester). For the description
of this species see page 137, Fig. 24.

Lankesteron sulfuratum (Warming), Ellis.

Syn. Bacterium sulfuratum (Warming). See page 138,
Fig. 25. Warming mentions Monas vinosa (Ehrenberg),
Monas erubescens (Ehrenberg), Monas Warmingii (Cohn), and
Rhabdomonas rosea (Cohn) as synonyms.

Lankesteron roseo-persicina (Zopf), Ellis.

Syn. Beggiatoa roseo-persicina [Zopi). There are numerous
phases in the life-history of this organism, and numerous
synonyms for these phases, as for example, Lamprocystis
roseo-persicina ; Protococcus roseo-persicina ; Pleurococcus roseo-
persicina ; Clathrocystis roseo-persicina ; Cohnia roseo-persicina ;
Thioroseo-persicina.

A description of this organism is given on pages 140-141,
Fig. 26.

Family 2. — Chromatace/E.

Free, motile, cells, normally of a cyhndrical-elliptical
shape. One polar cilium. Reproduction by transverse
fission.

Genus i. — Chromatium (Perty).

Literature. — Ehrenberg (i), 1838 ; Weisse, 1845 ; Perty,
1852 ; Cohn (i), 1875 ; Warming, 1875 ; Zopf
(l), 1882 ; Winogradsky (i), 1887, and (2), 1888 ;
Engelmann (3), 1888 ; Butschli (i), 1890, and
(3), 1896; Zettnow (i), 1891, and (2), 1897;

136

SULPHUR BACTERIA

P^P~-i

I

g-

Fig. 24.

RHODO^THIOBACTERIA 137

Forster, 1892 ; Beijerinck (i), 1893 ; Mitrophanow,
1893 ; Migula (2), 1895 ; Zopf (3), 1895 ; Ewart,
1897; Fischer (3), 1897; Butschli (4), 1902; Nadson
(2), 1903 ; Russel, 1909 ; Dangeard (i), 1909 ;
Strzeszewski (i), and (2), 191 3 ; Issatchenko (2),
1914 ; Buder (2), 1915 ; Lauterborn (7), 1915 ;
Metzner, 1920; Potthoff (i), 1921, and (2), 1922;
Gicklhorn (2), 1921 ; Bergey, 1923 ; Bavendamm,
1924 ; Scourfield, 1925-26 ; Buchanan, 1925 ; Elhs
(II), 1929.

Fig. 24. — Lankesleron riibescens (Lankester), Ellis.
Syn. Bacterium riibescens (Lankester).
Some of the forms of this organism as found on decomposing caddis

worms (after Lankester) : —

a. — A " sphaerous combination of cells forming a dark-coloured irregular
zoogloea mass." (So described by Lankester.)

b. — Acicular and sphaerous forms in attachment to another organism. The
extremities of the acicular forms showed oscillatory movements.

c. — Chains of cocci in filamentous formation. (Zopf records a similar forma-
tion resulting from the break-up of a filament into cocci.) This
" catenular aggregation," as named by Lankester, probably arose in
a similar way.

d. — Biscuit forms. Described as " loose and gloeoginous."

e. — Biscuit forms.

,''. — A globose aggregate of spherical units.

g. — Irregular forms.

h. — Reticulate aggregation of ellipsoidal cells. These recall the " Amoebo-
bacter " of Winogradsky (see p. 170).

i. — " Multilocular filament." The dense cell contents in such cases were
probably disposed so as to give the impression of a cell divided into
several compartments.

The bright colour imparted to the water in which Chroma-
Hum Okenii develops, and the interesting organism itself have
long made it a favourite object of study. It is the first to be
noticed in a group of different microorganisms on account of
its comparative largeness, its motility, and its bright colour.
The genus was established by Perty in 1852, although the type
organism Chromatiiim Okenii was recorded by Ehrenberg as
early as 1838, and named Monas Okenii, under the impression
that it belonged to the Monas group of Flagellates. Later,
other species were added, differences in size being the chief

138

SULPHUR BACTERIA

feature selected as a basis for division. At first three species
were distinguished and named as follows : —

Monas Okenii, 7-5 — 15/x X 5/x.

Monas vinosa, 2'^ijl long.

Monas Warniingii, somewhat longer than the first.

The genus was removed from the Flagellates, renamed
Chromalutm by Perty, and placed among the Bacteria. Perty

Fig. 25.^ — Lankesteron sulfuratum (Warming), Ellis.
Syn. Bacterlxtm sulfuratum (Warming).

Some of the hundreds of the different forms figured by Warming as
pleomorphic forms of this organism. The figure marked a is the
Rhahdomonas of later writers.

added to the genus two other species, which he named
Chromatium Weissii and Chroinathini violascens. Later the
number was added to by Winogradsky, who named Chro-
matium minus and Cliromaiium minutissimum ; and within
recent years Chr. Linsbaueri was added by Gicklhorn, Chr.
gracile by Strzeszewski, and Chr. Gobie by Issatchenko.

Description. — Coloured, free, motile cells, normally of ellip-
soidal-cylindrical form, but subject to considerable variations

RHODO'THIOBACTERIA 139

in shape, ranging from a very much elongated form to one
that is completely spherical. Multiplication is by simple
fission, the cell usually dividing at the middle. In one species
a process has been observed which may be sexual.

Chromatium Okenii (Ehrenberg), Perty.

Literature, — As for genus.

Description. — It is somewhat doubtful whether all the forms
described under the name of Chromatiimi Okeniihy different in-
vestigators are the same species. It is typically a free, wine-
red, coloured, motile organism, of an elliptical-cylindrical shape.
Motility appears to be due to the lashing of a single large wavy
cilium, which is readily made visible by staining with carbol-
fuchsin (Fig. 2'jh). However, it has been shown by Kolkwitz
that the apparently single cilium is compound and that it may
be composed of as many as forty cilia. He found that when
the slime which covers the cilia was removed, the individual
cilia came apart. When completely separated from the slime
they appear as very fine, wavy, filaments, that are on the
margin of visibility. Even in healthy cultures, some forms
may be observed which are strikingly different from the normal
shape. Thus bean-shaped or club-shaped individuals are not
uncommon in such cultures ; and there is often a noticeable
variation in their size. Scarcely two observers agree on the
range of its dimensions. The average is

7-5— iS^a X 5-0— 6-3/x.

Another range that has been given is

16/i, X 6-0 — 6-3/i.

Buder (i) has given lOyi as an extreme length for normal
specimens. Warming has shown that Chromatium Okenii
may exhibit a large range of pleomorphic forms ; and Zopf (i)
has come to the conclusion that so many intermediates may
be found between Chr. Okenii and Beggiatoa roseo-persicina
that these two apparently widely different organisms may be
regarded as the same species. The point cannot be settled
without further research.

140

SULPHUR BACTERIA

RHODO-THIOBA CTERIA

141

HA

3-5-
6.

7-

8-9.
10.
II.
12.
13-
14-15

Fig. 26. — Lankestei'on roseo-persicina.

(Diagrams after Zopf.)

Thread-form of this organism, x 300.

Small thread in undivided condition. X 300.

Thread fragments in process of transformation into cocci, x 900.

Large, branched and folded colony of cocci. X 500.

Net-form colony of cocci. X 250.

Various shapes and sizes of cocci. X 250.

Zoogloea of one coccus and three rod forms. X 900.

Zoogloea of large thick rods or short filaments. X 900.

Rod forms in massed formation, x 900.

Zoogloea of large cocci, x 900.

Various other forms assumed by this organism, x 900.

142

SULPHUR BACTERIA

P

'^f •s:% ..vi

#.

.J*' -^^

b

Fig. 27. — Chvomatium Okenii (Ehrenberg).
«.— Photomicrograph of Chv. Okenii, showing the inchisions of sulphur

grains, x 200.
6.— Another photomicrograph, one organism, showing the single large

compound cilium. x 500.
c— Diagrammatic representation of one of the cells, showing sulphur

granules, each showing as a thick black ring with a clear centre. X 1 6,000.

RHODO-THIOBACTERIA 143

Variant forms of Chromatiitm Okenii. — The tendency of
this species to vary both in size and in shape has been sufficiently
emphasized. The following organisms are, in the author's
opinion, merely variants of the highly pleomorphic Chromatium
Okenii.

(a) Chromatium Okenii forma Weissii.

Syn. Chromatium Weissii (Perty).

Literature. — Perty, 1852 ; Winogradsky (2), 1888 ; Miyoshi,
1897; Strzeszewski (2), 1913; Lauterborn (7), 1915 ;
Bavendamm, 1924.

Elongated form about ii-5ju. long and about 4-2/x thick.
Connected by transitional forms with the normal type of
Chromatium Okenii.

o o
0 O

o o

Fig. 28. — Chromatium Weissii, Chromatium minus, Chvomatium viriosum,
Chromatium minutissimum (arranged in order from left to right) .

[b] Chromatium Okenii forma minus.
Syn. Chromatium minus (Winogradsky).

Literature. — Winogradsky (2), 1888 ; Migula (3), 1897 ;

Strzeszewski (i), 1913 ; Bavendamm, 1924.
Described by Strzeszewski as an organism with numerous
intermediates bridging it to Chromatium Okenii forma Weissii.

[c] Chromatium Okenii forma minutissimum
Syn. Chromatium minutissimum (Winogradsky).
Literature. — Winogradsky (2), 1888.

Cells spherical or ellipsoidal, i — r2/x, in diameter. Indi-
vidual cells are colourless, but when seen in masses are of a
peach-fiower colour.

Issatchenko (4) records that this organism can be made to
grow in a test-tube filled with mud and water, forming a rose-
coloured film on the surface, which is identical with a similar

144 SULPHUR BACTERIA

formation that he observed at a depth of 13 metres in Lake
Mohilni. He adduces this fact, together with the fact that
sulphur bacteria collect round an algal filament which is
liberating oxygen, in proof of his contention that the genus
Chromatium is aerobic and not anaerobic, as reported by
Bredemann.

[d) Chromatium Okenii forma gracile.

Syn. Chromatium gracile (Strzeszewski).

Literature. — Strzeszewski (i), 191 3.

Ellipsoidal cells 2 — 6ft X I — 1*3/^. Single cells appear
colourless, but in masses, red.

Habitat. — Sulphur springs in Krakau (Poland).

Chromatium Warmingi (Cohn), Migula.

Literature. — Cohn (10), 1875 ; Warming, 1875 ; Engel-
mann (10), 1888 ; Ewart, 1897 ; Bavendamm, 1924.

Description.— This is a doubtful species. The name is
usually given to a Chromatium with the measurements 15 — 20/x,
X 8)u.. The shape is thus slightly different from that of the
normal Chromatium Okenii. Cohn named it after Warming,
but Warming himself sketches it as one of the many forms
assumed by Bacterium sulfuratiim. It is also claimed that its
motility is greater than that of Chromatium Okenii. Each
individual has a single large polar cilium, which is probably
compound. Bavendamm states that sulphur globules collect
in the cells only during division, when they increase in numbers,
and travel to the opposite ends of the dividing cells. Baven-
damm succeeded in obtaining pure cultures of this organism,
and as the cells in such cultures differed in size from the normal,
he named this variant Chromatium W armingii forma minus.
The same investigator also observed the development of buds
which in some cases had extended their lengths, and become
attached to similar buds from neighbouring cells. The pro-
cess is regarded by this writer as being possibly the first
phase of a sexual method of reproduction (see Fig. 29). The
probability is greater that the formation of the buds is a new

RHODO-THIOBA CTERIA

145

form of vegetative development that has come into being as
a result of new and unaccustomed conditions of growth, and
that the fusion is due to contact irritability. The new condi-
tions of growth are certainly responsible for the marked change
observed in the size of the cells. The buds are more likely to
be comparable to the " clamp connections " that are found
in some of the Fungi, and are, therefore, more of the nature of
simple vegetative fusions or mixing of plasma from different

Fig. 29. — Chromatium Warmingii forma minus (after Bavendamm), show-
ing the development of buds in artificial cultures of this organism.
The buds of different individuals are observed to be united. For
purposes of comparison of size a cell of the normal Chy. Okenii is
shown at circa X 2000.

sources. The unions observed in Bavendamm's cultures
appear to be similar to the H-connection in the Fungi and some
of the Floridese, which are not sexual.

Chromatium Linsbaueri (Gicklhorn).

Literature. — Gicklhorn (i), 1921 ; Bavendamm, 1924 ;

Scourfield, 1925 ; ElHs (11), 1929.
Description. — This species is distinguished from Chr. Okenii
by its Hme content, and more particularly by its peculiar

10

146

SULPHUR BACTERIA

D

Fig. 30.

RHODO-THIOBACTERIA 147

disposition of slime in the cell. A thin layer is formed immediately
inside the membrane (Fig. 30A), and, in addition, there are one
or two round patches of the same material, either together, or
separate. These patches may be situated in any part of the cell
(Fig. 31). This organism was found in a pool in Epping Forest,
the water of which is periodically coloured a blood-red by it.
Usually the red colour appears in autumn, and lasts for a few
weeks when the pool again becomes clear and remains so until
the f oUowingautumn. The Epping Forest overlies a bed of chalk,
so the water is rich in lime. The other microorganisms in the
same pond, however, did not appear to have absorbed the lime
in any quantity. The richness in the content of lime makes it
probable that the organism exercises a selective action in the

Fig. 30. — Chromatium Linsbaueyi (Gicklhorn).

A. Appearance of cell in certain conditions after treatment with carbol-

fuchsin. Immediately behind the membrane is a thin layer of slime
which extends round the whole cell inside this membrane. The
coloured globules are probably nitrogenous reserve material, and the
uncoloured ones lime corpuscles, x 5000.

B. The same cell unstained and observed " end on." The centre is

occupied by a large vacuole. All the globules are situated in the
peripheral plasma. The globules which appear to occupy the vacuole
in this diagram are in reality placed in the plasma on the far side of the
cell, which is here viewed " end-on ". x 6000.

C. The normal [b), and the pleomorphic type (a) of Chromatimn Linsbaueri.

D. Shows the stages in fission of this species.

absorption of this substance, but whether it is utilized in its
metabolism is not yet ascertained. The cells are ellipsoidal-
cylindrical, and measure up to 15/Lt in length and about 6 — 8/x
in breadth (Fig. 30). In the natural state, with one exception,
no marked deviation from the normal shape was observed.
This exception was a spiral form which appeared in some
cultures, and was in every other respect similar to the normal
organism. This is obviously a pleomorphic form. The
dimensions of the " Spirillum " were constant so far as this
could be determined from the small number of crude cultures
in which it appeared ; and there appeared in this case to be no
intermediate forms. The sudden appearance of this pleo-
morphic form was not a frequent phenomenon. Sometimes

10 *

148 SULPHUR BACTERIA

the cell is filled with the lime and the stainable granules, at
other times the cell is filled with sulphur globules (Fig. 30c).

f4>- - °

r; ^f% ' ^

%

sik^^^

'^\ <^ liH^

"mm

^t

• «.

Fig. 31. — Photomicrograph of Chromatiiini Linsbaueri (Gicklhorn). x 960.
(Photograph taken by Mr. H. W. Thorp, B.Sc.) The cells are almost com-
pletely filled with sulphur globules.

The intimate structure of the cell is described on pages 186
et seq. Gicklhorn, who was the first to describe this species.

RHODO-THIOBACTERIA 149

regarded its capacity for storing lime as an outstanding feature,
and considered it to be the first of a new class of bacteria, which
he named lime bacteria, because the globules are composed
of calcium carbonate. It must at present remain doubtful
whether lime takes an active part in the metabolism of this
organism, for its appearance seemed to be only occasional,
and to accompany the formation of the stainable globules
which arc evidently reserve material. It is possible that it
may be a waste product in the process which results in the
formation of the nitrogenous reserve material (the stainable
globules). The matter must await further investigation. The
cell contents vary somewhat, the cell being sometimes full of
sulphur granules, and at other times of lime granules and the
stainable corpuscles already mentioned. Nothing is at present
known of the conditions determining these differences, but
they are probably due to environmental changes. (Compare
Fig. 30A and Fig. 31.)

Chromatium violascens (Perty).

Literature. — Perty, 1852.

The violet colour is the distinctive feature of this organism.
It was found on the wall of a glass vessel containing decompos-
ing Chara. The cells are described as spherical or ellipsoidal,
2 — 3jL(- long, and very variable in size and shape.

Chromatium cuculliferum (Gicklhorn).

Description. — The cell is globular or slightly ovoid, and
measures 6 X 4jm. According to Gicklhorn its very small size
distinguishes the species from others of the same genus. It
possesses a single cilium, and rotates very slowly about its
longitudinal axis.

Description. — The sulphur globules are congregated at one
end, whilst the cilium is invariably attached to the other end
(Fig. 32).

The cells are colourless, and the size constant.

Habitat. — -In water containing decomposing Algee, taken
from Botanic Gardens, Graz.

The absence of colour is a feature which makes ques-
tionable its inclusion in the genus Chromatium. Baven-
damm, however, identifies it with Chromatium Warmiiwii

I50 SULPHUR BACTERIA

forma minus, chiefly on account of the characteristic massing
of the sulphur globules at one of the poles. He therefore
regards this organism as one potentially capable of developing
colouring matter, for Chromatium Warmingii forma minus
is the organism which was artificially cultivated by Baven-
damm, and in such cultures the colouring matter was richly
developed.

Fig. 32. — Chromaiiitm cuculliferum. X Oooo.

Rhabdomonas and Rhabdochromatium.

Literature. — Cohn (10), 1875 ; Warming, 1875 ; Zopf
(l), 1882 ; Mitrophanow, 1893 ; Zopf (3), 1895 ;
Biitschli (4), 1902 ; Nadson (2), 1903 ; Kolkwitz (3),
1909; Lauterborn (7), 1915 ; Gicklhorn (2), 1921 ;
Bergcy, 1923 ; Bavcndamm, 1924.

The term Rhahdomonas is of early origin, and was employed
by Cohn to designate those members of the Chromatium. group
which arc rod- or spindle-shaped. Rhahdomonas resembles
Chromatium in every respect except in shape. When the genus
Chromatium was transferred from the Flagellates to the Bacteria,
necessitating the discarding of the name Monas, the name was
changed to Rhabdochromatium. This term is now in general
use, although Bergey in his classification reverts to the older
name Rhahdomonas. As has already been shown (see pages
138-139) this peculiar shape of organism is linked up with the
normal Chromalimn by a very large number of transitional
forms, and so it cannot be regarded as a distinct species.

RHODO-THIOBACTERIA 151

Within recent years Bavendamm has succeeded in making
artificial cultures of a species of " RJiabdochroniathim.'" If
the spindle shape which is the distinguishing feature of this
organism were maintained in an artificial culture, a very
important fact would have been established, and the difference
of form, if permanent, would have sufficed to give this type
of organism generic rank. But Bavendamm describes the
organisms cultivated by him as made up of dull red rods of
irregular shape and of varying dimensions. The length
varied from short rods to lengths of 58/x. The spindle shape
is only one of numerous other forms. It seems probable,
judging from Bavendamm's figures and description, that he
had under observation an organism probably of the type of
Beggiatoa roseo-persicina that reacts to artificial conditions
by the production of a large number of pleomorphic forms.
Or, alternatively, they may have been variant forms such
as are commonly seen in the cultivation of the acetic acid
bacteria, such as, for example, Bacillus aceti. The terms
Rhahdomonas and Rhabdochromatiiini may have their uses for
descriptive purposes, but we are not justified in using them
with generic or even specific rank. In the following descrip-
tion the terms are retained merely as labels. They designate
organisms that in all probability are phases in the life-histories
of pleomorphic species, that have been described under other
names ; our knowledge is not yet sufficiently advanced to
determine the identity of the organisms of which they are
the phases.

Rhabdochromatium koseum (Cohn), Winogradsky.

Literature. — Cohn (10), 1875 ; Warming, 1875 ; Zopf
(i), 1882, and (3), 1895 ; Winogradsky (2), 1888 ; Mitro-
phanow, 1893; Migula, 1894, 1895, 1900; Butschli
(4), 1902 ; Nadson (2), 1903 ; Kolkwitz (3), 1909 ;
Lauterborn (7), 1915; Bergey, 1923.

Description. — ^This is the Rhahdomonas rosea of Cohn and of
Bergey. It is composed of spindle or rod forms, of unequal thick-
ness. Average thickness, 3 — 5jLt. Some attain the length of 35/i.

152 SULPHUR BACTERIA

Found in marine and fresh waters containing sulphuretted
hydrogen in solution.

Rhabdochromatium minus (Winogradsky).

Literature. — Winogradsky (2), 1888.

A smaller variety than the preceding, which it otherwise
resembles. Length, 5 — lO/x; maximum thickness, 2 — 9/m.
Colour, rose-red.

Rhabdochromatium gracile (Warming), Migula.

Literature. — Warming, 1875 ; Migula (3), 1900 ; Baven-
damm, 1924.

This is the Monas gracile of Warming, which was changed
to its present name by Migula.

Rod-shaped cells, in which one -end is frequently thicker
than the other. Length, up to 6o/x. Colour, rose-red. Found
in a fresh-water pond near Copenhagen.

Rhabdochromatium linsbaueri (Gicklhorn).

Literature. — Gicklhorn (2), 1921 ; Bavendamm (i), 1924.

This species differs from Chromatium Linsbaueri in being
spindle-shaped, and in being destitute of the slime patches
which are characteristic of that organism. As in other
respects the two forms agree, and as the marks of difference are
probably of a fugitive and unstable character it is highly
probable that the two organisms are both varieties of the same
species. This variety is described as being 30yu, long and 3 — 4/x
broad ; it is propelled by a cilium 20 — 30/x long. The colour
is wine-red. The cell contains calcium carbonate as well as
sulphur granules.

Habitat. — Found in a small pool near Graz (Austria).

Genus 2. — Thioporphyra (Ellis).

Literature. — Ellis (9), 1926.

Genus with one species. Cells spherical, or ovoid, motile.
Movement by one large cilium which is probably compound.
Colour from violet to mauve. Reproduction by transverse
fission, by budding, and possibly by endospores.

RHODO-THIOBA CTERIA

153

Thioporphyra volutans (Ellis).

Literature. — Ellis (9), 1926.

Description. — Mass cultures are usually found as a purple
mantle covering seaweed decomposing in shallow pools. Under
the microscope the large purple cells, of an ovoid or spherical
form, which are in active movement, are striking objects.
In addition, the cells contain numerous sulphur inclusions.

The organism is normally either a large unicoccus (Fig. 34a)
or a large diplococcus (Fig. 34^). Under certain conditions,

Fig. 33. — Thioporphyra volutans (Ellis).
a. — Tricoccus in triangular formation. x 3000.
b. — A tricoccus in linear formation. This form of tricoccus is exceptional.

X 3000.
c. — A diplococcus showing three of the structures which maj' be endospores.

In the cell a single sulphur globule, with its characteristic thick,

black, peripheral ring is shown, x 5000.

connected with reproduction, tricocci may be formed (Fig. 33
a and b). The plasma may occupy only the peripheral portion
of the cell, leaving a large vacuole in the centre (Fig. 34^^) ; or
it may be reticulate (Fig. 34^^), or peripherally placed but with
uneven distribution, the bulk of it being concentrated at one
end if the cell is ovoid (Fig. 34c). The average thickness of the
cells is 7ju,. The purple colouring matter is diffused throughout
the plasma. The cytoplasm is slightly granular, and is
readily stained by iodine, methylene blue and similar reagents.
It is bounded on the outside by a readily distinguishable

154

SULPHUR BACTERIA

Fig. 34.

RHODO-THIOBACTERIA 155

membrane. The colour varies from a deep violet to mauve.
Sometimes it is perceptible in single cells, but usually only when
they appear in groups. The separation of the cytoplasm from
the membrane can readily be brought about by the use of
concentrated reagents.

The sulphur granules may be few in number, or they may
completely fill the cell ; they may even be altogether absent,
even in specimens that appear to be perfectly healthy.

Fig. 34. — Thioporpliyra voliitans (Ellis).

a. — Spherical cell with reticulate plasma (diagrammatic), showing uniform
distribution of vacuoles. X 6000.

b. — Ovoid organism in process of division. Cell is constricted in the middle
preparatory to division. In the cell are four sulphur globules and two
globules from which the peripherally placed sulphur has been removed
by reagents. This core is organic. X 4000. '

c. — An ovoid cell in which the plasma is almost entirely disposed in the
form of a cap covering about three-quarters of the periphery of the cell
inside the membrane. Extensions from this cap to cover the rest of
the membrane on its inner side with an extremely delicate lining may
be demonstrated by special staining. The centre of the cell is
occupied by a large vacuole and at one end a large number of sulphur
granules are congregated. X 4000.

d. — An ovoid cell showing a third mode of distribution of plasma. The
centre of the cell is occupied by a large vacuole. The plasma is more
or less evenly distributed between the vacuole and the membrane.
X 6000.

e. — Sulphur crystals obtained by dissolving the sulphur globules with
aceto-carmine when they leave the cell and crystallize in the surround-
ing medium.

/.• — Cell showing its single long and wavy ciliimi.

g. — After treatment with aceto-carmine. Some of the sulphur globules
remain unaffected, in others the sulphur has disappeared, leaving a
coloured centrum. One sulphur crystal is shown, x 1000.

The membrane is readily seen without the use of stains.
As with all bacterial cells a layer of slime covers the membrane
at all times. Under some conditions the layer is too slight
to be perceptible without very careful staining. Under other
conditions slime formation is extensive and a thick layer covers
the membrane.

The cytology of the cell is treated with greater detail in
Chap. X.

156

SULPHUR BACTERIA
Methods of Reproduction.

(A) Fission. — The normal method of division is by simple
fission. In normal cultures uni- and diplo-cocci preponderate,

Fig. 35. — Thioporphyya volutans (El'is).

Stages in bud development — ■

a. — First stage. Formation of a peripherally placed clear region from which

the bud develops.
b. — Second stage. Protrusion of the bud.
c. — Third stage. Still further protrusion of the bud. Each contains a

single sulphur globule. The bud has no external covering, and

assumes a wavy outline when treated at this stage with staining

reagents.
d. — Fourth stage. Separation of the bud from the parent cell. Still

shows a wavy outline when stained.
e. — Fifth stage. The completed bud. Remains spherical when stained so

that probably a resistant covering is formed at this stage.

RHODO-THIOBACTERIA 157

the latter being evidently deri\-ed from tlie former. In division,
the coccus elongates slightly, and becomes constricted in the
middle (Fig. 34^ and Fig. 33tj. In many cells the process of
constriction does not proceed any further, and the cell con-
tinues its existence as a diplococcus. In others, the constric-
tion continues until complete separation of the two daughter
cells takes place. Whilst division is in process, the cells are
also increasing in size so that when separation take place, the
daugliter cells are as large as the parent coccus before division.
More rarely tricocci are formed (Fig. ^2)^ and h ; Fig. 2^6 at a).
The three units in a tricoccus are usually arranged in triangu-
lar formation (Fig. 33a; Fig. 36 at a) ; more seldom the linear
arrangement holds (Fig. 33^). It is probable that the formation
of a tricoccus from a diplococcus results from a process similar
to that by which a diploccocus is formed from a unicoccus.

(B) Budding. — This method of reproduction, normal to the
yeast plant, has not hitherto been recorded for bacteria.
Under natural conditions, the organism occasionally shows a
'very marked increase in reproductive activity, which is
marked macroscopically by an intense deepening of the
purple colour. During this period more individuals are formed
in a few days than are normally formed in several weeks. On
the occasion of such a marked increase, it was found that
the normal method of reproduction had been superseded by
a method of bud production. The buds are considerably
smaller than the normal form, and much more numerous. If
seen apart, and in aggregated masses, they would have been
denominated as a species of Lamprocystis. Compare Fig.
36 (i) with Fig. 36 (2).

Stages in Bud Formation.

(i) A more or less rounded space near the surface, marked
off from the rest of the cell by a difference in colour, is the
first indication that budding has commenced. This space is
clearer than the rest, and shows a greater resistance to the
penetration of stains (Fig. 35fl). We may apply the name
of Regional Rejuvenescence to this differentiation of the plasma, •

158

SULPHUR BACTERIA

T* • " V..' •' • V *

Fig. 36. — Thioporphyra volutans (Ellis).

Photomicrographs taken under the same magnification ( ■: 500) of the

normal and the pleomorphic varieties of this organism. The change of form

follows the change from reproduction by fission to reproduction by budding.

(i) Normal form. The majority of the individuals are unicocci. At b

. is shown a diplococcus, and at a n tricoccus.
(2) Pleomorphic variety. Large masses of small globules de^'eloped from
the normal form by budding.

RHODO-THIOBACTERIA 159

which is characteristic of Thioporphyra voliitans, for as far as
can be ascertained the bud is formed entirely from the material
in this differentiated region.

(2j A protuberance appears opposite the clear space (Fig.
35^), and a bud emerges which is devoid of a definite membrane,
and which is irregularly contoured. It was not possible to
ascertain whether the membrane burst to admit of the pro-
trusion of the bud, or whether the bud was abstricted. Usually
at this stage it contains one sulphur globule.

(3) Beyond this point the development does not follow a
uniform course. It may continue its growth but remain in
attachment to the parent cell, in which case a bud is not formed
but a unicoccus becomes a diplococcus, and a diplococcus a
tricoccus. Or the bud may separate early from the parent,
in which case the growth medium is occupied by a mass of
small non-motile globules, each containing a sulphur globule
(Fig. 35 d and e). The cells formed by budding are of uniform
size and shape within certain limits.

In addition to the above, structures have been found in
Thioporphyra vohUans which may be endospores similar to
those formed in the genus Bacillus, but no opportunities have
occurred to study them more closely (Fig. zy)-

ClLIATION AND MOVEMENT.

The movement is one of rapid translation and rotation,
by a long single cilium.

The pleomorphism of this organism has been considered
in Chap. I., and the intimate structure of the cell will be dis-
cussed in Chap. X.

CHAPTER IX.

RHODO-THIOBACTERIA (COLOURED SULPHUR
BACTERIA).

Family 3. RhodothiospirillacecB. Genus i. Riiodothiospivillum.

Family 4. RhodocapsacecB. Genus i. Rhodocapsa ; Genus 2. Rhodothece ;

Genus 3. Rhodothiosarcina.
Family 5. ThiocapsacecB. Genus i. Thiocapsa ; Genus 2. Thiocystis ; Genus

3. ThiosphcBrion.
Family 6. AmcebobacteriacecB. Genus i. Amcebobacter ; Genus 2. Thio-

dictyon.
Family 7. Thiopediacece. Genus i. Thiopedia.

Family 3. — Rhodothiospirillace.e. -
Spirally wound, motile, coloured organisms. Ciliation
polar.

Genus i.—Rhodothiospirillum (Ellis).

Literature. — Ehrenberg (2), 1838 ; Cohn (7), 1872 ; Wino-

gradsky (2), 1888; Butschli (i), 1890 ; Zettnow (2),

1897; Migula (3), 1900 ; Buder (2), 1915; Metzner,

1920.

Winogradsky's term Thiospirillum was designed to include

all spirilla with sulphur contents. The occurrence of these

organisms in sulphur waters has been known for nearly a

hundred years. Early descriptions of spiral sulphur bacteria

37. — Ophidonionas sanguinea.

are incomplete since no distinction was made between fat
globules, volutin, and sulphur globules.

In Winogradsky's genus, both coloured and uncoloured
spirilla were included, so that a change of nomenclature is

160

RHODO-THIOBA CTERIA

i6i

necessary to conform to the system which has been adopted
in this book. Hence the term Thiospirillum is restricted to
uncoloured organisms, whilst the term Rhodothiospirilhim is
reserved for the coloured ones. Winogradsky's genus includes
the spirilla described by Perty, 1852, and by Warming,
1875 ; and also Ehrenberg's two species Ophido7nonas jenensis
and Ophidomonas sangidnea (1838) (Fig. 37).

Rhodothiospirillum jenense (Ehrenberg), Ellis.

Literature. — Ehrenberg (2), 1838; Cohn (7), 1872; Biitschli
(i), 1890, and (4), 1892; Zettnow (2), 1897; Zopf (2),
1885; Migula (3), 1900; Buder (2), 1915; Metzner,
1920 ; Bavendamm, 1924.

Description. — Ehrenberg gave the name Ophidomonas
jenense to this organism, but it was changed by Winogradsky
to Thiospirillum jenense, and
for the reasons which are
given above it is now again
changed to Rhodothiospiril-
lum. Possibly some, if not
all, of the coloured spirilla
may be phases in the life-
history of some pleomorphic
organismlike Beggiatoa roseo-
persicina, for practically all
the spirilla have been named
from observations of the
adult specimens, without
reference to their life-
history.

Fig. 38. — Rhodothiospiyillum jenense.

Family 4. — Rhodocaps.acem.

Free cells, spiral, rod-shaped, or globular, surrounded by
a capsule of slime. On Hberation from slime the cells may
become motile. One genus with aerosomes.

H

i62 - SULPHUR BACTERIA

Genus i. — Rhodocapsa (Molisch).

Literature. — Molisch (3), 1907 ; Bergey, 1923 ; Baven-
damm, 1924 ;

One species found in Trieste marine water, containing de-
composing Zostera, to which it imparted a rose or peach-flower
colour. The organism is a rod-shaped, or sausage-shaped,
coloured bacillus. A shme capsule envelops each individual,
although during division there are temporarily several in-
dividuals in the same capsule (Fig. 39). The cells contain
peculiar structures called aerosomes, strongly refractive,
irregularly shaped, reddish bodies, the function of which, it
is claimed, is to keep the cell in suspension in the water. They
are also found in the Cyanophyceae. The cells also contain
sulphur granules. On liberation from the capsule, the cells
become motile, the movement being either a rapid rotation,
complete or partial, round a longitudinal axis, or rapid
oscillation of one of its ends.

7

Fig. 39. — Rhodocapsa suspensa. X 15. Fig. 40. — Rhodothece pendens.

Rhodocapsa suspensa (Mohsch).

Rod- or sausage-shaped, 3-5 — 180/A in length and 1-8 — 3-5/a
in thickness. Average length, 10 — 20fi.

Genus 2. — Rhodothece (Molisch), 1906.

Literature. — Molisch (3), 1907.

A genus of one species, which was found in a crude culture
prepared from decaying animal remains, and sea-water, from
Heligoland. The organism coloured the water as though with
rose-red milk of sulphur. Like the preceding genus the cells

RHODO-THIOBACTERIA 163

are enveloped in slime (Fig. 40), but they differ in being globular
in shape. They are arranged either as diplococci or as short
chains. Each cell measures 1-8 — 2-3/x in diameter, and its
slimy envelope 3 — 14/x.. Single cells do not show colour. The
colouring matter is stated to be a mixture of bacteriopurpurin
and bacteriochlorin (see Chap. XIII.). The cells are non-
motile. One species is known, Rhodothece pendens.

Genus 3. — Rhodothiosarcjna (Ellis).

Literature.- — Winogradsky (2), 1888 ; Schroeter (i), 1889 ;
Migula (3), 1897; Issatchenko (2), 1914; Bergey,
1923 ; Bavendamm, 1924.

One species in the genus. It corresponds to the Sarcina
of the Euhacteriacece, but differs from that genus in being
coloured, and in containing sulphur. The formation of
packets of cocci, resembling bales of cotton, is a distinctive
feature. So far as is known the organisms which are made
up of packets of cocci in regular formation do not exhibit
pleomorphic variations.

Rhodothiosarcina rosea (Schroeter), Ellis.

Described under the name of Sarcina rosea by Schroeter.
Cells 2 — 2"5jU. in diameter. Bright rose-red in colour.
Habitat. — In water containing H2S in solution.

Family 5. — Thiocapsacem (Winogradsky).

Literature. — Winogradsky (2), 1888.

Coloured sulphur bacteria, each being a community of
independent cocci enclosed in a common envelope of slime.
The members of this group are perhaps phases in the life-
histories of other organisms.

Remarks on the Division of Cocci.

It has been shown in Chap. VI. that organisms which
normally form clusters of globular cells, and apparently divide
in three planes, may under certain circumstances appear as
uni- and diplo-cocci, when the number of planes in which
division occurs is impossible to determine. Among the
Euhacteriacece an organism made up of cocci disposed in

i64 SULPHUR BACTERIA

clusters, regular or otherwise, would be placed in the genus
Sarcina, but under different conditions the same organism
may be found as uni- and diplo-cocci, when it could equally
find a place in the genera Micrococcus or Streptococcus. Hence
among the Coccacece an organism made up of groups of very
small denomination cannot be placed until it is determined
whether its division walls are found in three planes. In some
cocci this is undoubtedly the case, for the writer has observed
in some, three division walls, mutually perpendicular, to be
sinmltaneously present in the same coccus. It is felt, however,
that in the vast majority of cases, organisms have been
allocated to the genus Sarcina without ascertaining whether
division took place in three planes. It has been assumed
that because the organism was found in clusters it underwent
this form of division. No investigation of the number of
planes of division of any bacterial organism has been made to
determine the constancy of the number, and yet differences
in the number of planes of division have taken an important
place in the classification of bacteria.

The irregular arrangement of the globular cells of the
sulphur bacteria embedded in slime suggests the possibility
that these organisms may be classed as Sarcina, but since no
investigation of the number of planes of division has been made,
this is a mere speculation.

Genus i. — Thiocapsa (Winogradsky).

Literature. — Winogradsky (2), 1888 ; Migula (3), 1897 ;
Bergey, 1923 ; Bavendamm, 1924.

Description. — One species only, namely Thiocapsa roseo-
persicina. The cocci which make up the organism are en-
closed in slime and are about 2-8/x in diameter. The slime
ultimately liquefies and the enclosed individuals escape.
They are non-motile, and each settles down and forms a fresh
envelope of slime. Multiplication is by fission. The colour
is intense rose-red, and the cells are rich in sulphur inclusions.
Winogradsky observed the development of a zoogloea for a
period of six months, and found no change in its condition.

RHODO-THIOBA CTERIA

165

The growth of the zoogloea sometimes resulted in masses being
formed which w^ere several hundred microns in diameter.

The cells are presumed to divide in three planes by analogy
with the comparable genus Aphanocapsa (one of the Chroococ-
cacece) which was stated by Nageh to undergo " division
alternately in all directions of space, in the successive genera-
tions."

9^

Fig. 41. — Thiocapsa roseo-persicina. X 2000.

Genus 2. — Thiocystis (Winogradsky),
Literature.— Wmogradsky (2), 1888 ; Migula (3), 1897 ;

Bergey, 1923 ; Bavendamm, 1924.
Families of small cocci enclosed in slime. When the slime
disappears the cocci assume motility, and either separate and
swim away, or enter once more into the zoogloea condition.
In the former case, only a portion of the slime is liquefied,
sufficiently to permit the cocci to escape.

Fig. 42. — Thiocystis violacea. X 500.

Thiocystis violacea (Winogradsky).

Cells round, 27 — S-lfi in diameter, light rose-red or violet
in colour. Slime thick (Fig. 42).

Thiocystis rufa (Winogradsky).

Cells round, not greater than Ifx. Thick slime covering ;
cells deep violet-red, and occasionally black with sulphur
grains.

1 66

SULPHUR BACTERIA

Both these species have a strong resemblance to some of
the phases of Beggiatoa roseo-persichia which were regarded by
Zopf as " one of the many kinds of the zoogloea forms of
Beggiatoa roseo-persicina.'" Hence this genus is only pro-
visionally given a place in this classification.

a

Fig. 43. — (a) Thiosphcsrion violacea. (b) Species of ThiosphcBvion (unnamed).

In Fig. 43& is shown an organism in the zoogloea condition,
which appears to belong to the genus Thiosphcurioji. It is
made up of globular masses arranged in an irregular fashion,
the whole being slightly tinged with red. Each globular
mass is about 15/x in diameter, and is composed of a large

RHODO-THIOBACTERIA 167

number of small cocci, each containing one sulphur granule.
The coccus is roughly spherical (about l\yL in diameter) and
is devoid of a membrane. An opportunity has not arisen to
follow the life-history of this interesting species.

Genus 3. — Thiosph.^rion (Miyoshi).

Literature. — Miyoshi, 1897 ; Migula (3), 1897 ; Bergey,
1923 ; Bavendamm, 1924.

Description. — The outstanding feature of this genus of only
one species is the exactly spherical shape of the colonies, and
their violet colour. Migula and Bavendamm included the
genus in the Lamprocystece. Bergey removed it from this
group, and once more gave it generic status. The exactly
spherical solid masses of slime of a violet colour, arranged in
little groups in attachment to objects in the water, appear
to offer a sufficient distinction to merit generic rank.

Thiosph.erion viol ace a (Miyoshi).

Cells are spherical-ellipsoidal, 2-5 — i*8jLt, and violet in
colour. Sulphur inclusions are somewhat angular (Fig. 43a).

Habitat. — Sulphur springs in Japan. Found on threads
of Thiothrix.

Note on the Organism Thiosphjera gelatinosa (Miyoshi).

Literature. — Miyoshi, 1897; Migula (3), 1900 ; Bergey,
1923 ; Bavendamm, 1924.

All that is known of this organism is that it is made up of

ovoid cells (7/x X 5ju,) enveloped in a loose jgr-^i^jr*^-^

gelatinous envelope. Migula and Baven- ^ -n

damm placed it in the Lamprocystece, l' ®®®^ ® \

whilst Bergey gave it generic rank. As \- '^ (^ ^ ® '\

so little is known, and what is known is %. ®®(^^ ^|

a condition that may be a phase in %l:? '^■'^- -.J^

the life cycle of one of several organisms, •^■<i^-u-^

r 1 • 11-, Fig. 44. — Thiosphcsra

no useful purpose is served by its re- 7 .■

^ ^ J gelatinosa.

tention (Fig. 44).

i68 SULPHUR BACTERIA

Family 6. — AMCEBOBACTERIACEAi.

Literature. — Winogradsky (2), 1888; Migula (3), 1900;

Bergey (i), 1923 ; Bavendamm (i), 1924.
Cells united into clusters usually enclosed in slime. When
movement takes place the cells move in unison.

Genus i. — Amcebobacter (Winogradsky).

Description. — A peculiar group of three species. The cells
are enclosed in a slime sheath, and slowly move inwards or
spread outwards in unison inside the slime. As a result there
is a slow alteration in the shape and mass of the community
of cells. This united action of the cells was explained by
Winogradsky as being due to contractile plasma threads which
bound the cells together, but neither he nor Lauterborn was
able to see them. It is, however, difficult to explain these
co-ordinated movements except by postulating the existence of
such threads. There is also another character of interest. The
slime envelope is made up of two layers, an outer which is
strongly refractive, and an inner which is feebly refractive.
The cocci on the opening of the slime cyst do not separate, but
move out together, leaving an empty shell. They then attach
themselves to some object in the water and whilst in attachment
exhibit those slow changes of form which are characteristic of
Amceba. Also in each cell a perceptibly large vacuole can be
discerned.

Whilst in attachment, and before slime formation has pro-
ceeded very far, the cells close in until they are all huddled
together, and then they again spread out in extended formation.
It is not known whether the activating force of such move-
ments is spontaneous, or whether the movement takes place in
response to external injfluences. Winogradsky considers that the
presence of hydrogen sulphide in the water is responsible for
these movements, but has not adduced proof in support of his
statement. If the cells are connected by plasma threads, Amoeho-
bacter must be regarded as a loosely compacted coenobium.

Amcebobacter roseus (Winogradsky).
Literature. — Winogradsky (2), 1888 ; Macgregor Skene,
1914 ; Lauterborn (7), 1915.

RHODO-THIOBA CTERIA

169

Each cell is globular but capable of altering its shape.
Dimensions 2-8 — 3-4/^ in diameter. The cell elongates before
division to approximately 6/x. Colour a delicate rose (Fig. 45).

Habitat. — Found in waters containing sulphuretted hydro-

FiG. 45. — Amoebohacter roseus. {a) Encysted condition, [b) Emergence
of cells from cyst and formation of swarm cells, x 1500.

gen in solution. Skene obtained this organism from material
supplied from Kiel, and found that he could obtain impure
cultures by adding to the material sulphuretted hydrogen,
ammonia (as a source of nitrogen), and chalk (as a neutralizing
agent).

Amcebobacter bacillosus (Winogradsky).

Literature. — Winogradsky (2), 1888 ; Lauterborn (7), 191 5.
Cells rod-shaped, 2 — 4/x long and 17/i, thick. Colour red.
Habitat. — In waters containing sulphuretted hydrogen in
solution.

Am(Ebobacter granula (Winogradsky).

Literature. — Winogradsky (2), 1888.

Cells very small, about 0-5/x in diameter, and almost
colourless. Each contains a single sulphur globule so highly
refractive that it blurs the contour of the coccus.

Habitat. — In waters containing sulphuretted hydrogen in
solution.

Genus 2.- — Thiodictyon (Winogradsky).
Literature. — Lankester (i), 1873, and (2), 1876; Winogradsky
(2), 1888; Migula (3), 1900 ; Kolkwitz (3), 1909 ;
Bergey, 1923 ; Bavendamm, 1924.

Description. — An organism was sketched by Lankester in
1873 as a pleomorphic phase of Bacterimn riibescens which

lyo

SULPHUR BACTERIA

took the form of rod-shaped or spindle-shaped units, united
to form a net somewhat of the same kind as Hydrodictyon.
It differs from the preceding genus in the absence of a
well-defined substantial slime covering. The same or a
similar organism was found by Winogradsky, and given generic
rank under the name of Thiodictyon. If we accept the fact
of a wide pleomorphism in the sulphur bacteria we should rank
Thiodictyon as another instance of the phenomenon. Whilst,
however, it is possible that the majority of the forms observed
by Lankester on the caddis worms examined by him were
pleomorphic forms of one organism, it is also possible that
there may have been two or even three species present and that
the organism known as Thiodictyon was one of them.

Thiodictyon elegans (Winogradsky).
Literature.^ A?, for the genus.

Description. — The rods form at first a compact mass, and
are united to one another by their ends. Later they extend to

Fig. 46. — Thiodictyon elegans. Rod-shaped cells in process, spread to
assume arrangement shown in completed state in B. X 1500.

form a net-like arrangement (Fig. 46). Under unfavourable
conditions, the net arrangement is abandoned, and the units
move inwards so that compact formation is once more assumed.
Multiplication of the rods takes place by division, or a small
number of units may be liberated after division has taken place.
These grow into larger colonies. The cells are 5/i by I'^fx long
before division, but become longer during the course of the
division. They are coloured a very faint red. In the peripheral
plasma are a number of small sulphur granules.

RHODO-THIOBACTERIA 171

Habitat. — In sulphur springs, and other waters containing
sulphuretted hydrogen in solution.

Thiothece and Thiopolycoccus.

The genus Thiothece was founded by Winogradsky (2) to
include a single organism, which he named Thiothece gelatinosa
(Fig. 47). The cells of this organism are
small and either globular or ellipsoidal-
cylindrical. They measure 4*2^ in diameter,
and it is stated without experimental
evidence that they divide only in one
plane. For this reason the genus is placed
in the Amoshohacteriacece.

Winogradsky observed the emergence „ ^"^^^1 ■ ^
of the cells from the slime, and their gelatinosa. x 1000.
subsequent transition into the swarm con-
dition. Each cell has a single cilium. The colour of the cells
is an intensely grey violet, or pale rose, or even yellowish or
dirty green. The sulphur globules are located in the peripheral
layer. Winogradsky also noted the similarity of the organism
to one of the pleomorphic forms of Bacterium ritbescens, but
rejected the idea that the two were identical. Beggiatoa
roseo-persicina also exhibits a pleomorphic phase that appears
to be identical with the organism described by this investigator.
The figure which he gives of Thiothece is one which it would not
be difficult to match in the drawings of several of the better-
known bacteria. In addition, the name was given from
observation of only the adult condition of the organism, and
its life-history was not investigated.

The same objections apply to the genus Thiopolycoccus
of the same writer. This was stated to differ from Thiothece
only in the absence of cilia after release from the slime. The
differences between the two organisms which have given rise
to these two genera are not sufficiently marked to warrant
one being regarded as a variant of the other, far less suffi-
ciently marked to necessitate founding two genera. It is
considered that the objections just stated are such as to

172 SULPHUR BACTERIA

warrant the removal of both these organisms from a general
scheme of classification.

The Thiopolycoccus ruber of Winograclsky is composed of
small cells closely pressed together and non-motile. They are
spherical, l-2/x in diameter, and intensely red in colour.

Family 7. — Thiopediace.e (Winogradsky).

Literature. — Oerstedt (i), 1840 and 1841 ; Rabenhorst

(2), 1865; Warming, 1875; Winogradsky (i), 1887;

Utermohl (i), Migula (3), 1900 ; Kolkwitz (3), 1909;

Bavendamm, 1924.

Description. — The single species of which this family is
composed has been known for ninety years. Oerstedt named
it Erythroconis littoralis, but its best-known earlier name was
Merismopedia littoralis. Warming was of the opinion that it
was either a variety of Bacterium sulfuratum, or a small colony
of Clathrocystis. In Migula's classification Merismopedia
littoralis was placed among the sulphur bacteria. It was
regarded as identical with the Thiopedia rosea described by
Winogradsky, and so the term Merismopedia was dropped
and the term Thiopedia took its place. The genus Merismo-
pedia of to-day belongs to the Cyanophycece. The American
Society of Bacteriologists has rejected Thiopedia as an in-
dependent genus. Its rejection is not justified, because the
regularity of formation of the plates of cells is a striking
feature, which is absent from all the other genera of the sulphur
bacteria, and the regularity indicates a first step towards the
colonial habit. This organism is thus essentially different
from the other slime-enclosed bacteria in which the association
of the component cells is accidental and in which the cells
behave as independent units.

Genus i. — Thiopedia (Winooradsky).

Literature. — As for family.

Thiopedia rosea.

Globular cells, i-i — 2/x in diameter, and arranged in
the slime in regular plates. Later, the regular arrangement

RHODO-THIOBA CTERIA 1 73

is discarded and reproductive activity is intense. Colour,
red (Fig. 48).

Habitat. — Widely distributed in waters containing hydrogen
sulphide.

\

0® "^
0®

r 0© I

I- ^ ^^ i

Fig. 48. — Thiopedia rosea, x 750.

Note on "Genus" Lamprocystis.
The following " species " are given for purposes of reference.
All except the first were found by Miyoshi, and brought under
Lamprocystis by Migula.

Lamprocystis roseo-persicina.

Literature. — Kiitzing (i), 1833, (2), 1843, and (3), 1849;
Cohn (4), 1864; Fleischer (i), i860; Rabenhorst (2),
1868; Cohn (10), 1875; Warming, 1875 and 1876;
Zopf (i), 1887; Winter (i), 1884; Winogradsky (i),
1887, and (2), 1888; Engelmann (3), 1888; Migula (2),
1895 ; Bergey, 1923 ; Bavendamm, 1924.

Spherical-ellipsoidal cells, 2-i — 2-5ju. in diameter. Colour
intense violet (Fig. 49).

Lamprocystis violacea (Miyoshi), Migula.

Literature. — Miyoshi (i), 1897 ! Migula (3), 1900 ; Bergey,
1923 ; Bavendamm, 1924.

Named Thiosphcerion violaceum by Miyoshi. Spherical-
ellipsoidal cells, 2-5/Lt X i"8/x,, and capable of motility. Found
in solid families surrounded by slime. Colour violet. Angular
sulphur granules. See page 167.

Habitat. — Sulphur springs in Japan.

174

SULPHUR BACTERIA

Lamfrocystis gelatinosa (Miyoshi), Migula.

Literature. — As for preceding.

Named Thioderma gelatinosa by Miyoshi. Spherical-
ellipsoidal cells, 7/x X 5/x. Light violet in colour with covering
of slime loosely surrounding the family.

Habitat. — Sulphur springs in Japan.

Fig. 49. — Lamprocystis. A. Zoogl. of L. roseo-persic ; B. same 3 weeks
later; C. same after i month, x 1000.

Lamprocystis rubra (Miyoshi), Migula.
Literature. — As for L. violacea.

Named Thioderma ruhrum by Miyoshi. Ellipsoidal
cells, 4ju, X 2ju,. Light red in colour and surrounded by

RHODO-THIOBACTERIA 175

peach-flower red, detachable, sHmy membrane. Cocci are
motile.

Habitat. — Sulphur springs of Japan.

Lamprocystis rosea (Miyoshi), Migula.

Literature. — As for L. violacea.

Named Thioderma roseum by Miyoshi. Ellipsoidal cells,
2-5 X l'5/A. Light red in colour and covered by a thin
detachable membrane of a dull purple red colour. Angular
sulphur granules.

Habitat. — Sulphur springs of Japan.

The reasons for discarding this genus are given on page 89.

CHAPTER X.

THE INTIMATE STRUCTURE OF THE CELL IN THE
SULPHUR BACTERIA.

Introduction.

In the Eiibacteria the cell structure of the Bacillus and the
Coccus differs essentially from that of the Spirillum. In the
first two there is a definite peripheral cell membrane* which can
be separated by plasmolysis from the rest of the plasma. The
method of division is one characteristic of plant cells. When
division takes place, a transverse membrane is formed across
the plasma, and separation of the dividing cells is effected by
an equatorial fission of this transverse membrane. In the
Spirilla, division is effected in a far simpler fashion. The
plasma retracts from a zone which extends across the middle
of the cell. This appears as a hyaline space, and is occupied
by slime. The daughter cells are already organically separated,
but are held together by the slime. Complete separation is
effected by the two daughter cells pulling in opposite directions,
thereby causing the binding slime to stretch out to the point
of separation. This method of division is typical rather of
the simpler animal cells than of the simpler plant cells.
Indeed the genus Spirillum shows more affinities to the animal
than to the vegetable kingdom. The distinction may appear
to many to justify the separation of the genus Spirillum from
the class of Bacteria, but it must be borne in mind that all
bacteria are so low in the scale of life that the distinction
between an animal and a plant is not so hard and fast as in
higher organisms ; and so it is not so inconsistent to place into

* The membrane in the genus Bacillus is not Ufeless. It is the outer-
most, somewhat transformed but Hving layer of the plasma,

176

THE INTIMATE STRUCTURE OF THE CELL 177

the same group organisms, some of which show plant-Hke
affinities, whilst others are more nearly akin to animals.

Beggiatoa alba (Trevisan).

The filaments vary in length, and may be motile or non-
motile. The 7nembrane is well defined and stains more deeply
than the internal plasma. It is plastic, and offers no resistance,
either to the bending of the filament, or to the escape of solid
cell inclusions when these are subjected to outwardly directed
pressure. Plasmolysis has not been observed.

The plasma is reticulated, the meshes being irregular in
form and variable in size. Very minute granules can be
distinguished in its substance which, since they appear to be
used up during growth, are probably reserve materials compar-
able to the basophile granules of the yeast cell. The plasma
consists of strands which extend throughout the cell (Fig. 6^).

Sheath formation. — The outer layers may undergo trans-
formation into a mucilaginous material which first hardens,
and then separates from the rest of the organism. It forms a
hollow cylindrical covering to the rest of the cell, and is known
as the sheath. The formation of a similar sheath has been
observed in the life-history of the two Iron bacteria, Cladothrix
dichotoma and Crenothrix polyspora. In these it is formed
early in development, persists through life, and subsequently
hardens, thereby forming a sheath around the cells. In
Beggiatoa alba, on the other hand, although sheath formation
can be demonstrated in motile threads, its amount is so very
small and insignificant that it can be recognized only with
difficulty. It reaches full development when conditions of
life become unfavourable ; and its formation precedes the
dissolution of the cells (see p. 94). When the protoplast
under unfavourable conditions breaks up into a string of
segments, sheath formation occurs between the segments,
and when the latter ultimately disappear a ragged hollow
cylindrical sheath is left with occasional transverse bridges
of sheath material running across the hollow interior
(Fig. 6h). A similar formation of mucilage, which similarly

12

lyS SULPHUR BACTERIA

undergoes a hardening process, is characteristic of yeast cells
under certain unfavourable conditions. Former investigators
of Beggiatoa alba have not distinguished with sufficient clear-
ness between sheath-walls and cell membrane. To sum up,
the cell membrane is a living, peripheral, plasmatic covering,
whereas the sheath is a non-living transformation of the latter.
Sulphur globules of an oily consistency and high refractive
power arc found throughout the cytoplasm. The number of
globules varies, and they may even be entirely absent. The
variation in their number is evidently connected with metabolic
changes, and their absence from the filaments usually indicates
unfavourable conditions. They vary considerably in size,
some being mere points whilst at the other extreme globules
of 2/A in diameter may be found. They present a very
characteristic appearance ; a thick black ring sharply defined
appears to enclose a clear interior (Figs. 6a, 6g). Each gives
an appearance under the microscope comparable to a thick
ring of black ink drawn on white paper. It is highly probable
that the development of a sulphur droplet is preceded by the
formation of a vacuole in the cytoplasm into which secretion
takes place. Arthur Meyer and the author (Ellis (i)) have
shown that the development of the bacterial spore takes
place by secretion into a previously prepared vacuole. The
development of the sulphur globule appears to follow similar
lines. In the investigation into the intimate structure of the
cell of Thioporphyra volutans, it is shown that the sulphur
globule possesses a centrum of organic matter.

Tests for Sulphur. — Sulphur globules in plant cells have a
characteristic appearance. See Figs. 27 {a and c), 31, 32. The
centrum is hyaline. When the cells are treated with aceto-
carmine, the sulphur is dissolved and crystallizes outside the
cells in octohedra (see Figs, lyd, 34^) typical of this element.
When treated with a concentrated solution of sodium-nitro-
prusside, Na2[Fe(NO)(CN)5] . 2H2O, the rings of sulphur as-
sume a blood-red colour.

There is no evidence of a nucleus, and no colouring matter
is developed.

Motility. — The cause of movement is at present unknown.

THE INTIMATE STRUCTURE OF THE CELL ijg

It is probable that cilia are not formed, and that motility is
due to the power of contractibility which is inherent in
protoplasm, and which here finds expression.

Thioporphyra volutans (Ellis).

Each organism is composed of either a single cell, or less
frequently, of a diplococcus, or very rarely a tricoccus. The
single cell is either spherical or ovate, and its diameter is
approximately 7/x.

The membrane is well defined, and appears to be the outer
limiting layer of the cytoplasm ; it takes the colour more deeply,
apparently because its consistency is denser than that of the
cytoplasm. Its outer layers turn readily to a slimy material
which forms a loose irregular mantle enveloping the coccus.
The mantle is best observed when the cell is treated with the
Giemsa stain. It is too thin and too delicate in texture to be
regarded as a regular sheath, and it may be remarked that
most, if not all, bacterial cells of the Euhacteria possess a
similar covering. As is the case with Beggiatoa alba, solid
objects such as sulphur globules readily pass out of the cells.

The cytoplasm is reticulate and readily stained. The dis-
position of the protoplasmic matter is described on page 153.
It is finely granular, and diftused through its substance is
a purple colouring matter which varies in tint from a deep
violet to a mauve colour. It is visible in individual cells only
if the pigment is intense. There is no nucleus.

Motility is effected by means of a single long and strongly
developed cilium (Fig. 34/). Probably, as in Chromatiiim, this
cilium is composed of several cilia united by a common
envelope of slime.

The sulphur globules which are formed in the cytoplasm
may be scattered throughout the cell, or grouped at one spot.
If the cell is ovoid, the grouping is at one of the poles (Fig.
34c). In appearance, size, and reactions they resemble the
sulphur globules of Beggiatoa alba (see Fig. 34 a — d, Fig. 35 a — e).
It is only the outer portion of the globule that is composed of
sulphur. Each contains a central core of organic matter which

12 *

i8o SULPHUR BACTERIA

is stained by aceto-carmine. Compare with sulphur globules
of Thiophysa volutans. This reagent dissolves the sulphur
and stains the core after twenty-four hours. The core can
also be stained with carbol fuchsin and other reagents. It
is probably a nitrogenous reserve material of the organism.
The sulphur may be dissolved by chloroform, hot potassium
nitrate, or 50 per cent, acetic acid ; in the last named an ex-
posure of twenty-four hours is necessary. In strong picric
acid solution the globules tend to fuse to form larger irregular
drops, but complete solution does not take place. They are
unaffected by strong hydrochloric acid, and are insoluble in
water. When dissolved, and free from the cell, they frequently
combine to form typical sulphur crystals (Fig. 34^).

Thiophysa volutans (Hinze).

This is a colourless, motile organism, spherical in shape,
which was found in the Gulf of Naples, near a submarine
sulphur spring. The cocci measure 7— 18/i. in diameter

(Fig. 17)-

The membrane is double contoured and o-jjx thick. It
gives the reaction for pectin. Three distinct layers can be
distinguished with Delafield's hgematoxylin.

The protoplast consists of a very delicate hyaline layer on
the inner side of the membrane, and from it radiate fine threads
of plasma, the whole forming a delicate network. The central
portion is occupied by a large vacuole. The cytoplasm can
be separated from the membrane by plasmolysis.

The sulphur globules are insoluble in mineral acids and
alkalis, they are partly soluble in acetic acid, and very soluble
in alcohol and chloroform. In concentrated glycerine they
crystallize out, and form monoclinic crystals outside the cell,
several droplets uniting to form one crystal (Fig. lyd).

Sulphur Builders. — These are bodies ranging from 4/x to a
size when they are scarcely visible. They are ovoid or round,
are of a dull green colour, and occupy the vacuoles in great num-
bers. They stain with methylene blue, Congo red, and other
reagents ; are insoluble in i per cent, potassium hydrate,

THE INTIMATE STRUCTURE OF THE CELL i8i

and in lO per cent, sodium chloride. Hinze regarded them as
sulphur builders because they resemble the remains which are
found after the sulphur droplets have been dissolved out. His
conclusion that they are " builders " of sulphur is conjectural
(compare the sulphur globules of Thioporphyra volutans).

Other statements made by Hinze require confirmation,
especially where he distinguishes in the cytoplasm a distinct
wall which his figures do not show% and where he refers to small
bodies embedded in the wall as being chromatin. If this is so
it demonstrates the essentially living nature of the membrane.
The author (p. 189) has shown that the cilium of Chro-
matium Linshaueri is directly connected with the membrane
and does not pass through, a fact which is confirmatory of the
same conclusion.

Beggiatoa m IRA bills (Cohn).-

This organism was described by Cohn (5), by Warming from
material found on the Danish coast, and
by Engler who found it in the Kiel
Canal. It is doubtful whether all three
have described the same organism. All
describe a species that is relatively very
large, for its thickness may reach up to
45jU,, and so it is exceptionally favour-
able for the study of the intimate
structure of the cell. Cohn sketches
definite transverse membranes of a
typical plant character (Fig. 8c) ; whilst
Warming states " j'ai pu alors distin-
guer des cloisons avec des interstices
de 2-5 a S'S/u,," and depicts threads
(Fig. 8 a and b and Fig. 50) in which
it appears as though the division
of the thread into segments was not
effected by transverse walls, but rather
by plates of mucilaginous matter such
as are observed when segmentation occurs in threads of

'vd.'o','

^---.^v^^'

^m:^-^

Fig. 50. — Beggiatoa

mirabilis (Warming).

X 750.

i82 SULPHUR BACTERIA

Beggiatoa alba. Also, if Warming's drawings are correct, the
threads divide by simple fission, a process in which the forma-
tion of transverse walls plays no part.

Hinze (i — 2) examined an organism under this name which
evidently tallies with the Beggiatoa mirabilis of Cohn, but not
with that of Warming. The membrane is double contoured,
and is not composed of cellulose. The transverse walls are
thinner than the outside walls and stain with ruthenium red,
safranin, and other reagents that stain pectin bodies. Inside
the wall is a peripheral layer of plasma (Rindenschicht) in
which are numerous fine red granules which stain with hasma-
toxylin and alleged by Hinze to be chromatin. Inside this is
a large vacuole occupying the chief space of the cell and
containing a large number of solid granules which often show
molecular movements. The plasma is granular, homogeneous,
and contains numerous sulphur granules.

Chromatium Okenii.

The observations on the intimate structure of this well-
known and much-studied organism are few and unsatisfactory.

Dangeard states that a colourless membrane encloses an
alveolar cytoplasm, and that the colour is diffused throughout
the cytoplasm. He made out a " Centralkorper " which
corresponded to the Centralkorper of the Cyanophycece, and
regarded this as the nucleus of the cell. Attempts to dis-
tinguish in bacterial cells in general between a peripheral and
a central body (Rindenschicht and Centralkorper) date from
Biitschli's investigations, and his conclusions are nowadays
not accepted. If we may judge from the structure of the
allied species Chromatium Linshaueri it seems as though
Dangeard's conclusions were influenced more by Biitschli's
conception of the cell than from the evidence of structure
presented by the cell itself.

AcHKOMATiUM OXALIFERUM (Schewiakoff).

Under the various names of Achromatiimi oxaliferum
(Schewiakoff), M odder ula Hartiengi (Frenzel), and Hillhousia
mirabilis (West and Grifiiths), the intimate structure of the

THE INTIMATE STRUCTURE OF THE CELL 183

cell of this species has received close attention, especially at
the hands of West and Griffiths, and of Virieux.

The cell wall is well defined, double contoured, and is not
composed of cellulose. It is easily separated from the proto-
plast by plasmolysis. It measures 2 — 3/x in tliickness and can
be made to swell by the use of various reagents. When thus
treated the swollen outer layers show, with high magnification,
a mass covered with minute granular objects and numerous
short filaments. These filaments were at first considered to

-^,-i

\^

a b

Fig. 51.

a. — Achromatium oxaliferuni. Figure given by West and Griffiths as a copy
of one of Schewiakoff's figures of Achromatium, in which is shown a
marked distinction between peripheral and central cytoplasm.

6.— Figure oi Achromatium oxaiiferum as given by Virieux in which no such
distinction between the peripheral and central positions is shown.

be short peritrich cilia arranged in the cell somewhat after the
manner of the cilia arrangement in the Diatoms. Virieux
found, however, that they are an artefact. The cell wall is
lamellose, consisting of several firm layers with intervening
layers which become gelatinous on the addition of carbolic
acid. From its behaviour with certain chemical reagents
West and Griffiths concluded that the cell wall is composed of
a substance analogous to fungus cellulose.

The protoplast is uniform and composed of a reticulum
evenly distributed throughout the cell. In the work of earlier

1 84 SULPHUR BACTERIA

workers the protoplast of Achromatium oxaliferum is shown as
two well-marked regions, an outer which was considered as the
Rindenschicht (peripheral layer), and an inner region which was
regarded as the Centralkorper (central body). Later workers
consider that this distinction does not exist, and that the earlier
workers made the same error as we have noted above in the
investigations of Chromatium Okenii.

In the organism examined by West and Griffiths, by
Virieux, and by the author, the protoplasm showed no such
distinction (Fig. 51). The meshes are occupied each by a large
globule, varying from 6)u- to lOju, in diameter, of a calcium salt.
These are steel blue in colour, and highly refringent. Outside
the cell they crystallize as fiat rhombohedra or rhombic prisms.
West and Griffiths consider the salt to be calcium carbonate,
but Virieux states that when treated with hydrochloric acid,
it disappears without effervescence, and so considers it to be
calcium oxalate.

Virieiix's Investigation of Achromatium Oxaliferum. — This
writer gives the name of granules to the calcium globules
which fill the alveoles, and states that when they are
dissolved, refringent particles which are neither chromatin
nor metachromatic granules appear in the plasma. The
metachromatic granules are soluble in dilute acetic acid,
whereas the refringent particles are insoluble in that fluid.
These particles are further distinguished by turning red when
the cell is treated with haemalum, the rest of the cell being
violet. He gives the name corpuscles to the refringent particles
and considers that they are composed of sulphur, in spite of
the fact that his microchemical reactions for sulphur did not
give positive results. He bases his conclusion on the fact that
they increase in number when the organism is supplied with
sulphuretted hydrogen. They vary from 0-5ju, to 2/a in diameter

(Fig. 5I&).

In addition, Virieux maintains that grains of chromatin
are present which are so small that they are not visible unless
the magnification is greater than lOOO diameters. The sum
total of these particles is, according to him, the nucleus.

THE INTIMATE STRUCTURE OF THE CELL 185

AcHROMATiUM MOBILE (Lauterbom), syn. Microspira
VACiLLANS (Gicklhorn) (see Fig. 16).

The general appearance of the organism is described on
page 121. The cell is limited by a distinct membrane, which
can be separated by plasmolysis, and is almost completely
filled by two or a very small number of large globules. In
addition, there are five to fifteen smaller globules. Gicklhorn
considers the smaller to be composed of sulphur, but is uncertain
of the constitution of the large bodies. Each of these appears
to be formed by secretion into a previously formed vacuole,
which is limited by a wall. The formation of a vacuolar
wall is unknown in plant cells, and the observation needs
confirmation.

The plasma is stated to be confined to the periphery of
the cell, but there must be bands of plasma running across,
otherwise it would be difficult to account for the vacuoles ;
there must be a plasma to form a matrix in which the vacuoles
can form, for each must be sufficiently large to admit of the
formation inside it of one of the " large bodies." The cell
does not contain a well-defined nucleus.

Thiovulum Mulleri (Warming), Lauterborn.

This peculiar organism was investigated by Hinze (6) from
material obtained from the Gulf of Naples (see Chap. VIL),
and differs essentially from any of the organisms hitherto
considered, by exhibiting longitudinal cell division. The cell
is bounded by a delicate membrane, and the plasma consists
of delicate strands which, however, are completely absent
from the centre of the cell, which is occupied by a large oval
vacuole. At the thicker end of the cell is an aggregation of
plasma in which numerous sulphur globules are placed (Figs.
18-19).

In addition to sulphur, Hinze found large grey-green struc-
tures of fugitive existence which he regarded as a form of
reserve material (Fig. 18). Further, he found in the plasma
very fine granules which colour in the same way as the nucleus,
and which also he regarded as reserve food.

i86 SULPHUR BACTERIA

Cell-juicleiis. — The most remarkable feature in the cell is
a body which when stained with Delafield's haimatoxylin
exhibits a coloured centrum. This organ is regarded by
Hinze as the nucleus, and the coloured centrum as the
nucleolus. Hinze further stated that the nucleolus breaks
up into fragments before division and that nuclear division
follows the longitudinal cell division characteristic of this
organism, Hinze's figures are somewhat unsatisfactory, and
confirmation of his interesting statements is required.

Thiovulum majus (Hinze).

The cell is ellipsoid in form, and possesses a sharply
contoured membrane. Inside it, and penetrating in all
directions, are delicate plasma strings. In some cells the
plasma is massed at one of the ends, and a large vacuole
occupies the greater part of the cell. Chief among these
plasmatic inclusions are the sulphur globules, which are
soluble in 90 per cent, alcohol, and insoluble in hydrochloric,
nitric, and acetic acids. In the layer of protoplasm lining the
wall are found dull green, round, oval, or sometimes angular
plates, of varying size. They appear to increase in quantity
as the sulphur contents decrease, and are not present in all
individuals. Hinze regards them as reserve material, but
of a different nature from that found in Monas Mulleri.
They stain with Sudan, di-methyl-amido-azo-benzol, iodine
and methylene blue ; with Delafield's hsematoxylin they stain
a blue violet. The material is soluble in caustic potash, and
also in a I per cent, solution of nitric, sulphuric, and hydro-
chloric acids, but is insoluble in acetic acid.

There are also in the plasma numerous small particles which
Hinze names " chromatin granules," and which react in a
similar manner to similar particles in Thiophysa volutans and
Beggiatoa alba to which he has given the same name.

Chromatium Linsbaueri (Gicklhorn).

An actively motile pinkish-red organism, ovoid in form,
and measuring up to i5/x in length. It grows in a pond in

THE INTIMATE STRUCTURE OF THE CELL 1S7

the Epping Forest, near London, and in autumn imparts a
red coloration to the water.

Outer protoplast. — The cell is apparently delimited on the
outside by what appears to be a membrane, but as it reacts
to stains in the same way as the rest of the cell, it must be
regarded as the outer somewhat denser part of the protoplast.
Its living nature is indicated by the attachment of the cilium
to it ; if it were not living, one living part of the organism
(the cilium) would be completely separated from the remainder.
On physiological grounds this is impossible.

It is possible to detach the two protoplasts by treatment
with a saturated solution of picric acid for two weeks, when
the outer part expands, as shown in Fig. 52^. By further
treatment with weak methylene blue, the connection of the
cilium with the outer protoplast is made evident. Further,
during this procedure the inner protoplast contracts slightly,
and assumes a rounded shape, as is shown in Fig. 52 h and /,
leaving the outer protoplast as a delicate loosely folded
covering.

When the cell is pressed gently downwards, what appears
to be a darker layer at the periphery of the outer protoplast
disappears, an added proof that there is no real membrane at
the periphery of the organism, and that what appears to be
a membrane is due to the manner in whicli the light falls on
the edge of the organism. The pressure of the cell causes a
rearrangement of the light, and the " membrane " disappears.

The slime layer is from iju. to 2/x in thickness. A distinguish-
ing feature of the structure of this organism is the occurrence
in the outer plasma of one or two gaps filled with slime (Fig.
52a, e, /). They occur in any part of the cell and may be
together, or separated by the length of the cell. Although
exceptionally there may be only one of these gaps, usually
there are two, and apparently their number never goes beyond
this. Each is about 4/Li — 6/x. in diameter and oblate-spheroidal
in shape (Fig. 52^). As at such parts of the cell the inner
plasma is separated from the outside only by a layer of slime
it is possible to cause the inner plasma to project through these
slime areas by setting up differences of pressure inside the cell.

SULPHUR BACTERIA

^>i^?

-'5^i*

/

'^■^k\:yA

Fig. 52.

THE INTIMATE STRUCTURE OF THE CELL 189

An example after this treatment is shown in Fig. 526". Some-
times this can be accompHshed merely by pressing the cover-
slip laterally downwards, sufficiently firmly to cause the cell
to become somewhat flattened out. The effect of iodine on
the cell is most marked, for it causes the outer plasma and its
attached cilium to swell up in a very irregular fashion, and if
the reagent be removed before it has affected the inner plasma
it is possible to distinguish between the inner and the outer
plasma with great clearness (Fig. 52 b and/). By allowing the
cells to remain in picric acid for two weeks and then staining
with dilute methylene blue the separation of the tw^o plasmas
may be made complete as is shown in Fig. 52t/. In this in-

FiG. 52. — Chromatium Linsbatieri (Gicklhorn). x 1000.

a. — Unstained individual showing a single siime area at one of the ends.

b. — Cell treated with picric acid for two weeks, and then stained with dilute
methylene blue. The outer plasma is shown with the cilium in
attachment and the widely separated inner plasma has contracted
and resumed the rounded form natural to the organism.

c. — Cell which has been treated to make a part of the inner plasma project
through the slime areas.

d. — Cell treated with iodine, which causes irregular swelling of the outer
protoplast. The irregularity in the swelling is noticeable even in the
cilium.

e.— Normal organism showing its single polar cilium and one slime area.

/. — Cell in which the treatment with iodine has been advanced a step
further than is shown in [d). The inner plasma has begun to disin-
tegrate. The large irregular empty space marks the position of a
slime area.

stance the outer plasma with its attached cilium has evidently
been destroyed by the acid, but the inner plasma, protected
probably by the slime layer, has not succumbed but has con-
tracted and rounded itself within the destroyed outer plasma.
It is noteworthy that when this takes place the contracted
inner plasma appears to be bounded by a cell membrane.
This effect is an optical one, as can be seen when the cell is
pressed flat.

It is highly probable that the inner and outer protoplasts
are connected by plasma strands, otherwise the organism could
not function as a physiological unit. It is possible, however,
that the slime layer may in reality be a somewhat tenuous

igo SULPHUR BACTERIA

plasma, in which case it is not necessary to postulate the
existence of connecting plasma threads. The inner proto-
plast is about 4ju- in thickness, whilst the centre is occupied
by a large vacuole. The inner protoplast is not reticulated,
and frequently contains a large number of small round globules
of calcium carbonate (see Fig. 30 a and b). When the organism
is slowly rotating it can be seen that these bodies are confined
entirely to the inner protoplast. As already stated, Gicklhorn
suggests that this is an example of a new class of organisms
which he proposes to name lime bacteria. All the round globules
are not composed of lime, for whilst some (the lime globules)
do not stain with methylene blue, others do take up the stain.
The globules that take up the colour arc probably reserve
material. This is supported by the fact that their number
fluctuates, suggesting that they are first stored, and then used
up during metabolic processes.

There are also very small granules distributed in the
cytoplasm but their nature is unknown.

The sulphur globules are distributed in the plasma and when
present their number varies considerably.

The colouring matter is pinkish-purple in tint and diffused
uniformly throughout the plasma, a detailed account of the
pigment is given in Chap. XIII,

Summary and Conclusions.

The intimate structure of the cell in the sulphur bacteria
shows the same primitiveness as the rest of the Schizophyta.
There is less differentiation than is found in the cells of the
higher Fungi, or of the Algas. With one exception [Beggiatoa
mirabilis), the outer envelope or "membrane" is plasmatic,
and distinguished from the inner plasma only by its greater
density. It takes up the same stains, and in the one species
tested, namely, Chromaiium Linsbaueri, is the base to which
the cilia are attached.

Division throughout the group is by the simple fission
characteristic of the Schizophyta. The cells when divided
maintain connection for a period by a bridge of slime, but

THE INTIMATE STRUCTURE OF THE CELL 191

later draw away, and effect complete separation. The trans-
verse walls that are seen in such organisms as Beggiatoa alba
and Thiothrix are not plasma-derived. They are transverse
bands of slime that have formed between the dividing cells,
and owe their origin to the transformation of the outermost
layers of the cell into slime.

Slime Formation. — This is well developed in many of the
sulphur bacteria, although when the organisms arc actively
motile its amount is very small. The slime, when formed,
covers the whole organism with a closely folded mantle, which
subsequently hardens. In Thiothrix the slime hardens early
and forms a permanent sheath enfolding the inner cells, which
continue to grow, and to be successively thrust out of the
sheath. In Beggiatoa alba excessive sheath formation seems
to occur only under unfavourable conditions. The production
of slime by a mass of clustered cocci leads to the zoogloea
condition, and when the cocci multiply inside the slime an
appearance is presented of a totally different organism from
that from which the cocci were derived. This has led, in the
author's opinion, to the erroneous formation of new species,
and even of new genera.

Slime formation in some cases has become fixed to the
extent that the cells of an organism pass their whole existence
inside a slime covering. This is the condition of such bacteria
as Thioploca, Thiopedia, and Thiodictyon. It may be presumed
that a continuance of this colonial habit would in time lead to
the differentiation of the cells, and the evolution of a multi-
cellular organism. Such an advance, however, has not been
accomplished by any of the sulphur bacteria.

The Question of the Nucleus. — There is no readily demon-
strable nucleus in any member of the sulphur bacteria. Two
claims have been made, both of which need confirmation. It
will not be possible to prove or disprove Virieux's claim until
the behaviour of the small bodies, alleged to be the nucleus,
is observed during cell division. Hinze's claim that a nucleus
is present in Monas Miilleri rests on a somewhat sounder
foundation, and if it can be confirmed that the body that he
regards as the nucleolus breaks up into fragments during cell

192 SULPHUR BACTERIA

division, and that such fragmentation is associated with cell
division, his view would be considerably strengthened. This
organism, however, is far from being a typical sulpliur microbe,
and in all probability should find a place among the Flagellates.
It is noteworthy that even in such large cells as Beggiatoa
mirabilis, in which, if present, the typical nucleus would be
a prominent object, there is no trace of it.

The cytoplasm does not call for special mention, except to
stress the fact that in no single instance is there a differentiation
between a peripheral and a central cytoplasm based on a marked
difference in the size of the vacuolar meshes.

Cell Inclusions. — Sulphur appears to be the inclusion which
is common to all these organisms. As they flourish under
marine, brackish, and fresh water conditions, it is probable
that the total number of metabolic products is very large,
and that the different organisms show considerable variety in
this respect. The chemical analysis of the sulphur bacteria
on a large scale has yet to be made.

Interesting inclusions are the lime granules of Chromaiium
Lmshaiieri. In this organism also are small round bodies
which may be stained with methylene blue, and which are
probably protein reserve products. The minute granules
that are found in Beggiatoa mirabilis, Beggiatoa alba, and
Thiovulum majiis, are probably of the same nature.

The core to the sulphur granules which was found in
Thioporphyra volutans appears to be the same as the " sulphur
builders " which Hinze found in Thiophysa volutans. It is
probable that in all sulphur bacteria the sulphur globule has an
organic core, and that it plays an important role in the meta-
bolism of the sulphur bacteria.

The apparent secretion of the sulphur globule into a vacuole
recalls the mode of formation of the spore in the genus Bacillus ;
see A. Meyer, and Ellis (i), and the formation of the spore in
the genus Sarcina (Ellis (i)).

CHAPTER XI.

IRRITABILITY; INFLUENCE OF LIGHT;
CHEMIOTACTIC PHENOMENA.

Irritability.

hitrodiiction. — Irritability, or the response of a living
organism to external influences, is characterized by a move-
ment of an organism as a whole, or in part, either towards
or away from the source of influence. Capacity for move-
ment is one of the properties of protoplasm, and is as general
among the lower plants as the lower animals. The higher
plants are rooted by their habit to the soil, and so are bound
by their physical conditions, but even they make a partial
response to external influences. For example, when young
cress seedlings are made to grow at the dark side of a room
their stems assume a horizontal position in their endeavour
to reach the light. Movements either of attraction or of
repulsion may be caused by light, gravity, water, injury, and
various chemical substances. The movement is usually, but
not invariably, one that is distinctly beneficial to the plant.
As an exception may be mentioned the movement of certain
bacteria towards corrosive sublimate which is toxic to them.

Much has yet to be learnt about the response of the sulphur
bacteria to external influences, but there are considerable data
on the effect of light and of various chemicals in inducing
movements. Some of the coloured bacteria of this group are
probably the m.ost sensitive of all organisms to light, and the
response both of coloured and of uncoloured sulphur bacteria
to certain chemical compounds is very marked.

193 13

194 SULPHUR BACTERIA

Methods of Investigation.

Light. — The procedure in determining the influence of light
is simple. The organisms are exposed to light of varying
intensity and direction. By projecting a spectrum on to the
organisms their movements through the different colours
may be noted. In various simple ways it is also possible to
expand or contract the length of the component colours of the
spectrum, and to alter their intensity. In the examination of
the effect of colour elaborate apparatus has been used which
will be explained later.

Chemicals. — Pfeffer's Capillary Tube Method is used for the
determination of the effect of chemicals on the sulphur bacteria.
A piece of capillary tube of approximately 0-05 mm. bore by
I cm. long is sealed at one end, filled with the salt solution under
investigation, and completely immersed in the fluid containing
the bacteria. If sensitive, the organisms move either towards
the open end of the tube, or away from it. Some of the
movement is due to diffusion between the two fluids, but its
extent can be estimated by control experiments. The move-
ments usually occupy one to several days before they are
completed, and by a system of controls it is possible to allow
for all other influences except that due to the fluid under
investigation. By using a fluid which does not contain
nutrient matter multiplication of the bacteria is avoided. The
entrance of the bacteria into the capillary tube can be easily
followed when viewed under the microscope.

Influence of Light on the Coloured Sulphur Bacteria.

The colourless sulphur bacteria are indiftcrent to light,
whether they are cultivated in the dark or in the light. On
the other hand, the coloured sulphur bacteria are extremely
sensitive. Light affects plants in various ways. A distinction
must be made between the tonic, the directive, and the photo-
synthetic effect of light. An organism like Volvox, for example,
is influenced by light in three different ways. Light is necessary
for its continued health, and so exerts a tonic influence ; its
direction of movement is determined by the line of incidence

IRRITABILITY ; IXFLUEXCE OF LIGHT 195

of the light ; and, finally, light supplies the energy for_ the
carbon-assimilation which is characteristic of Volvox as of
all green plants. The photosynthetic effect of light is not
a phenomenon of irritability, and is discussed on page 204.
There have been no specific investigations into the tonic eftect
of light on the sulphur bacteria, but it may be presumed that
it operates in producing a state of well-being in the coloured
sulphur bacteria, for, so far as is known, no multiplication and
no pigment formation take place in the dark. It is impossible
to separate altogether the tonic from the photosynthetic effect
of light. The absence of colouring matter in the sulphur
bacteria from which light has been withheld may be the cause
of their lack of tone. It is responsible for their failure to
multiply, and so it is impossible in many cases to state whether
there is a tonic effect of light on these bacteria apart from
the deleterious efiect produced by the absence or loss of pig-
ment formation. In some cases, however, a distinct tonic
action of light may be noted. Thus it is shown in Engel-
mann's investigation that when motile coloured sulphur
bacteria lose their motility through the withdrawal of light,
they do not assume motility when they are once more exposed
to light if they have been kept too long in the dark. This
failure is to be attributed to the loss of tone by the withdrawal
of light, and to the same cause must be attributed the fact that
exposure to constant light intensity is injurious, for motile
organisms stop moving altogether unless slight changes are
made in the intensity of the light.

The most notable investigation on the reaction of the
sulphur bacteria to light-intensity and colour is that of Engel-
mann (3).

Engelmann's Investigation : Effects of Changes in the
Intensity of Light.

Engelmann used for his experiments a number of species
which cannot now be identified. Among these was the very
sensitive Bac. photometricum (Engelmann), which was possibly
a pleomorphic form of Lankesteron.

13 *

ig6 SULPHUR BACTERIA

The following is a summary of his conclusions : —

1. Within certain limits, a direct correlation exists between
the velocity of the organism and the intensity of light. The
dependence of one factor on the other is more marked at
low oxygen pressures.

2. The movement completely stops when the organism is
left in the dark beyond a certain period, and if kept in the dark
beyond a further period it will not resume movement when
again exposed to light.

3. Different species show responses which differ both in
kind and in degree. An increase of intensity produces different
rates of increase of velocity in different species. In some
species an increase of intensity may even result in a decrease of
velocity.

4. The change of velocity is influenced by the amount of
HgS present.

5. Exposure to light of constant intensity ultimately
effects a stoppage of movement. When a quiescent organism,
kept in the dark, is exposed anew to light, movement does not
begin until a certain interval elapses. Engelmann called this
delay in response Photokinetic Induction. The converse pro-
cess, namely, the cessation of movement as a result of the
removal of light also takes place after a slight time interval.
This was named Photokinetic After-effect.

These delays in response to a given stimulus are comparable
to the " presentation time " which elapses in roots of higher
plants before geotropic curvature takes place.

Shock Movements (Schrenk Bewegungen).

The curious effect was observed in many of the sulphur
bacteria that the sudden removal of light resulted in first a
stoppage, then a slight backward movement, and finally a
forward movement once more, but with diminished velocity.
The organisms react as though they had received a slight shock
from the sudden change. The shock becomes weaker on repe-
tition, and is much less pronounced when it takes place in
a medium rich in oxygen. The extent of the reaction differed

IRRITABILITY : INFLUENCE OF LIGHT 197

according to the suddenness of the change from Hght to dark-
ness. Again all the individuals were not affected to the same
extent, and some failed to respond altogether. Similar
effects were obtained by causing the organism to traverse
from one colour to another. If the intensity of light in the
spectrum is great, the organism travels from one end of the
spectrum to the other with undiminished vigour, but if the slit
be made very narrow, shock movements are observed in
passing from one colour to another. They were observed in
the passage from beyond infra-red to infra-red ; from infra-red
to red ; from yellow to red ; from yellow to green ; from green
to blue ; from blue to violet.

With one exception the reactions occurred when the
organisms passed from a colour of greater to one of lesser wave-
length. Engelmann did not apparently observe the effect
of a passage from a colour at one end of the spectrum direct
to one at the opposite end, but he noted that shocks were
observed in passing from any colour to the dark, and that the
shock was not so pronounced when the organism entered the
dark from either extremity of the spectrum.

Shock Movements a form of Preservation :
Valve Action.

Engelmann maintained that the shock movements exercised
a self-preserving effect on the organisms, as their tendency was
to prevent their passage into the
dark. Thus an individual moving in
the dark along the line ah (see Fig.
53) experiences no shock on reaching
the light, but when it crosses the
area of light and enters the dark
zone on the other side a shock
movement occurs which brings the
individual back once more to the pj^ _^

lighted region. The influence of

light will now operate to prevent it once more taking
up the path ah which would lead it into the dark. If the
Hght is strong enough the reversed movement caused by the

igS SULPHUR BACTERIA

shock can be maintained and the individual remains in the
hght. When next it crosses the hghted area and once more
reaches the dark zone on the other side a similar shock
causes it to reverse again, thereby preventing its passage
into the dark zone. The effect of the operation of these
factors may be observed by covering a microscope field
containing motile purple bacteria with a disc which darkens
the whole field except for a restricted region. The organisms
tend to collect in this region to the exclusion of the rest of
the field. Engclmann compared these shock movements to
the action of a valve, presumably because they exercise a
certain measure of control on the direction of movement. The
analogy is not a happy one, for the essence of valvular action
is regulation, and there is no question of regulation of move-
ments in the shock phenomena. It is doubtful whether the
incidence of these movements is a factor of sufficient importance
to affect materially the natural striving of the organism to-
wards the light when it is about to pass from the light into
the dark.

MoLiscH ON Shock Movements.
Molisch experimented with Rhodospirillum photomelricum*
an organism so sensitive that the effect of passing the hand
between the light and the mirror produced a shock movement.
Molisch was convinced that the colouring matter was closely
concerned in the production of these movements, and that in
a field of various microorganisms, including the purple bacteria,
these could be recognized by their shock movements, even
although the colouring matter was not present in sufficient
quantity to be perceptible. He confirmed Engelmann's
statement that shock movements are more pronounced in an
atmosphere containing only a very small quantity of oxygen.

Distribution of the Purple Bacteria in the various
Colours of the Spectrum.
Engelmann found that when a spectrum was projected on
to the microscope field, the purple bacteria in that field

* This organism was probably some pleomorphic form of one of the
better-known sulphur bacteria.

IRRITABILITY : INFLUENCE OF LIGHT 199

arranged themselves in greatest concentration in the infra-red
(between hnes * 80 and 90). There was a distinct but smaller
concentration in the orange-red (between lines 54 — 64), and a
still smaller gathering in the green (between lines 52 — 55), whilst
a few also collected in the hlue-violet. The organisms avoided
the other colours. It is shown below (p. 200) that the region
of maximum concentration of these bacteria, namely the infra-
red, is also the region of maximum absorption of energy. Engel-
mann considered that the correspondence was significant,
and that oxygen was liberated in that zone because photo-
synthesis was carried on by the purple bacteria. Here,
however, different wave-lengths were utilized for the absorption
of light energy. The significance of these results will be dis-
cussed later.

Effect of Colour on the Absorption of Light.

In order to examine the absorption spectrum of the pigment
found in purple bacteria, it is necessary either to prepare an
emulsion of the organisms in water, or to extract the colouring
matter with a suitable solvent. An extract with chloroform
or carbon bisulphide is purple in colour ; with ether or alcohol
it is yellow or yellowish-green.

Engelmann used an aqueous suspension of Bac. photo-
metricum and determined the exact amount of absorption at
different wave-lengths by the use of Langley's Bolometer.f

Figures on the following page show the results obtained
by him.

A gives the wave-length of the different colours.
E gives the percentage of energy that is transmitted after
the light has traversed the coloured fluid.

* See page 200, footnote.

t The Bolometer is an instrument used for estimating the total energy
contained in any part of the spectrum. As it travels along the spectrum
it picks up the energy, with the result that it causes an alteration in the
resistance of an electrical wire with which it is in attachment. The amount
of alteration in the resistance is measured by a galvanometer.

SULPHUR BACTERIA

infra-red -

A.

E,

I -bo*

94-4

1-40

Q4-8

I-OO

78-3

•95

69-5

•qo

44-2

•^5

2-91 -^ ist max.

•So

30

•70

69

•68

77

•66

80

•64

84

•62

77

•60

40

•59

27 > 2nd max

•5«

28

•57

28

•55

18

•53

9-5

•5^

40-5

•50

Q-O

•48

IO-5

•46

I2-0

•44

17-0

Thus the point of maximum absorption is at wave-length
85 in the infra-red, which is beyond the end of the visible

100
90^-

80

GO

50-

40|-

30

20

10

. I I I I [1 Y"

HO

130

110 100

90 m

>A

70 60 50 40

Fig. 54. (A.fter Engelmann.)

spectrum. A second maximum, and a third, appear at 59
and at 50 — 52, in the orange and green-blue, respectively.

In the accompanying graph (Fig. 54) the ordinate gives
the energy absorbed (E'), In Engelmann's table the figures
give the proportions transmitted (E).

*The figure i-6o = i-6 x 10,000 Angstrom units. A = 16,000 x
io~^ cm.

IRRITABILITY : INFLUENCE OF LIGHT 201

Liberation of Oxygen by the Purple Bacteria on
Exposure to Light.

Whilst direct proof of the Hberation of oxygen is still
not available, strong presumptive evidence of its liberation
is supplied by the following facts, which were elicited by
Engelmann.

I. A colony of sulphur bacteria in the zoogloea condition
was introduced, together with a strongly aerobic
spirillum, into a drop of water. The spirilla collected
round the colony if the drop was poor in oxygen,
but did not do so if the drop was well aerated. If
the space surrounding the drop was filled with
hydrogen the spirilla showed a greater tendency to
collect around the zoogloea.

Fig. 55.

2. Variations in the pressure of oxygen were correlated with

variations in the activity with which the movement
of the spirilla towards the zoogloea took place.

3. In one observation of a drop of water which had been

kept for some time in the dark and then brought
into the light, a number of spirilla were observed in
position round the circumference of an air bubble,
and near the bubble there was also a purple organism
[Monas vinosa) (marked a in diagram). When the
drop was rich in oxygen the purple organism had no
particular attraction for the spirilla (Fig. 5 5 A),
but when it was poor in oxygen they distributed
themselves as shown in the B position.

4. Many of the actively motile sulphur bacteria are

attracted by oxygen. If a drop of water containing

202 SULPHUR BACTERIA

the bacteria is placed under a coverslip and left in
the dark, the organisms take up a position varying
from \ mm. to 2 mm. from the edge. If the drop is
now exposed to the light, the bacteria scatter in all
directions. This is consistent with the explanation
that they had produced oxygen in the light, so that
their movements were less restricted.
5. A fluid containing purple bacteria was placed in a glass
tube 5 cms. high, and exposed in a vertical position.
After five months the fluid was colourless except at
the bottom, where there was a layer of the bacteria
2 mm. high. By attaching the tube to a hydrogen-
generating apparatus the pressure of oxygen was
diminished. If this apparatus was now placed in
the dark the organisms scattered throughout the
field, but if in the light they collected at the bottom.
In this case the inference is that in the light so much
oxygen is developed that its concentration is greater
than the optimum for these bacteria, and so they
move as far as possible from the surface. The effect
in this experiment of introducing oxygen is to cause
repulsion of the bacteria, a result which is the
opposite of that obtained in the previous experiment.
It must, however, be borne in mind that the sulphur
bacteria are attracted to oxygen up to a certain
concentration, and therefore it must be presumed
that in the previous experiment the concentration
of oxygen was below the optimum, whilst in the
present experiment that point had been passed.
The facts, however, have been challenged by subsequent
investigators. Molisch (3), working with pure cultures of
purple bacteria found that oxygen was not liberated in
sufficient quantity to collect in fermentation tubes. Further,
he found that the purple bacteria had no noticeable effect on
motile bacteria sensitive to the presence of oxygen, when such
bacteria were cultivated in association with purple bacteria.

He also stated that the oxygen which is necessary for the
manifestation of phosphorescence by phosphorescent bacteria

IRRITABILITY : INFLUENCE OF LIGHT 203

cannot be supplied by growing the purple bacteria along with
these organisms.

Both Molisch and Winogradsky have concluded that the
oxygen in Engelmann's experiments was derived from green
organisms accidentally present in the medium. It is evident
that the whole subject must be reinvestigated, before Engel-
mann's observations can be accepted. In his favour it may
be mentioned that Molisch's results were all negative, whilst
those of Engelmann were positive. In one experiment at
least the effect of contamination with green alga; can have no
part ; in the third experiment mentioned the bacteria are
shown clustered round a single individual of the purple
bacteria, and here there can be no question of the interference
of green organisms.

Relationship of Light to the Growth of the Purple

Bacteria.

Engelmann sought to establish a relationship between
growth and light from the following experiment. When
tubes containing sea-wrack infected with purple bacteria are
left in the dark they remain colourless. When other tubes
of a similar kind are exposed to light the colour deepens.
When now the first set is exposed to the light the colour
develops after only a few days, and if the second set is now
placed in the dark the colour disappears. It is possible to
put another interpretation upon these results, for the mass of
individuals of the purple bacteria may remain stationary in
point of numbers, and develop or lose colour according to
whether they are placed in the light or the dark. Thus proof
is supplied of the dependence of colour on light but not neces-
sarily of the measurement of growth by colour. However,
Winogradsky (i), Molisch (3), and Skene have shown con--
clusively on other grounds that the growth of these organisms
is dependent on light. The dependence is established by the
fact that the spread of the purple colour to cover fresh material
readily takes place in the light but not in the dark. Skene
cultivated purple bacteria in different coloured lights and

204 SULPHUR BACTERIA

showed that the relative efficacy of daylight, red and blue
light was about as 6 : 3 : i respectively.

The Function of Light : Photosynthesis.

It is universally agreed that light is essential to the sulphur
bacteria, but there is considerable diversity of opinion as to
its precise function. It has been maintained by Engelmann
and others that light is a source of energy for an anabolic
process comparable to the photosynthesis of green plants. It
will be recollected that the chlorophyllous plant tissues utilize
solar energy for the synthesis of carbohydrates from carbon
dioxide and water. During the process oxygen is liberated
as a waste product.

Now Engelmann has demonstrated that the purple bacteria
liberate oxygen in the light, and it is from this that he deduces
that a similar photosynthetic process takes place in them.
Both Engelmann and Molisch have made the liberation of
oxygen the test of photosynthesis, whereas the essential proof
is the demonstration of the synthesis of more complex from less
complex compounds. Engelmann proclaimed the occurrence
of photosynthesis chiefly because of the liberation of oxygen,
whilst Molisch denied its occurrence because he considered
oxygen was not liberated. It is possible to imagine a photo-
synthetic process in which oxygen is not liberated.* Hence
as neither advanced proof of synthesis the question must
still be regarded as an open one. Subsequent opinions on
the occurrence of photosynthesis have mainly centred on the
question whether oxygen is liberated, and are therefore not
relevant.

Whilst definite proof of photosynthesis is not available the
following facts form strong indirect evidence of its occurrence
in the purple bacteria.

* As an analogous case may be mentioned the fact that respiration
is in essentials the katabolism of complex compounds, and not only may the
process be imagined without the intake of oxygen, but there are actually
organisms (the anaerobic bacteria) in the respiration of which oxygen
is not taken in.

IRRITABILITY : INFLUENCE OF LIGHT 205

1. The indispensability of light for the growth of the

purple bacteria, and for the development of the
colouring matter.

2. The fact that carbon dioxide is the source of carbon of

these organisms.

3. The fact that the part of the spectrum which absorbs

the greatest amount of energy is also the part at which
it is highly probable oxygen is evolved.

4. The known use of the light in this manner in green plants.
Molisch (3) has advanced the opinion that light is used

to break up the organic matter which he claims is absorbed by
the purple bacteria. He brought no experimental evidence
in support, and the opinion is evidently unsound, for the
purple bacteria do thrive in the complete absence of organic
matter, as has been shown by the investigations of Skene,
Bavendamm, and others, and the necessity of light is as great
in the absence as in the presence of organic matter.

If photosynthesis does occur in the purple bacteria it is
of special interest, for whilst in the green plants the red and
the green constituents of light are the most effective, in these
bacteria the most active rays are in the infra-red. It is of
interest that di|Terent organisms use light of different wave-
lengths and suggests that light of other wave-lengths may
be utilized by other organisms of whose physiology we have at
present little knowledge.

The Directive Effect of Light.

Engelmann stated that light exerted no directive influence
on the purple bacteria, but Winogradsky maintained that
there was such an influence, because the purple bacteria
collected on the side of the culture vessel which was turned
towards the light. This statement is, however, incomplete,
for it is a matter of common observation that in intense light
they collect with equal readiness on the side removed from the
direction of light. Also it may be pointed out that the
appearance of colour does not alone indicate movement,
for it may appear as a result of the development of pigment

2o6 SULPHUR BACTERIA

in organisms that were hitherto colourless or in which the
colour was feebly developed. In such a case there would be
a development of colour without mass movements of the
organism in any particular direction. Hence colour may
develop on the side of the culture vessel towards the light
because the individuals on that side have been stimulated
by the light to develop the pigment to a stronger degree.
Winogradsky also attributed the shock movements to the
directive effects of light, but the statement was made from
general observation, and the experimental data given above
do not support this view. Beijerinck investigated a species
of Chromatiuni and found that it swam towards the point of
maximum light intensity. This observation is not correct,
for, as already stated, in bright light the bacteria collect on
the side of the culture vessel which is away from the light.
Beijerinck's statement would be true only if he experimented
in light of low intensity. Molisch (3) came to the conclusion
that light exercised no directive influence on the purple
bacteria.

The whole subject needs reinvestigation.

Summary of the Effects of Light on the Purple Bacteria.

The ionic effect is indicated by the necessity of light for the
development of colour, and for the growth of the bacteria, and
by the fact that the resumption of movement in the light is
not possible if the organisms are previously kept too long in
the dark. Also, the need of a slight change in the intensity
of light to keep the bacteria at their highest speed is indicative
of this tonic effect.

The directive or phototactic eft'ect of light on organisms in
general is of two kinds. There is a directive effect which
makes the organisms change the inclinations of their direc-
tions so as to bring them either nearer or farther away from
the source of light. There is no evidence that the purple
bacteria respond in this way. On the other hand, there is
ample evidence that changes in the intensity or in the colour
of light produce the second effect, namely, a complete

IRRITABILITY ; INFLUENCE OF LIGHT 207

reversal in the direction of movement. The conditions deter-
mining such reversals have been described above.

The photo synthetic effect of light has not been proved.
Reasons have been given for , the conclusion that in all
probability some form of synthesis does take place through
the agency of light.

The Effect of Chemical Substances in Changing the
Direction of Movement.

Aliyoshi's Experiments. — The table on next page gives the
results of this investigator with Pfeffer's Capillary Tube
Method.

The absence of controls in these experiments somewhat
impairs the value of these results, but there is no doubt of the
sensitiveness of the bacteria to peptone and flesh extract
with which the most marked results were obtained.

Bengt Lidforss found that even very minute quantities
of ethyl alcohol attractecj certain bacteria. He also found that
a colourless sulphur spirillum was attracted by a dilute
solution of sulphuretted hydrogen, but that this compound
was poisonous in concentrated solutions. It is inferred that
the organism was presumably repelled by more concentrated
solutions. A substance may, however, be poisonous to bac-
teria and yet exercise attraction for bacteria, as for example
corrosive sublimate.

Sodium thiosulphatc was also found to attract bacteria
in dilute solution.

Molisdi's Experiments. — The results obtained by Molisch
gain an added value from his rigorous use of conti'ols.
The methods of experimentation were the same as those
used by Miyoshi. He estabhshed the important point that
ditierent purple bacteria may react differently to the same
reagent. Thus Rhodospirillum giganteiim is attracted by
weak hydrochloric acid, whilst Rhodospirillum photometricum
and Chromatium are repelled. Again, Rhodospirillum gigan-
teiim is sensitive to carbon dioxide, hydrochloric acid,
dextrin, sucrose, and peptone, whilst Chromatium is in-

2o8

SULPHUR BACTERIA
MIYOSHI'S TABLE.

Concentration of

Name.

Solution
per cent.

Result.

Remarks.

Sulphuretted

Weak

Indefinite

hydrogen

Strong

+

Results sometimes negat-
ive : must be regarded as
inconclusive.

Pot. nitrate

0-3

+

Slow entrance : crowded

Other strengths

Indefinite

mass of bacteria moved
slowly inwards at rate of
1-5 mm. per hour.

Am. tartrate

0-5

+

Bacteria appeared at
mouth, then moved slow-
ly inwards. Many re-
mained outside. After a
month in the dark inside
the tube, the bacteria
mo^•ed still farther up
when exposed to the light.

Pot. dihydrogen

phosphate

0-3

+ + +

(KH.POJ

0-8

Doubtful

Sod. chloride

0-3
o-S

0-3

+

Am. chloride

+

0-3

—

Am. sulphate

0-3

+

Mg. sulphate

0-3

—

Peptone

0-5

+ +

Fle.sh extract

0-5

+ + +

Sucrose

0-5

+ +

Glucose

0-5

+ +

Asparagin

0-5

+ +

Glycerine

0-5

+ +

Baking soda

0-3

—

Pot. chlorate

0-3

_

Alum

0-3

—

Acetic acid

+ +

Lactic acid

+ +

Mono-, di-, and tri-

hydric alcohols of

fatty series

+ +

Aldehydes

+ +

Ketones

+ +

Tri-hydric alcohols

Tetra- and Penta-

hydric alcohols

O

The -\- sign indicates a movement towards, the — sign a movement
away from, the source of stimulus.

different to all these substances. Further, Rhodospirillum
giganteum is attracted, but Chromatium repelled, by sulphur-
etted hydrogen (concentration not stated). He also established
the important fact that for every reacting substance there is a

IRRITABILITY

IXFLUEXCE OF LIGHT

log

particular range of concentration within ivhich the organism is
sensitive. Above this range the fluid may injure, or even
destroy, the organism, whilst below this range there is no
reaction. These observations are in general agreement with
the results of similar tests on the effect of chemical substances
on spermatozoids, and small organisms. Molisch's results
are tabulated below. The organism used for experimentation
was Rho do spirillum giganteum. In the third column the
signs +, — , and O, have been inserted from Molisch's descrip-
tion, and were not so set down by the investigator himself.

.\ame.

Percentage Strength
of Solution.

Reaction.

Remarks.

Carbon dioxide

+ +

+

The bacteria stream in and
remain inside for some
hours, after which they
stream out again until
the distribution is uni-
form throughout.

Hydrochloric
a.cid

0-005
o-oi

+ +1

+ 4-

Rhodosp. photometricum is

O* I

1 1

L

negative at concentra-

Greater
concentrations

.

tions of O-OI — o-ooi per
cent.

Sulphuric acid

0-005

+

=:

Rhodosp. photometricum is

0-0025

H-

>•

indiflerent at all con-

o-oi

0

centrations.

Nitric acid

001

+

Acetic acid

o-i

O-OI

+

"1

Reactions variable, some-

0-005

_u

/

times caused repulsion.

Caustic potash

O-I

—

Same result with Chrom-
atium.

Potassium

I -00

+

Chromatium weakly posi-

chloride

tive.

Dextrin

I-O

+ + + +

Chromatium weakly posi-

tive.

Cane sugar

I-O

+ +

+

Chromatium indifferent.

Peptone

10

+ +

+

Chromatium indifferent.

Sulphuretted

not given

+ +

+

Chromatium (marine

hydrogen

species) indifferent.

Irrit.ability and Environment.

It was maintained by Kniep, who experimented with a
colourless sulphur organism and with Spirillum rubriini,
that the reaction of an organism to a particular salt is in-
fluenced by the presence of other salts. Thus Spirilhim

14

2IO SULPHUR BACTERIA

riibruni is sensitive to sodium chloride, and also to ammonium
chloride. But if, in Pfeffcr's experiment, the water in the
capillary tube contains both i/iOO sodium chloride and l/iOO
ammonium chloride, and if the bacterial suspension outside
the capillary tube contains l/iOO sodium chloride, there is no
attraction. The presence of sodium chloride has destroyed
the efficacy of ammonium chloride. Similarly ammonium
chloride neutralizes the efficacy of sodium chloride. The same
held true for other combinations of salts, for example, potassium
sulphate and ammonium sulphate.

Buder's Researches.

[a] Ciliary Movements of the Sulphur Bacteria. — -Very
interesting results were obtained by this investigator in

Fig. 56. — Positions of the cilium of a spirillum moving forward with the
ciliiim attached behind. For explanation see text.

his examination of the cilia and the movements of Thio-
spirilhmi jenense (see p. 161), and a species of Chromatium.
Buder found that the single cilium shows a rotary movement.
The positions of the cilium when the organism is moving
forward with the cilium behind are shown in Fig. 56.

The shaded parts show a section of the space enclosed
during the revolution of the cilium. It will be seen from the
diagram (Fig. 56) that the shape of this space is, during move-
ment, continuously undergoing alteration, and that the
alteration takes place in a definite order. In No. 4 of the series
the cilium is rotating almost at right angles to the spirillum.
This is the position which it assumes prior to that indicated in

IRRITABILITY : INFLUENCE OF LIGHT 211

Fig. 57- The cilium then revolves round the hinder part of
the body and propels the spirillum in the reverse direction.

[b) Reaction to Light.- — The reaction following the exposure
of an organism to light of a given intensity depends on the in-
tensity of the light to which the organism was previously ex-
posed. There may be, for example, a reaction when the liglit
intensity is increased from 18 units to 20 units (units arbitrarily
chosen), but not from 40 to 42, although the difference in inten-
sity, namely, 2 units, is the same. Before a reaction due to a
change of intensity occurs, the new intensity must be a certain
percentage greater than the original intensity, and so the greater
the original intensity the greater the number of units which nfiust
be added to raise the percentage to the required point. The
susceptibility of the human eye to a change in the intensity of

Fig. 57.

light is formulated in Weber's Law, which states that before
the eye can appreciate a change in the intensity, the increment
of light units must bear a certain ratio to the original intensity,
and this ratio is independent of the value of the intensity.
Weber's Law does not hold for the reactions of Thiospirillimi
jenense to light, for whilst in some cases an increase of 10 per
cent, on the original intensity was required to produce the effect,
in other cases as much as 25 per cent, was necessary. Again,
according to Buder, the changes in the direction of move-
ment in response to changes in the intensity of light are not
invariably as sudden as was claimed by Engelmann. The
length of time required for the completion of the reaction
depended on the velocity of the organism at the moment, the
length of time taken by the light to change its intensity, and
on other factors, so that under some conditions quite an

14 *

212 SULPHUR BACTERIA

appreciable interval of time elapses before the reaction is
completed.*

[c] Experiment on Shock Movements.- — -An interesting con-
tribution to our knowledge of shock movements was made
by Buder by an experiment which is illustrated in Fig. 58. In
No. I a spirillum with a single polar cilium is shown moving
towards a dark patch. Nos. 2, 3, and 4 show its further progress.
At No. 4 three-quarters of the organism, but not the sensitive
region, which is at the base of the cilium, is inside the dark patch.
In Nos. 5, 6, 7, and 8, the dark patch is made to move in the

^^

9 10 11

' ! )

Fig. 58.

same direction as the spirillum, to prevent the sensitive zone from
entering the dark patch. No shock movement occurs, because
the sensitive zone is still outside the dark patch. At No. 9,
however, the sensitive zone is shown for the first time inside
the dark patch. The result is shown in No. 10, a shock move-
ment having caused a strong recoil into the light. Whilst
the spirillum! is recoiling the dark patch is made to follow at
a greater rate than the spirillum, and thus to overtake the
organism. This phase is shown in Nos. 11-15. At No. 15
the dark patch overtakes the sensitive zone. The result here
was that the organism executed another shock movement,
which caused it to move forward again even although by so
doing it moved still farther into the darkness. Hence shock

* The author considers that only failure will result from the attempt
to express vital phenomena in terms of a strict mathematical law.

IRRITABILITY : INFLUENCE OF LIGHT

213

movements do not invariably have the effect of preventing
the organism from moving into the dark.

{d) Localization of Area of Sensitiveness. — Buder has deter-
mined the part of the organism which is sensitive to Hght
by the following experiment. A microscope field containing
Thiospirillum jenense was completely darkened except for a
small rectangular area. By an ingenious arrangement this
patch of light could be set in

any part of the field, and
made to move in any part of
it. In the illustration two
spirilla (A and B) are shown
(see Fig. 59). In B the cilium
is in front, in A it is behind.
In one experiment the lighted
patch was made to move to
the left at a greater speed
than that of the spirilla mov-
ing in the same direction, with
the result that the line CD
overtakes the spirilla from
behind. When this line over-
takes an organism like A with

the cilium behind, the response is sooner than when it over-
takes one like B with its cilium in front. But in both cases
the response, which in this case is a faster movement in the
same direction, follows the passing of the line CD over the
base of the cilium. Buder thus demonstrated that the base
of the cilium is the seat of sensitiveness to changes in light
intensity.

Interpretation of Results.

The experiments which are illustrated in Figs. 58 and 59
show that under normal circumstances shock movements are
such as bring the organism back into the light when it enters the
dark, or when a dark patch passes over it. The circumstances
were exceptional in the experiment illustrated in Fig. 58, and

Fig. 59.

214 SULPHUR BACTERIA

cannot therefore be regarded as typical, for here a second
shock movement was superimposed upon the organism before
it had recovered from the effects of the first shock movement.
Buder's own explanation is probably correct. When an
organism passes into the dark it recoils in order to get back
into the light, the mechanism of the process, for Thiospir ilium,
being the rotation of the cilium round the hinder part of the
organism. It considers, as it were, the end in view, and if
this can be attained without a shock movement, then such a
movement does not take place. Buder thus regards the laws
of shock movements as aspects of a wider biological law,
operating to preserve the organism from destruction. Whilst
in agreement with the general view, it must be added that all
the details of his experiments do not lend support. Thus in
the experiment illustrated in Fig. 58 the second shock move-
ment which propelled the organism farther into the darkness
should, according to Buder's view, not have taken place, for
it was one which was injurious to the microbe. As this
particular movement, however, was made under abnormal
circumstances, its significance cannot be unduly stretched,
particularly as there are other determinants of movement
besides these shocks. Proof is still wanting, however, of the
withholding of a shock movement when the withholding would
be advantageous to the organism. It would, however, be
difficult to stage the conditions for such an occurrence.
Another detail which lacks conformity with Buder's view is
the fact that all the individuals did not exhibit the movement
when they passed from the light into the dark, a fact which
seems to diminish their importance as vital factors in the life
of the organisms. Here again it would be easy to imagine
the prevention of the movement by the intervention of other
factors. The conditions of movement are similar to the con-
ditions determining any other physiological function. It
takes place when several contributory factors are working in
unison. A general review of the facts leads to the conclusion
that shock movements are broadly of a protective nature, but
that as movements are conditioned by several factors, the
incidence of a shock does not invariably produce a protective

IRRITABILITY : INFLUENCE OF LIGHT 215

movement because of the contrary influence of other factors
under abnormal circumstances.

Jennings in his description of the movements of the Flagel-
lates states that these organisms have two movements, one
the normal, and the other, which is brought into play only
when the organism is abnormally placed. The idea seems
fanciful.

CHAPTER XII.

THE MECHANICS OF CILIARY MOVEMENT.
ACID BACTERIA. THE PHYLOGENY
SULPHUR BACTERIA.

THIONIC
OF THE

The Mechanics of Ciliary Movement.

Spirilla exhibit both rotatory and translatory movements.
These result from the movements of the cihum which in the
spiral sulphur bacteria assumes the helicoid form. It is
possible by a mathematical treatment of the forces which
come into action when this kind of cilium is set in motion, to
show that the organism must necessarily exhibit translatory
and rotatory movements.

Axis of
rotation

Fig. 6o.

In Fig. 60 is shown a helically wound cilium rotating in
a clockwise direction.

The cilium, by its whipping action, leads to a forward
force on the spirillum, which may be considered the resultant
of a tluid pressure.

The forces on each point on the cilium system can be
analysed. Suppose D' to be any point in the systemu Let

2T6

THE MECHANICS OF CILIARY MOVEMENT 217

a'D' represent tlic force on D' due to the reaction of the hquid
to the motion of the cihum. This force is normal to the axis
of the ciUum and tangential to the cyHnder of rotation, and
may be divided into two components, one h'D' along the
axis of rotation, the other 6-'D' in a direction perpendicular
to the plane of this axis and O'D'. Similarly at any other
point D" the force may be split into the two components,
b"Yy" and c"D".

The sum of the component forces ^'D', b"T)" , etc., for
every point on the cilium may be represented as BD, such that

LbVi - BD.

Similarly, the sum of the tangential components c'D',
c"D", etc., for every point on the cihum may be represented
as CD, such that

UcD -- CD.

The latter forces have a moment about the axis of rotation
which tends to produce rotation
of the body about this axis.
Their summation gives a re-
sultant couple F X MN (Fig. 61),
such that

6-D X OD -^ F X MN.

Consider the effect of these
two systems of forces on the
body of the spirillum (Fig. 62). Fig. 61.

Fig. 62.

The resultant OB represents the total effective force act-
ing upon the body from the water, probably through slight
pressures established by the motion given to the water as
a result of the whipping action of the cilium. The motion

2l8

SULPHUR BACTERIA

gives rise to liead resistance and frictional forces, p, the magni-
tude of which are dependent upon the speed of the body
through the hquid. These tend to resist the motion and act
in the opposite direction.

Then QB = M/ + kv,

where M -- mass of body,

/= acceleration,
/c = a constant,
V =^ velocity.

The above equation shows that any increase in the velocity
will produce a corresponding increase in the resistance.
Movement will be uniform after a balance has been attained
between the applied force and the opposing frictional force.

OB- W

where V is the ultimate velocity.

Let us now consider the condi-
tions determining the rotational
equilibrium (Fig. 63).

The couple F X MN tends to
turn the body of the organism in the
direction opposite to that in which
the helix is turning. The body will
set itself to counteract this force,
and in consequence a redistribution
of forces from the water will follow
until the couple is balanced. This
presumably cannot be accomplished in a straight cylindrical
body with very soft membranous outer layers without altering
its shape, and so if tiic spirillum is at rest it will at once assume
the spiral form on the resumption of movement. The forces
which here come into play may be illustrated by the following
example. If the elbow is placed on a table while a long stick is
held between the fingers and set into a whipping action, it will
be found necessary to counteract the resulting torque reaction
by offsetting the elbow to some extent in relation to the
shoulder. Any increase in the force of the whipping, or in the
addition of more weight to the stick will result in a corre-

THE MECHANICS OF CILIARY MOVEMENT 219

spending increase in the offset of the elbow. This gives us a
possible explanation of the observed behaviour of the spirillum
when in motion. This force is not exerted when the spirillum
is at rest. The natural condition of a spirillum at rest is the
straight rod shape, and many are observed to have this shape ;
but we may suppose that the continuous assumption of the
spiral shape when in motion, accompanied probably by a
very slight increase in the outer membranous layers, has made
most of the organisms retain the spiral form permanently.

When the ciliary impulse has been communicated to the
body of the organism, and the latter has assumed the spiral
form, there will be a similar torque between the body of the
organism and the surrounding liquid medium, and the extent
of this reaction will probably control the degree of spiral for-
mation. As the body of the spirillum moves through the
medium the reaction pressures of the liquid upon the organism
will have a resultant which will be tangential to the body,
and so a rotatory movement will be imparted to the body in
addition to its translatory motion.

It is clear from the above dynamical treatment of the
subject that a helicoid movement of the cilium ought to
produce translatory and rotatory movements in the spirillum.
As such movements do, in fact, take place in the spirilla, the
observations of the helicoid whiplike action of the cilium
described by Buder (p. 2io) are supported.

The following hypothesis is suggested in explanation of the
ciliary movement. The impulse, a vital action, comes in the
first case from the end of the body to which the cilium is
attached, and is similar in its nature to the elbow action in the
above experiment. The vital action is communicated to the
cilium, which then takes up the rotating helicoid form, thereby
giving to the organism a rotational and translatory movement.

The following facts lend support to this hypothesis : —

1. The cilium is not an independent unit, but is in pro-
toplasmic union with the cell.

2. It has been proved that the sensitive seat of the organism
regulating movements in response to changes in the intensity of
light lies at the end of the body to which the cilium is attached.

220 SULPHUR BACTERIA

3. It is frequently observed that spirilla at rest are straight,
and assume the helicoid form only after the organism has
begun to move.

It is not possible at present to state whether the vital
action which brings the cilium into the helicoid form is preceded
by a circular movement of the end of the body which ex
hypothesi is the seat of movement.

The Thionic Acid Bacteria.
Introduction : Methods of Culture.

The thionic acid bacteria, like the sulphur bacteria in
general, oxidize sulphur compounds, but they differ from the
sulphur bacteria in that they do not store sulphur in their cells.
Strictly speaking, therefore, they are not sulphur bacteria,
but it is appropriate to consider them briefly here.

These organisms oxidize thiosulphates to tetrathionic and
sulphuric acids. They were first isolated by Nathansohn
from a crude culture of organic debris and calcium sulphide
in sea-water, prepared for the investigation of sulphur bacteria.
Nathansohn isolated instead small actively motile thionic
acid bacteria, which were readily found in the white sulphur
surface scum.

He recommended the following medium for their culture : —

NaCl,

3-0 per cent.

MgCl,,

0-25

KNO3,

o-io

NagHPOj,

0-50

MgCOa,

Excess.

When this mixture was inoculated with mud containing
these organisms a white scum appeared on the surface after
a few days. This consisted of oily amorphous sulphur in the
substance of which various bacteria were found. Among
them were the thionic acid bacteria, which were isolated
without difficulty. According to Nathansohn the reaction
which takes place is as follows : —

3Na2S203 +50 = Na2S406 + 2Na2S04.

THE TH IONIC ACID BACTERIA 221

Their development is possible only when the CO2 of the
atmosphere is freely admitted ; or, failing this, a free supply
of carbonates. Nathansohn considered that they were not
able to utilize the carbon of organic matter.

These organisms were also isolated by Beijerinck (5) from
material taken from the sea on the Dutch coast, and cultivated
in fresh water. The medium used by him was made up as
follows : —

NaaSgOg . 5H2O, 0-5 per cent.

NaHCOg, o-i

K2HPO4, 0-02 „

NH4CI, o-oi ,,

MgClg, o-oi ,,

A culture made up of these ingredients, and inoculated with
the appropriate material, became covered with a film of sulphur
in which were swarms of bacteria, including the thionic acid
bacteria. These were isolated and grown in pure culture.
The chemical reaction was stated to be as follows : —

NaaSgOg + O = NagSOj + S.

The sulphur was not deposited inside the cell, but in the
surrounding medium. In a similar fashion, though with
greater difficulty, it was found possible to oxidize tetra-
thionic acid with these organisms, according to the following
reaction : —

Na2S406 + NaaCOa + 0 = 2Na2S04 + CO2 + 2S.

Pure cultures were obtained by adding the appropriate
amount of agar-agar to the fluid culture, and plating in the usual
manner. The colonies which form on the plates are distin-
guished by the large amount of sulphur which collects on their
surfaces.

The thionic acid bacteria have been placed by Beijerinck
in one genus, which he names Thiohacillus* One of them,
Thiobacilliis thioparus, is made up of small thin rods, 0-3 — 0-5/Lt
in length, and is very motile. It is non-sporing. Another
somewhat similar form has been named Thiobacilliis denit-
rificans.

* See page 226.

222 SULPHUR BACTERIA

When first investigated, the thionic acid bacteria were
believed to be obligate autotrophs, obtaining their carbon
from the carbonic acid of the atmosphere, and their nitrogen
from nitrates or ammonium compounds. It was emphasized
that the source of their energy was the oxidation of thiosul-
phatc to the normal sulphate. Later investigations have
not borne out these statements. Thus Issatchenko and Salis-
mowskaja found that not only inorganic salts like ammonium
chloride, ammonium phosphate, and potassium nitrate, but
also organic compounds like asparagin and peptone could be
utilized as sources of nitrogen. It has also been necessary to
revise the opinions that were at first held regarding the
secretion of sulphur. The last-named investigators, working
with material from the salt marshes of the Crimea, from the
neighbourhood of Odessa, and from the Black Sea, found that
the secretion of sulphur was not a necessary accompani-
ment of the activities of these bacteria. This was con-
firmed by Trautwein who divided them into two classes
according to their capacity or incapacity for secreting free
sulphur.*

Probably the chemical activities of these bacteria are not
as simple as they are represented in the equations formulated
by Beijerinck, and this is particularly true of those members
that do not secrete sulphur.

Trautwein has shown that they are capable of considerable
adaptation, for he has isolated an organism of this class with
denitrifying powers. It reduces nitrates to nitrites and nitro-
gen. Further, the ferments trypsin, lipase, catalase, diastase
and oxidase have been identified in its cultures. With ex-
tended investigations the number of bacteria belonging to
this group has been increased. In addition to the two already
mentioned three species were isolated by Issatchenko and
Salismowskaja, and were named

Thionsdure-hacteriimi Beijerinckii,
,, Nathansohnii,

,, Beijerinckii f. Jacobsenii.

*See page 226 on the nomenclature of these bacteria.

THE TH IONIC ACID BACTERIA 223

These are regarded as related to, but not identical with,
Thiohacillus ihioparus. Another is that isolated by Waksman
and Joffe and named Thiohacillus thiooxidans. This organ-
ism has the following characters : It is rod-shaped and very
small, being less than i^ long and about 0-5/x in thickness, non-
motile, Gram positive, aerobic. It grows readily on solid media,
and oxidizes sulphur to sulphates. Carbon is obtained from
the CO2 of the atmosphere, whilst nitrogen is best supplied by
ammonium compounds. Growth is stimulated by the addition
of such organic compounds as glycerol, alcohol, mannitol, and
glucose, and certain inorganic compounds like thallium nitrate,
aluminium sulphate and manganese sulphate. The optimum
temperature is 28°-30°C. The culture medium is acidified
by its development, and the amount of thiosulphate increases
steadily with the growth of this species. It is stated that this
organism does not cease its development until the acidity
reaches the low value of pYi 0-6 — ro. This is probably the
most acid condition under which any microorganism has been
known to exist.

H. D. Brown has obtained from sewage and activated
sludge an organism which appears to be identical with Thio-
bacillus thiooxidans, and Waksman has recommended a med-
ium in which its cultivation may be best secured: —

Na^S.Oa,

5 gra

KH2PO4.

NH4CI, .

I ,

MgCl^,

I ,

CaCla,
Agar,

25 ,
20 ,

Distilled

water,

1000 c.c.

The physiology of this organism has been investigated by
Starkey, who finds that a parallel may be drawn between the
rapidity of growth and the extent to which sulphur oxidation
has been accomplished. He therefore argues that the oxi-
dation of sulphur is an essential process in the metabolism
of Thiohacillus thiooxidans. Starkey also found that whilst

224 SULPHUR BACTERIA

the addition of 3 per cent. NagSgOg induces vigorous growth,
10 per cent, of this substance in a culture medium is inhibitory.
Still another member has been isolated which is closely associ-
ated with alkali deposits, and which is particularly abundant
in the sandy loam soils of the "black" type. This has been
provisionally named Thiohacillus B. It oxidizes the thiosul-
phate with liberation of sulphur, and the latter is then oxidized
to the sulphate. Its growth is best in a medium with a pW
value of 6 — 10.

The activity of the thionic acid bacteria in furthering
changes of economic and biological importance in the soil is
considerable. It was estimated by Ames that 50 per cent, of
the sulphur combined in soil to the extent of 0-5 gram sulphur
per 500 grams of soil was changed into the sulphate when the
soil was inoculated with these organisms, and that even as
much as 70 per cent, was possible if the amount of sulphur
was present to the extent of 2-0 grams per 500 grams of soil.
As the higher green plants assimilate sulphur in the form of
sulphates, the agricultural importance of these organisms is
obvious. They are also important in another aspect, for the
greater acidity in the soil which follows their multiplication
enables the higher green plants to absorb a larger amount of
the sulphate, and a greater variety of soil constituents, than
is possible in a less acid medium. Many salts which would
otherwise be insoluble are thus made available to crop plants.
There is, however, a limit to the acidity beyond which harm
rather than good would be achieved.

The two organisms Bacterium crystalliferwn (Gicklhorn)
and Bacterium retifor^nans (Gicklhorn) must also be assigned
to this group. They were isolated from garden soil that had
been covered with water containing potassium sulphide in
solution. After three weeks the surface of the water was
covered with snow-white points, each being a colony of one
or other of these organisms. Between the individual colonies
were numerous minute particles of sulphur, evidently derived
from the sulphide by oxidation.

Bacterium crystalliferum is i — 2/i long and 0-3 — 0-5/^ broad,
and is non-motile. The individuals are closely pressed to-

THE THIONIC ACID BACTERIA 225

gether and covered with a very thin layer of sHme. Pure
cultures were not obtained.

Bacterium retiformans is 2 — 4'5/x long and 0-5 — i-o^ broad.
It develops as zoogloea colonies adhering to the walls of the
culture flask. It is doubtful whether these two organisms
belong to the genus Bacillus (or Bacterium).

Pseudomoiias bipunctatiis (Gicklhorn) is provisionally
placed in this group, but it is doubtful whether this organism
should be classed in the group of Bacteria. Its characteristics
are rather those of the simpler Flagellates. Furthermore, its
physiology differs somewhat from that of the thionic acid
bacteria. It is composed of a single colourless transparent
cell, which becomes ovoid during division. Its length is
8 — I2/X and breadth 3 — 5jLt. It possesses a single, delicate
polar cilium, about I2ju. long, which propels the organism from
behind at the rate of 600/L1 per minute. Each cell contains
two, sometimes three, highly refractive drops. These resemble
the large drops found in Microspira vacillans. Their com-
position has not yet been ascertained. They are not composed
of sulphur, and the sole justification for the inclusion of the
organism in the sulphur bacteria rests on the fact that the
organisms show a preference for sulphuretted hydrogen.

After division, many of the daughter cells are temporarily
pear shaped, and sometimes even show pointed ends (Fig. 23c).
Pure cultures have not been obtained.

Habitat. — Found in foul mud from the Botanic Gardens
of Graz.

Pseudomonas hyalina (Gicklhorn) resembles the preceding
in general form and ciliation, but is smaller, measuring 4 — 6jain
length and 2 — 2-5^ in breadth. The reason for its separation
from the preceding species lay in the absence of intermediate
sizes. As both were found in the same medium, and at the
same time, the probabilities are in favour of the two organisms
being the same species.

Habitat. — As the preceding species.

15

226 SULPHUR BACTERIA

Nomenclature of the Thionic Acid Bacteria.

All the members of this group are rod-shaped, and therefore
may be included either in the genus Bacillus, or the genus
Pseudomonas. It is unfortunate that Beijerinck should have
used the term Thiohacilliis to designate these organisms, for
the prefix thio in all morphological classifications is used to
designate the bacteria with sulphur contents, and the thionic
acid bacteria either do not secrete sulphur, or if they do it is
eliminated from the cell. The cumbrous name Thionsdure-
hakteriiim (Eng. Thionic acid bacterium) has been suggested
and used as a generic title by Issatchenko ; and lastly the
name Sulfonionas has been proposed. In both these changes
it would appear as if each innovator had held in view only
the needs of his own particular branch of research without
reference to the effect which the innovations would have on
the efforts of the systematists who have to pass in review
all the groups of bacteria. The generic terms Bacillus and
Pseudomonas are sufficiently clearly defined to be used without
fear of confusion, and all the known thionic acid bacteria can
readily be included in one or other of them. A loose nomen-
clature based on unstable physiological functions can only
lead to ultimate confusion.

The Phylogeny of the Sulphur Bacteria.

The most primitive members of the group are contained in
the genus Lankesteron, and of these Lankesteron roseo-persicina
is probably the most primitive. The majority of the sulphur
bacteria are probably derived from some such form. This
species reproduces by simple fission, and has not an external
plasmatic membrane differentiated from the rest of the cell,
and in addition it is highly pleomorphic. The more various
the number of pleomorphic phases in an organism, the greater
its chances of success in a changing environment. Probably
the first stage in the advance of such an organism would be the
assumption of a stable form under a new set of conditions.
If the new conditions persisted for a sufficiently lengthened
period a morpliologically stable organism could evolve, which

THE PHYLOGENY OF THE SULPHUR BACTERIA 227

in its turn could serve as a starting-point for a further
advance.

From the ease with which it is possible to effect slight
morphological and physiological changes in bacterial species it
may be argued that the rate of advance of bacterial organisms,
and probably of all lowly organisms, is greater than in more
complicated forms. Indeed, it is not beyond possibility that
some of the organisms described in the early days of Bacteri-
ology may have completely disappeared, and their places taken
by species that were not in existence at that time.

The following factors appear to be those most concerned
in the evolution of the group : — ■

A. Slime Formation. — In varying degree all the sulphur
bacteria form slime from the outermost layers of the cell. In
Beggiatoa alba, even in motile individuals, a thin layer of slime
is always present. Under unfavourable conditions of growth
its amount may be considerable.

In Thiothrix, on the other hand, slime development is
normal, and begins at an early stage in its growth. As the
slime subsequently hardens, and as the cells enclosed by it
continue their growth before the slime has completely har-
dened, the structure peculiar to this organism is developed.
The filament continues to grow inside a hollow sheath, and
its fission is limited to the apical parts that have emerged
from the sheath. The fissured fragments when short may be
regarded as the precursors of the conidia of more highly
developed plants. It is of interest to note that a similar line
of development appears to have been followed in the related
Iron Bacteria in such organisms as Cladothrix dichotonia and
Crenothrix polyspora.

The zoogloea condition is also in some phases a characteristic
of Lankesteron roseo-persicina. If this condition is one that
is peculiarly favourable to a new environment, a condition
temporary under normal conditions may well have become
permanent. This probably occurred in the evolution of such
organisms as Thiocystis violacea in which the cocci are em-
bedded in a mass of slime of a permanent character. The
cocci escape in this organism at a definite point in the slime.

15*

228 SULPHUR BACTERIA

In Thiobacillns Bovistus a further step is marked by a slightly
greater interdependence of the enclosed cells. In Thiohacillus
thiogenus the advance has taken the form of a more complex
covering of slime, an inner and an outer layer being distin-
guishable, the outer with an obviously protective function.
An interesting extension of this line of development is that
shown in Thioploca, for here the movements of the filaments
inside the slimy colony are not unrelated, showing that
the units have already lost their independent character.
This is the last stage in the evolutionary series along this
line.

B. Enlargement of the Coccus. — Large cocci are occasionally
formed as pleomorphic phases of Lankesteron roseo-persicina,
and it is possible that the evolution of such organisms as
Chromatium, Achromatium, and Thioporphyra may have re-
sulted from the stabilization of similar large forms. Under
unfavourable conditions both Chromatiinn and Thioporphyra
tend to revert to smaller cocci.

C. Shortening of the Filament. — The greater freedom of
movement possible to short rods in which cilia have developed
indicates another line of development. The filaments of such
primitive forms as Lankesteron roseo-persicina readily break up
into shorter lengths ; these are probably the precursors of
such bacteria as Thiobacillus and Thiopseudomonas. Thio-
hacillus Bovistus appears to be an intermediate species in this
line of development, for in it there is considerable slime for-
mation, whilst the cells on the other hand have attained a
certain freedom of movement.

D. Colonial Habit. — In Thiopedia the cocci, although
somewhat loosely arranged, develop symmetrically. In
Rhodolhiosarcina a further advance is noted, as the cocci are
closely associated in regular formation, and whilst in Thio-
pedia slime formation is regular, in the other it has become
entirely suppressed. The ease with which the disruption of
the units of a Sarcina can be brought about so that they exist
as uni- and diplo-cocci shows that the grouping of the cells to
form colonies is not a racial habit of long duration as time is
reckoned in evolution.

THE PHYLOGENY OF THE SULPHUR BACTERIA 229

E. Colour. — The following facts are important in esti-
mating the phylogenetic importance of colour : —

(i) There is little, if any, to choose in point of morphological
complexity between the coloured and the uncoloured sulphur
bacteria.

(2) The occurrence of colour is not correlated with the
possession of any other feature either in structure or in life-
history.

(3) There is no distinction in the quality of the pigment
between the most primitive and the more highly developed
members of the group.

It may be concluded from the third statement that the
coloured sulphur bacteria were just as highly coloured on their
emergence from still more primitive conditions. Hence we
must conclude either that the uncoloured forms were once
coloured, and have lost their pigments, or that the sulphur
bacteria are of polyphyletic origin. As there are very strong
grounds for the belief that photosynthesis occurs in the col-
oured sulphur bacteria a polyphyletic origin is very probable.
There is nothing in common between the two groups apart
from what is common to all the microorganisms classed under
the Schizophytes, except a common facility for oxidizing
hydrogen sulphide to elementary sulphur. In view of the
advantage which would be derived from photosynthesis, it
is not probable that organisms that flourish in the full light
of day, as do the uncoloured sulphur bacteria, would abandon
such an obvious advantage. It is much more probable that
their ways have lain apart from very early beginnings,
and that the uncoloured sulphur bacteria have been derived
from still more primitive uncoloured forms. Issatchenko (4),
who accepts the photosynthetic view of the coloured sulphur
bacteria, in consequence regards the origin of the group as
polyphyletic. He states that coloured bacteria like Lampro-
cystis roseo-persicina thrive in a purely mineral solution,
drawing their carbon from the carbon dioxide of the atmo-
sphere, and considers that this is possible only to organisms
that are capable of photosynthesis.

When the mode of life and the general structure of

►30

SULPHUR BACTERIA

Laniprocystis is compared with those of an organism such as
Beggiatoa alba, which hves in a medium rich in organic matter,
the contrast is so great that it is scarcely possible to imagine
two organisms at this phase of existence that are so diamet-
rically opposed.

The general conclusion is drawn that the coloured and the
uncoloured forms began their present stage of existence as
coloured, and as uncoloured organisms, respectively, and that
in their further development colour has played no part.

F. Reproduction. — In all the sulphur bacteria which have
been investigated the prevailing method of reproduction is a

Thiovulum

Thiophysa

Chrotnatium
Thioporphyra ^
Achromatium

Thiospirillum -«—

(Rhodo-thiospirillum)

Thiopedia -*-

I

Thiosarcina
(Rhodo-thiosarcina)

Primitive highly
pleomorphic org-anism

Filament

Coccus

Rod form -

— Spiral form

Filament in
zoog-loea condition

_ Coccus in
zoogloea condition

Rods in
zoog-loea condition

Fig. 64.

^Beggiatoa alba
Thiothrix

■^^»-Thiobacillus
^Thiopseudomonas

^_^ Thioploca
-^Rhodocapseae
— -^Th iocap s e ae
^^Thiosphaerion

Thiobacillus

Bovistus

simple fission followed by the growth of the separated portions.
This method is followed even in such organisms as Beggiatoa
alba, in which the cell has a slightly differentiated peripheral
layer. In Beggiatoa mirabilis the formation of a new cell is
preceded by the development of a transverse cell membrane
as in the higher plants. This, however, is not associated with
any other complexity in Beggiatoa mirabilis, notwithstanding
its relatively gigantic size.

The occurrence of structures that may be endospores in
Beggiatoa alba and in Thioporphyra vohitans is at present too
problematical for discussion.

THE PHYLOGENY OF THE SULPHUR BACTERIA 231

The bud formation of Thioporphyra volutans appears to
be not an advance in methods of reproduction, but rather the
recurrence of what is probably a more primitive method of
reproduction, for the products of multipHcation are of a lower
type. Support is given to this view by the fact that it occurs
under unfavourable circumstances. The formation of zoo-
spores in Achroniatimn oxaliferutn marks a decided advance,
but much stress cannot be laid on the fact because it is found
only in one organism, and this, one which in its general
structure is widely different from the other sulphur bacteria.

These suggested lines of development are schematically
represented (Fig. 64).

Summary.

The coloured sulphur bacteria have probably all arisen
from the development along several lines of one (or a few)
highly pleomorphic primitive organism of a type which is
represented among modern forms by such organisms as
Lankesteron roseo-persicina. It is conjectured that with changes
of environment certain pleomorphic forms became stabilized
on account of their greater fitness for the changed environ-
ment. The uncoloured sulphur bacteria have probably
sprung from similar types that had no colouring matter.
The sulphur bacteria as a whole are polyphyletic in origin.

The following lines are suggested as having resulted in the
modern forms : —

1. Development of hardened slime at an early stage in
development, resulting in a hardened sheath surrounding the
organism.

2. Fixation of the zoogloea condition with the consequent
retention of the units within the slime.

3. The enlargement of the coccus with freedom from
excessive slime formation, and greater freedom of action.

4. The contraction of the filament to a length suitable for
free and active movement.

CHAPTER XIII.

THE COLOURING MATTER OF THE SULPHUR
BACTERIA.

Introduction.

All the chromoparous sulphur bacteria arc purple, and
range from the pink-purple colour of Chromatium to the plum-
purple colour of well-developed cultures of Thioporphyra
voliitans. The same culture does not invariably retain the
same shade of colour, but frequently passes from one extreme
of purple to the other. These shades are due to varying
proportions of a purple pigment, named hacteriopurpurin,
and a yellow-green pigment, bacteriochlorin. Again, these
two pigments are compound, not simple substances. Their
chemical composition has not yet been determined.

Historical.

Ray Lankester (i — 2) was the first to investigate the colour-
ing matter of the purple bacteria, which he named bacteriopiir-
piirin. This substance was stated to be insoluble in water,
alcohol, ammonia, acetic acid, and sulphuric acid. In one
place the pigment is said to be insoluble in chloroform, and in
another that this reagent changes it to an orange-brown colour,
which gradually dissolves. In point of fact the pigment is
soluble in chloroform in the dry state, insoluble in the wet
state. It is also soluble in alcohol.

Ray Lankester records the following results of his
spectroscopic analysis : —

1. End absorption in violet.

2. Two absorption bands, one near the D line, and the other
near the E line (see Fig. 65, I).

Interest in the subject was quickened by Engelmann's

232

COLOURING MATTER OF SULPHUR BACTERL4 233

discovery that certain purple bacteria, particularly Bacterium
photometriciim, when exposed to light congregated more densely
at tliat part of the spectrum where the absorption of light was
greatest (see Chap. XI). He recorded two absorption bands : — -

I. 61A — 57A (orange to green) ; sharply defined) .

2- 55A— 52A (green) ; less well defined J &• 5,

A darkening was observed in the violet end, beginning near
50A, and a slight darkening in certain other parts of the
spectrum. The maximum light intensity lay in the red at
62A — 63A. It is thus seen that this pigment differs spectro-
scopically from the chlorophylls and the carotinoids of higher
plants.

Biitschli first observed that there were two pigments,
one soluble in alcohol, and the other only slightly so. The
former, which imparted a purple colour to the alcohol, formed
crystals which turned blue with sulphuric acid, and blue-green
with iodine. This occurs also with the colouring matter of
Eugenia sanguinea. He therefore regarded the colouring
matter of both of these organisms as being a form of carotin.

Arzichowsky claimed to have discovered two red colouring
matters, which he named hacieriopiirpurin and bacterio-
erythrin respectively. Unfortunately the first of these names had
been used in a different sense by Ray Lankester. His claim for
the presence of two colours rested on the fact that a wet
filter-paper dipped in the purple extract first took up a rose-
red colour, which was a different tint from that left behind.
Further, the separation could be eft'ected by shaking the
extract with carbon bisulphide when the bacterioerythrin
separates from the alcohol, imparting a raspberry colour to
the solvent. Molisch (3) experimented with pure cultures
of Rhodobacilliis palustris. He also found two colouring
matters : —

1. A green colour which he named bacteriochlorin.

2. A red colour which he named bacteriopurpitrin.

The name bacteriopurpitrin is used here by Molisch with
still another meaning.

Bacteriochlorin is soluble in benzene, olive oil, turpentine
oil, and chloroform.

Fig. 65.

Absorption Spectra of the Sulphur Bacteria.
I. The bacteriopurpurin of Ray Lankester.
II. Absorption Bands (after Engelmann).

III. Bactenochlorin in absolute alcohol (after Molisch).

IV. Bacteriopurpurin a. in chloroform (after Molisch).
V. Chlorophyll.

VI. -VIII. Thioporphyra volutans. — Three spectra obtained on different
days from the same carbon bisulphide extract.
IX. Thioporphyra volutans. — Spectrum of coloured extract, taken
from culture on cardboard.
X. Phycoerythriu.

COLOURING MATTER OF SULPHUR BACTERL4 235
Spectra.

Bacteriochlorin. — Solution in absolute alcohol :

^^^. , , 1 n • 1 rAbsorption at red end from 65A.

With coloured fluid . , .

... J ,, ., violet ,, 52'5A.

4 mm. thick U 1 , , ; , ^ ^

I Dark band, 61A-57A.

Absorption at red end^

With coloured fluid

i

10 mm. thick

from 65A
Absorption at violet end

from 5 3 -5 A
Dark band 6r5A-56"5A

big- 65, III.

Bacteriopurpurin. — Molisch distinguishes two purple pig-
ments :

Bacteriopurpurin a. — Solution in chloroform :

With coloured fluid fBand I. 56A-SSA 1 t^. ^ ttt
\ J JO \Y\g. 6s IV.

10 mm. thick iBand II. 52A-48-5AJ "

Bacteriopurpurin. ^. — Solution in carbon bisulphide :
With coloured fluid (Band I. 56A-53-5A.
8 mm. thick iBand II. 51A-49A.

Solution in chloroform :
With coloured fluid fBand I. 53A-51A.
2 mm. thick iBand If. 50A-48A.

With concentrated solutions Molisch found what appeared
to be a third band, but he was not certain of the fact.

He obtained crystals of bacteriopurpurin with the following
properties : —

1. Insoluble in water and in glycerine.

2. Slightly soluble in cold absolute alcohol.

3. Insoluble in cold, soluble in hot, glacial acetic acid.

4. Blue to violet colour in pure sulphuric acid.

5. Blue in concentrated nitric acid.

6. Dirty green in iodine and potassium iodide solution.

7. First blue then colourless in strong bromine water.
From these facts he concludes that bacteriopurpurin, as

defined by him, is identical with carotin, using this term in a
general sense to cover all the carotinoid pigments.

236 SULPHUR BACTERIA

Methods of Investigation.

1. A sample of pond water containing purple bacteria in
sufficient numbers to colour the water may be used. Light
is passed through a thin layer of fluid, and examined in the
spectroscope. This was the method of the earlier investigators,
for example, Lankester and Engelmann. The obvious dis-
advantage of the method consists in the impurity of the fluid.
Green Algae are frequently present and even mere traces of
chlorophyll affect absorption spectra.

2. The colouring matter may be extracted by solvents,
and the absorption spectra of the coloured fluids examined.
This method has the same disadvantage as the first.

3. The most reliable results are obtained when the colouring
matter is extracted from pure cultures of the organisms.
Only absorption bands of the non-sulphur purple bacteria
have liitherto been examined by the third method, because
until quite recently pure cultures of the sulphur bacteria have
not been obtained. This method, however, is not free from
objections, which are those that are inherent in all spectro-
scopic methods of examination. The chief objection is the
extreme unreliability of the evidence obtained in this way
when applied to the investigation of complex organic matters.
Such evidence must be limited to very wide generalisation,
for the following reasons : — ■

(i) Increase in the concentration of a fluid giving absorption
bands leads to a widening of the bands, not to an
intensification of their opacity.

The range of wave-lengths occupied by the bands of
strong solutions may be considerably greater than those
occupied by the bands of weaker solutions, and may
extend to several colours of the spectrum. The difference
in the range may be so great that whilst four or five
dift'erent colours are affected by the bands of strong
solutions, only two, or at most three, are affected by
those of the weaker solutions. It is therefore not possible
to fix a definite range of wave-length as characteristic
of the bands of any particular coloured fluid.
(ii) This difficulty would not be so great if it were always

COLOURING MATTER OF SULPHUR BACTERIA 237

possible to fix the same middle point for the bands both
of weak and of strong solutions of the same fluid. This
is, however, not the case, for the absorption bands change
their position even with changes in the specific gravity of
the solvent, as was pointed out by Kraus.
(iii) The number and intensity of the bands vary with the
solvent.

The Colouring Matter of Thioporphyra volutans.

The cultures vary from a pinkish-purple to a deep violet-
purple. The same culture frequently shows a variation of colour
during its periods of active multiplication. Like other coloured
sulphur bacteria it is aftected by the intensity of the light to
which it is exposed. Bright light is deleterious to its pigment
as it is to chlorophyll. On the other hand, during periods when
the intensity of daylight is low, as in the winter season in north
temperate climates, the colour is not developed. It begins to
form in the early summer and persists until late autumn.
This seasonal appearance is doubtless correlated with such
factors as temperature, presence of dissolved organic matter,
etc., which determine the growth of the organism. The
absence of colour is not due to the absence of colouring matter,
but to the absence of the organism in an active state of multi-
plication.

Spectroscopic Examination of the Colouring Matter
OF Thioporphyra volutans.

By the use of different solvents two coloured substances
were separated :—

1. A purple colouring matter, soluble in chloroform and
in carbon bisulphide.

2. A brown or greenish-brown colouring matter, insoluble
in chloroform and in carbon bisulphide, but soluble in alcohol
and readily soluble in petroleum ether.

The purple colouring matter is unstable, and oxidizes in
a few clays to a brownish-purple colour.

When the alcohol extract is shaken with petroleum ether
a yellowish substance separates from the alcohol, and tinges

238 SULPHUR BACTERIA

the ether with a yellow colour. Hence the alcohol extract
contains a component which is more soluble in petroleum
ether than in alcohol. The following experiment shows that
the bacteriopurpurin obtained by Molisch from the non-
sulphur purple bacteria is identical with the purple colouring
matter of the sulphur bacteria.

When the purple chloroform extract of Thioporphyra
volutans was filtered and evaporated, coloured crystals and an
oily substance separated out. The crystals were then ex-
tracted with cold water, filtered and again evaporated. The
residue was a thin brown skin which formed an intense reddish-
brown solution with carbon bisulphide. The crystals were
similar to those obtained by Molisch. The experiment also
showed that in the plant the purple colouring matter is in all
probability dissolved in the oil, from which it cannot be
extracted by water, but that with the removal of the oil they
can be taken up by water.

The spectroscopic examination of coloured extracts from
material growing on seaweed was unsatisfactory, for the
disintegrated Fucus cells were a source of chlorophylls and
carotinoid pigments. The spectrum of the purple fluid
exhibited the characteristic bands of chlorophyll ; and this
occurred even when, owing to careful scraping, at least 90 per
cent, of the colour was derived from the microbe.

The organisms were then grown on cardboard in culture
pools and the coloured material was collected from the deposit
which formed thereon. The absorption spectrum showed a
complete absence of absorption bands and localized darkened
areas, and the same results were obtained with both the purple
and the brown-green fluids. The complete absence of chloro-
phyll was shown by the absence of the very distinctive dark
band in the red between B and C (see Fig. 65, V). There was a
certain amount of end-absorption, particularly on the violet
side, when the purple fluids were used, and the extent of the
end-absorption was dependent on the thickness of the fluid.

This unexpected result appears to place the colouring
matter of Thioporphyra volutans in a different category from
those of the other sulpliur bacteria, but it is considered that in

COLOURING MATTER OF SULPHUR BACTERIA 239

spite of these spectroscopic differences the colouring matter of
this microbe is not essentially different from that of the other
sulphur bacteria. Its similarity is shown by its behaviour to
the various solvents, and by the shape and colour of the
crystals formed from the purple extract.

To the doubts that have been cast on the value of absorption
spectra as a mode of investigation, the following must be added.
When the same purple fluid, containing a preponderating
percentage of colouring matter from Thioporphyra, was daily
examined, the spectrum gradually changed, so much so, that
quite a different spectrum was presented at the end of a week.
This might be attributed to chemical changes during the period,
although such were not apparent, but when the same prepara-
tion was again made from the same material and using the
same solvents there was frequently no similarity in the ab-
sorption spectra. (Compare VI, VH, VIII, Fig. 65.) When
cultivated on cardboard the absorption spectrum shown in
Fig. 65, IX, was obtained. In this there is nothing to observe
except a general darkening on both sides, which is more
pronounced on the violet end. The spectra obtained by
cultivation on cardboard differed from those obtained by
cultivation on Fucus. These spectra, however, showed
that in the colouring matter of Thioporphyra vohitans both
chlorphyll and phycoerythrin (Fig. 65, X) are absent.

Spectro-photometric Method of Examination.

By the use of the apparatus described below it is possible
to measure the amount of light absorbed at any desired wave-
length. Two spectra, one above the other, are viewed at the
same time by an arrangement which may be understood by
reference to the accompanying diagram (Fig. 66). The light
in one passes through the solution, in the other it does not.
By means of a cap the whole light can be cut off except for that
which comes through a small vertical slit with which the cap
is fitted, and which exposes a small area of each spectrum.
As the cap can be made to travel over the whole length of the
spectrum a comparison can be made of the intensity of the
light from the two spectra at any selected point.

The light from Li passes through a glass screen (AJ, the

240 SULPHUR BACTERIA

prism (R), the collimator (C), and the prism (P), by which it is
refracted and passes into the telescope (T). It enters the lower
part of the slit at S and forms the upper spectrum. The light
from L2 passing through a glass screen at Ag is reflected by the
prism R and enters the upper part of the slit and forms the
lower spectrum. In comparison with the refracted light
the reflected light loses 8 per cent, of its intensity, which must
be allowed for in the calculation. In order to produce spectra
of the same intensity, the light Lj is kept fixed and the light
Li moved backwards or forwards until both spectra appear to
be precisely similar.

nd

ti
position^ HL

" 'jst position

Fig. 66.

Let Ij and l^ be the respective intensities of L^ and L2.
Then if d^ and d^ be their respective distances when similar
spectra are produced, we have

idy- [d^ ■ 100- • • • ^^>

The coloured fluid is now placed in position (F), and
reduces the intensity of the lower spectrum. Next the light
Lj is moved backwards to some position at a distance D^ at
which the two spectra are once more of the same intensity.

The insertion of the coloured fluid alters the intensity of
the light from Lg. Let its new value be Ig'.
. I2' 92 Ii

• {d^Y ' 100 (D,)^

(2)

COLOURING MATTER OF SULPHUR BACTERIA 241

From equations (i) and (2) we get

Hence

I2 (Di)^-
•I'-i- ^

(3)

Or the fraction of the Hght absorbed by the coloured fluid
is thus

Thus the effect of the insertion of the coloured fluid on the
intensity of the light can be accurately calculated for each
part of the spectrum.

Results with Thioporphyra volutans.

Confirmation was obtained of the absence of definite
absorption bands and localized darkened areas in the ab-
sorption spectra of this organism.

Summary.

The colouring matter of Thioporphyra volutans is similar
to that of other sulphur bacteria in being composed of two
groups of substances, one of which forms a purple colour
in some solvents, e.g. chloroform, whilst the other forms a
brownish-green colour in other solvents, as e.g. ether. The
former does not appear to be identical with the phycoerythrin
of the Floridese (Fig. 65, X), nor does the latter show any
relationship with chlorophyll. Spectroscopic analysis show
the absence of absorption bands and of localized darkened
areas in cultures on cardboard, but these were found in
cultures on Fucus. The want of stabihty of such bands and
areas in the absorption spectra makes this method of inves-
tigation very uncertain. At present it is not possible to
associate the colouring matter of Thioporphyra volutans with
any arrangement of dark bands in its absorption spectrum.

16

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INDEX OF AUTHORS' NAMES.

Agardh, C. a., 104.

American Society of Bacteriologists,

The, 67, 78, 88, 172.
Ames, J. W., 224.
Andrussow, N., 28.
Arzichowskv, W., 6, 233.
Atkins, W. R. G., 44.

Baas-Becking, L. G. M., 6, 22-23,

29-33. 44-45- 46, 47. 50-52-

Bavendamm, VV., 11, 14-15, 17, 38, 39,
40-41, 42, 47, 62, 63, 93, lot, 104,
107, no, 113, 115, 119, 120, 123,
124, 126, 128, 129, 132, 135, 137,
143, 144, 145, 149-150. 151. 152, 161,
162, 163, 164, 165, 167, 168, 169, 172,
173. 205.

Beijerinck, M. W., 6, 22-23, 24, 76, 137,
206, 221, 222, 226.

Bergey, D. H., 88, 93, 135, 137, 150,
151, 162, 163, 164, 165, 167, 168, i6g,

173-
Bersa, E., 121, 122.
Bredemann, 144.
Breed, 88,
Brown, H. D., 223.
Brussilowsky, 22.
Buchanan, R. E., 69, 78, 85-88, 107,

135. 137-
Buder, J., 6, 137, 139, 160, 161, 210-215,

219.
Biitschli, O., 93, loi, 117, 135, 137,

150, 151, 160, 161, 182, 233.

Chester, 80.

Cohn, F., 6, 93, loi, 103, 104, 105,

135, 144, 150, 151, 160, i6r, 173,

181, 182.
Corda, A. J. C, 6, 104.
Cornil-Babes, 80,
Corsini, A., 93, 107.
Cranston, J. A., 46, 50,
Crow, W. B., 6,

Dangeard, p. a., 137^ 182.
De Toni, 80.
Doss, B., 28.
Duclaux, E., 24.
Duggeli, M., 6.

Ehrenberg, C. G., 6, 135, 137, 139,

142, 160, 161.
Elenkin, A. A., 6.

Ellis, D., 6, 1719, 25-29, 40, 47, 62,
63-66, 69, 70, 73, 74, 76, 88 91, 93,
loi, 110-112, 128, 129, 130, 132, 134,
135. 137. 138, 143. 145. 152-159. 160,
161, 163, 178, 179, 191, 192, 237-241,

Engelmann, T. W,, 135, 144, 173,
195-206, 211, 232-235, 236.

Engler, A., 6, 68, loi, 104, 181.

Ewart, A. J., 6, 137, 144.

Fischer, A., So, 93, 137,

Fleischer, 173.

Fontan, A., 6.

Forster, F., 135.

Frenzel, J., 116, 117, 119, 120, 182.

Gerardin, 6.
Gertz, O., 6.
Gicklhorn, J., 6, 121, 128, 132-133, 137,

138, 145, 14S-149, 150, 152, 185, i85,

189, 190, 224-225.
Griffiths, B. M., 68, 116, 117, iig, 121,

182-184.

Hammer, 88.

Hansgirg, A., 80.

Harder, E. C, 28.

Harrison, 88.

Hinze, G., 6, 8r, loi, 103, 104, 122,

123, 124, 125, 180-181, 182, 185-186,

191, 192.
Hirsch, B., 6.
Holschewnikoff, 23.
Hopkins, F. G., 32.
Hunton, 88.

ISSATCHENKO, B. L., 6, 44, I37, I38,

143, 163, 222, 226, 229.

Jegunow, M., 6, 58-59.
Jennings, 215.
Jensen, O,, 76, 83-85.
Joffe, 223.
Joly, N., e.

255

256

SULPHUR BACTERIA

Keil, F., II, 39, 43, 56-57, 62, 63, 93,

94. 107.
Kendall, E. C, 20.
Kendall, 80.
Klein, E., 6.
Kniep, 209.
Kolkwitz, R., 6, loi, 115, 116, 122, 127,

128, 139, 150, 151, 169, 172.
Koppe, P., 94, 104, 113, 114, 115, 121,

128.
Kraus, 236.
Kiitzing, F., 104, 107, 173.

Lankester, R., 6, 14, 88, 134, 135, 137,

i6g, 232, 233, 236.
Lauterborn, R., 83, 99, 113, 114, 116,

117, 120, 121, 123, 128, 137, 143, 150,

151, 168, 169, 185.
Lehmann, 80.
Lewis, G. N., 32.
Lidforss, B., 207.
Lieske, R., 44, 48, 62.
Lloyd, B., 46, 50, 63.
Ludwig, 80.

Massart, J., 105, 116, 120.
Meneghini, 93, 105.
Metzner, P., 137, 160, 161.
Meyer, A., 68, 178, 192.
Migula, W., 68, 70, 73, 80, 107, 123,
124, 129, 130, 135, 137, 143, 144, 151,

152, 160, 161, 163, 164, 165, 167, 168,
169, 1/2, 173, 174, 175.

Miquel, 24.

Mitrophanovv, P., 93, 137, 150, 151.

Miyoshi, M., 6, 143, 167, 173, 174, 175,
207-208.

Molisch, H., 39, 40, 42, 47, 55-56,63,
80-82, 109, no, 121, C23, 124, 126,
127, 130, 132, 162, 198, 202, 203,
204, 205, 206, 207-209, 233-235, 238.

Nadson, G. a., 6, 22, 39, 40, 42, 119,

122, 123, 137, 150, 151.
Nageli, 165.

Nathansohn, A., 84, 220-221.
Naumann, E., 6.
Neumann, 80.
Nicolet, B. H., 20.

Oerstedt, 107, 172.

Omelianski, W,, 6, 28, 76, 126, 127.

Perfiljeff, 126, 129.

Perty, 135, 137, 138, 139, I43> I49. 161.

Pfeffer, L., 194, 207, 210.

Pollini, 107.

Potthoff, H., 137.
Pribram, E., 88.

Rabenhorst, L., 94, 104, 107, 172, 173.
Randall, M., 32.
Rey-Pailhade, J. De, 24.
Russell, W., 137.

Salismoskaja, 222,

Schewiakoff, W., 116, 117, 119, 182-

183.
Schroeter, 163.
Scourfield, D, J., 137, 145.
Selinsky, 22.
Selk, 94.
Skene, M., 6, 39, 40, 42, 47, 59-62,

168, 169, 203, 205.
Starkey, R. L., 223.
Sternberg, 80.
Stoddart, J. H., 63-66.
Strzeszewski, B., 6, 137, 138, 143, 144.
Swellengrebel, 107.
Szafer, W., 6.

Thienemann, 5.

Thorp, H. W., 148,

Trautwein, K., 222.

Trevisan, V., 6, 80, 92-93, 105, 177.

Utermohl, H., 121, 172.

Vaucher, J. P., 93.

Virieux, J., 116, 117, 120, 183-184, igr.

Waksman, S. A., 6, 49, 223.

Warming, E., 6, 14-15, 88, 93, 99, roi,
103, 104, 116, 117, 123, 124, 134, 135,
138, 139, 144, 150. 151, 152, 161, 172,
173, 181, 182, 185.

Weisse, J. F., 6, 135.

West, G. S., 68, 116, 117, 119, 120,
182-184.

Wieland, H., 32.

Winogradsky, S., 6, lo-ii, 15-17, 38,
41-42, 47, 53-55, 66, 72, 78-80, 82,
84, 85, 93, 94, 95, 98, 99, 105, 107,
108-109, 135, 137, 138, 143, 151, 152,
160-161, 163, 164, 165, 168, 169, 170,
171-172, 173, 203, 205-206.

Winter, 94, 173,

Wislouch, S. M., 113, 114, 115, 126,
127, 128.

Zacharias, O., 6, 116, 117.
Zettnow, E., 135, 160, 161.
Zopf, W., 6, 15, 89, 93, 98, 99, loi,

134. 135. 137. 139. 141. 150. 151. 161,

165, 173.
Zuelzer, M., loi.

INDEX OF ORGANISMS.

Pact's in clan'iuloii denote an illustration.

Achroinatium mobile, 121, 185.

— oxalifernin, 116-121, I20, 122,

184, 183,231.
Amoebobacter bacillosus, i6g.

— graniila, 169.

— roseiis, 168-169, 169.

Bacillus aceti, 151.

— cholera, 76.

— megatherium, 71.

— subtilis, 13.

— thiogcnns, 132.

— vulgaris, 22.
Bacterium Bovista, 130.

— cerinum, 70.

— crystalUferum, 132, 224.

— filamentosum, 70.

— hirtum, 70.

— hydi'osulfurcum, 22.

— hydrosulfuricum ponticum, 23.

— photometricum, 195, 199, 233.
— • retiformans, 132, 224, 225.

— rogusum, 70.

— rubescens, 14, 134, 135, 1 36, 169,

— sulfuratum, 14-15, 71, 74, I34(

138, 144, 172.

— tomentosum, 70.

Beggiatoa alba, 15, 38, 45, 54, 72
93, 94-99, 96, 100, loi, 103,
127, 177-179, 182, 186, 191,
227, 230.

— arachnoidea, 94, 104-105.

— leptomitiformis, 105.

— major, 72.

— media, 72, 93, 99.

— minima, 72, 93, 99.

— mirabilis, 75, 101-104, I02, iSi

190, 192, 230.

— nivea, 107.

— punctata, 93.

— rosco-persicina, 15, 74, 134, 135,

151, 161, 166, 171.

Chromatium cucullifcrum, 149, 150.

— Gobie, 138.

— gracile, 138, 144.

— Linsbaueri, 18, 47, 138, 145-149,

148, 152, 182, 186-190, 189.

— minus, 138, 143.

182-

171.
135,

, 73,
105,
192,

182,

139,

146,

Cliromatinm minntissimum , 138, 143.

— Okenii, 39, 47, 137, 139, 142, 143,

144, 145, 182, 184.

— — forma grcuile, 144.

— — — minus, 143.

— — — minutissum, 143.
Weissii, 143.

— violascens, 138, 149.

— Warmingii, 41, 144.

forma minus, 17, 144, 145, 149,

150.

— Weissii, 138, 143.

Cladothrix dicholoma, 18, loi, 127, 177,

227.
Clathrocystis rosco-persicina, 135.
Cohnia rosco-persicina, 135.
Conferva alba, 107.
Crenothrix polyspora, 12, 19, 94, 177, 227.

Erythroconis littoralis, 172.
Eugenia sanguinea, 233.

Hillhousia mirabilis, 117, I18, 182.
Hygrocrocis nivea, 107.

— Vandellii, 93.

Lamprocystis gclatinosa, 174.

— rosea, 175.

— roseo-persicina, 135, 173, 1 74, 229.

— rubra, 174.

— violacea, 173, 174, 175.
Lankesteron roseo-persicina, 135, 1 40 = 141,

226, 227, 228, 231.
— ■ rubescens, 135, 1 36.

— sidfiiratum, 135, 136.
Leptomitus, 107.
Leucothrix Mncor, 107.

Merismopedia littoralis, 172.
Micrococcus citreus, 70.

— grossus, 70.

— helvolus, 70.

Microspira vacillans, 121, 185, 225.
Modderula Hartwigi, 117, 182.
Monas erubescens, 135.

— gracile, 152.

— Miilleri, 124, 186, 191.

— Okenii, 137, 138.

— vinosa, 135, 138, 201.

— Warmingii, 135, 13S.

257

17

258

SULPHUR BACTERIA

Ophidomonas jenensis, i6i.

— sanguinctmi, l6o, i6i.
Oscillaria alba, 93.
Oscillatoria arachnoidca, 104.

Pleurococcis roseo-persicina, 135.
Proteus vulgaris, 22.
ProtococcHs rosco-pcrskina, 135.
Pscudoiiionas bifuncfatus, 131, 132-133,
225.

— hyaluia, 132, 133, 225.

— retijorriiaiis, 1 31.

Rhabdochvomatium gracilc, 152.

— Linsbaucri, 152.
— • minus, 152.

— roseum, 151.
Rhahdomonas rosea, 135, 151.
Rhodobacillns palustris, 233.
Rhodocapsa suspensa, 1 62,
Rhoduipi rill inn gigantenm, 207-209.

— photoinctricuni, igS, 207.
Rhodothece pendens, 162, 163.
Rliodothiosarcina rosea, 163.
Rhodothiospirillum jenense, 161, 210,

211, 213, 214.

Sarcina aurescens, 70,
— • fimentaria, 70.
■ — Jlava, 70.

— jlavescens, 70.

— fusccscens, 70.

— gasoforntans, 70.

— marginata, 70.

— tnobiUs, 70.

— olens, 70.

— pulmonuin, 70.

— 7'0S(:fl, 70, 163.

— striata, 70.

— ventriculin, 70.

— vcrmiforniis, 70.
Spirillum rnbrum, 55, 209.

— volntans, 73.
Spirophis minima, 99.
Streptococcus pallidus, 70.

— pyogenes, 70.

— tyrogeims, 70.

Thio bacillus B., 224.

— Bovistus, 130, 131,228.

Thiobacillus denitrificans, 221.
• — thiogencs, 132, 228.

— thionxidans, 130, 223.

— thiopants, 221, 223.
Thiocapsa roseo-persicina, 164, 1 65.
Thiocystis riifa, 165.

— ■ violacca, 17, 1 65, 227.
Thiodcrnia gclatinosa, 174.

— roseum, 175.

— rubra, 174.
Thiodictyon clegans, 170.
Thionsdure bacterium Beijerinckii, 222.
— • — Beijerinckii f. Jacobsenii, 222.

— — Nat/iansohnii, 222.
Thiopcdin rosea, 172, 173.
Thiopliysa macrophysa, 123,

— volutans, 81, 84, 122, 123, 180, i85,

192.
Thioploca ingrica, 115.

— mininia, 115.

— mixta, 115.

— Schmidlei, 113, 114, 115.
Thiopolycoccns ruber, 172.
Thioporphyra volutans, 17-18, 38, 40,

43, 45, 52, 62, 63, 64, 74, 153-159.

153-158, 178, 179-180, 181, 192, 230,

231, 232, 237-239, 241.
Thioroseo-persicina, 135.
ThiosphcEra gclatinosa, 1 67.
Thiospliarella amylifera, 123.
ThiosphcErioH violacca, 166, 167.

— violaceum, 173.
Thiospirillum agilis, 128.

— agilissimum, 128.

— ■ bipHiictatum, 127, 1 28.

— clongata, 129.

— granulatum, 127.

— Winogradskii, 1 27.
Thiothece gclatinosa, 171.
Thiothrix annulata, 109, no.
— • marina, 109, no.

— nivea, 107-109, 108, no.

— tenuis, 107, 109, no.

— tenuissinia, 107, 109.

— violacca, 110-112, III.
Thiovolum majus, 124, 186, 192.

— minus, 124.

— Miillerii, 124,' 1 25, 185-186.

Vibrio hydrosnlfurcus, 23, 25.

GENERAL INDEX.

Absorption spectra, 236 et seq.

Aerosomes, 162.

Albuminous substances, decomposition

of, 20.
Arabia, 6.
Assimilation of hydrogen sulphide,

46-47.
Autolysis, 94-98.

Bacterial plate, 58.
Bacteriochlorin, 232, 233.
Bacterioerythrin, 233.
Bacteriopurpurin, 232, 233, 235.

— a, 235.

- i3, 235.

Black sand of Clyde estuary, 25-29.
Bud formation, stages in, 157-159.
Budding, 157.

Carbon, the source of, 40-60.
Cell division, 69, 74, 77.

— inclusions, 192.

— intimate structure of the, 75, 77,

176-192.

— nucleus, 186.

— size, 71, 77.

— wall, 183.
Ceylon, 6.

Cilia insertion, 73, 77, 159.
Ciliary movement, mechanics of,
216-220.

— — of the sulphur bacteria, 210.
" Clamp connections," 145.
Classification of the sulphur bacteria,

67-91.

— — — attributes used in,

6978.

— Buchanan's, 69, 85-8S.

Ellis', 88-91.

— — ■ — Engler's, 68.

■ Jensen's, 83-85.

Meyer's, 68.

Migula's, 68.

— — — Molisch's, 81-83.

• Winogradsky's, 78-81

— principles of a natural, 67.

Classifications, review of previous,

78-8S.
Colonial habit, 228.
Colour, 75, 77, 229.
Colouring matter of T. voliitaus, 237

ct seq.
Conidium, 107.
Copenhagen, 5.
Culture methods, Jegunow's, 58.

Keil's, 55.

• Molisch's, 55.

Skene's, 59.

Winogradsky's, 53-55.

— of the sulphur bacteria, 53-63.
Cysteine, 7, 20, 32.

Cystine, 7, 20.
Cytoplasm, 179, 192.

Denitrifying sulphur bacteria, the,
48.

— thiosulphate bacteria, 3.
Denmark, 6.

Egypt, 6.

Endospores, 98-99, 102, 159.

Fermentation, 36.

Ferment snlf-Jiydriqtie, 24.

Ferrous sulphide, formation of, 27, 59.

Fission, 98, 112, 119, 156.

Food requirements of the sulphur

bacteria, 3-1-36.
Fountain plate, 59.
France, 6.

Geographical distribution of the

sulphur bacteria, 4-7.
Germany, 6.
Glutathione, 20, 32.
Great Britain, 6.
Growth, 94.

— relationship of light to, 203.

Habif, diversity of, 76, 78.
Holland, 6.

259

17

26o

SULPHUR BACTERIA

Hydrogen-ion concentration, 44-46.
Hydrogen sulphide, assimilation of,
46-47.

— — equilibiium in water of, 29-33.

Inorganic sulphur compounds, reduc-
tion of, 22-23.
Intimate structure of the cell, 176-192.
Irritability, 193-205.

— and en\'ironment, 209.
Italy, 6.

Jamaica, 5.
Japan, 6.

Light absorption, effect of colour on,

199.
• — directive effect of, 194, 205-206.

— Engelmann's investigations, 195-196,

198-202.

— function of, 204.

— influence on coloured sulphur

bacteria of, 194.

— photosynthetic effect of, 194, 207.

— phototactic, effect of, 206.
■ — tonic effect of, 194.
Limans, the, 25, 58.
Lime-bacteria, T49.

Metabolism of the sulphur bacteria,

34-46-
Methods of investigation, lo-ii.
Mineral matters, 43-44.
Motility, 12, 70, 77, 159, 178, 179.
Movement, effect of chemicals on

direction of, 207-209.

Natural classification, principles of a,

67-69.
Nitrogen, source of, 60.
Nucleus, rgi.

Organic matter, 38.

— — in sulphur waters, 50-52.
Oxygen, 41.

— liberation from purple bacteria on

exposure to light, 201, 203.

Pffffer's capillary tube method, 194.

Phi lot/lion, 24.

Photokinetic after-effect, 196.

— induction, 196.
Photosynthesis, 204.

Phylogeny ot the sulphur bacteria,

226-231.
Physico-chemical speculations, 50-52.
Pleoenergism of the sulphur bacteria,

47-
Pieomorphism, 12-19, 74, 77, 99, i47-

— author's investigations. 17-19-

— evidence of occurrence, 14-19-
Poland, 5.

Pure cultures, 11, 14.

— — of Beggiatoa, 57.

— • — — Thioihrix, 56.

Purple bacteria, effect of light on, 206.

— — liberation of oxygen on exposure

to light, 201-203.

— — relationship of light to growth,

203.

— sulphur bacteria, necessity of HoS

to, 61.

— ■ — • — light to, 6 1.

Pyrenees, 6.

Reduction of inorganic sulphur com-
pounds. 22-23.

— — sulphates, 22.
sulphites, 23.

— — thiosulphates, 23.
Regional rcjuvc7iesccnce, 157.
Reproduction, methods of, 98-99, 112,

119, 156, 230.
Respiration in the sulphur bacteria, 37,

41-43-
Rod-Gonidium, 107, 112.
Russia, 6.

Saprophytic bacteria, 4.
Sheath formation, 94, 177.
Shock movements, 196-198.
Buder's researches, 212.

— — Molisch on, ig8.
Siberia, 6.

Slime formation, 12, 13, igr, 227.

Spectra, absorption, 236 ct seq.

Spectro-photometric method of examina-
tion, 239-241.

Spectroscopic examination of the colour-
ing matter, 237.

Spore germination, T^-TJ.

Sulphate-reducing bacteria, 4.

Sulphates, reduction of, 22.

Sulphites, reduction of, 23.

Sulphur, tests for, 21, 178.

— bacteria, area of sensitiveness, 213.

— — attributes used in the classifica-

tion of, 69-78.

— — ciliary movements, 210.

— — classification of the, 67-91.

— — colouring matter of the, 232-241.

— — connotation of the term, 3.

— — culture of the, 53-63.

— — denitrifying, 48.

— — distribution in spectrum colours,

198.

— — food requirements of the, 34-36.

— — geographical distribution of the,

4-7.

— — intimate cell structure, 176-192.

— — metabolism of the, 34-46.
phylogeny of the, 226-231.

— — pleoenergism of the, 47.

— — reaction to light of the, 211.

— — respiration in the, 37-43.

GENERAL INDEX

261

Sulphur cycle in nature, the, 7-10.
— waters, organic matters in, 50-52.
Sulphuretted hydrogen, 38.

natural sources of, 20-29.

production of, 20-29.

— under marine conditions,

24-29.
Sweden, 6.

Thionic acid bacteria, the, 3, 220-226.

nomenclature of the, 226.

Thiosulphate bacteria, 3.

Thiosulphate bacteria, denitrifying, 3.
Thiosulphates, reduction of, 23.

United States of America, 6.

Water, changes effected by T. volnians,

63-
West Galicia, 7.

ZooGLOEA condition, 13, 15,
Zoospores, 120.

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