I.C.D. LI

MICRO-ORGANISMS

AND

FERMENTATION.

BY

ALFRED JORGENSEN,

Director of the Laboratory for the Physiology and Technology of
Fermentation at Copenhagen.

NEW EDITION.

TRANSLATED FROM THE RE-WRITTEN AND MUCH
ENLARGED THIRD EDITION IN GERMAN BY

ALEX. K. MILLER, PH.D., F.I.C., AND E. A. LENNHOLM,

AND REVISED BY THE AUTHOR.

Witlj 56 IUtt«it*tt<rtr*.

LONDON:
PUBLISHED BY F. W. LYON,

EASTCHEAP BUILDINGS, E.G.

1893.

LIBRARY

PREFACE.

THE present book gives an account of the morphology and
biology of the micro-organisms of fermentation, and it thus
forms a complement to the text-books which treat mainly of
the chemical side of the subject.

I have attempted to give a general review of all the
knowledge we possess in the above-mentioned field, and have
described the various methods of investigation which in the
course of time have proved of importance.

In discussing the organisms of fermentation and their
relation to industry, there are two names which in a high
degree especially attract our attention, namely, Pasteur in
the older literature, and Hansen in the more recent literature
of our subject. Since this book is intended to give an
account of the present stand-point of the science, it is
evident that the investigations from the Carlsberg Laboratory
must occupy an important position. In Chapters V. and VI.
will be found an accurate description of Hansen's theoretical
investigations on the alcoholic yeasts, of his methods for the
pure cultivation and analysis of yeast ; likewise an account
of the practical employment of his pure yeast, and of the
results obtained with it in breweries, distilleries, and pressed
yeast factories, and in the preparation of wines from the grape
and other fruits.

tl f* ft I

IV PREFACE.

This book thus appeals to chemists, botanists, and biologists,
likewise to those technologists who are engaged in the branches
of industry named.

In the bibliographical list I have included all important
works of the older and more recent literature which are of
interest to the scientist and technologist.

In its present form this book in the main has the same
scope and contains the same matter as the third completely
revised Grerman and the French editions.

ALFKED JORGENSEN.
Copenhagen,
May, 1893.

TABLE OF CONTENTS.

CHAPTER I.

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION.

PAGE

1. Microscopical Preparations, Staining, and Microchemical

Examination . . . . . . . . . . . . 1 — 5

2. Biological research by means of the Microscope ; moist

chambers . . . . . . . . . . . . . . 6 — 9

3. Sterilisation (Spallanzani, Scheele, Appert, Schulze,

Schwann, Schroder, Dusch) . . . . . . . . 9 — 14

4. Disinfection . . . . . . . . . . . . . . 14 — 17

5. Flasks : Pasteur, Chamberland, Freudenreich, Hansen,

and Carlsberg Flasks 17—21

6. Nutritive Substrata '21—22

7. Preparation of the Pure Culture 22—32

(a) Pure Cultures for Morphological Investigations ;

Ehrenberg, Mitscherlich, Kiitzing, F. Schulze,

Tulasne, De Bary, Brefeld 23—24

(b) Pure Cultures for Physiological Experiments

with Mass-cultures ; Pasteur, Cohn, Lister, ,

Hansen, Schroeter, Koch . . . . . . 24 — 32

8. Counting the Yeast-cells 32—35

CHAPTER II.
EXAMINATION OF AIR AND WATER.

Tyndall, Miquel, Koch, Hesse, Hueppe, v. Schlen,

Frankland, Petri, Pasteur, Hansen . . . . . . 36 — 47

Hansen's Zymotechnical Analysis of Air and Water . . 47 — 50

CHAPTER III.
BACTERIA.

Forms 51—53

Anatomical Structure, Colouring Matters, Phosphorescent

Bacteria, Locomotion . . . . . . . . . . 53 — 54

Multiplication, Growth-forms, Endosporous and Arthro-

sporous Bacteria, Zoogloea . . . . . . . . 54 — 56

Anaerobic and Aerobic Species . . . . . . . . 56

VI TABLE OF CONTENTS.

PAGE

1. Acetic Acid Bacteria 56—61

2. Lactic Acid Bacteria . . . 61 —66

3. Butyric Acid Bacteria 66 — 70

4. Kephir-organisms, Ginger-beer Plant . . . . . . 70 — 73

f). Slinae-forming Bacteria . . . . . . . . . . 73 — 76

6. Bacteria exercising an inverting, diastatic or peptonising

action . . . . . . . . . . . . . . 76 — 77

7. Sarcina Forms. . .. .. .. .. .. .. 77 — 80

8. Crenothrix 80—81

CHAPTER IV.
THE MOULD-FUNGI.

Occurrence in nature . . . . . . . . . . . . 82 — 83

1. Botrytis cinerea 83—86

2. Penicillium glaucum . . . . . . . . . . 86 — 89

3. Eurotium Aspergillus glaucus . . . . . . . 89 — 92

4. Aspergillus Oryzas 92—93

5. Mucor Mucedo' 93—96

Mucor racemosus, Mucor erectus, Mucor circinelloides,

spinosus, stolonifer . . . . . . . . . . 96 — 99

Fermentative action of the Mucorinae . . . . . . 99 — 100

6. Monilia Candida 100—103

7. Oidium lactis . . . . 103—106

8. Fusarium . . . . . . . . . . . . . . 106

9. Chalara Mycoderma 106—107

10. Dematium pullulans . . . . . . . . . . . . 107 — 109

11. Cladosporium herbarum .. .. .. .. .. 109 — 110

CHAPTER V.

ALCOHOLIC FERMENTS.

INTRODUCTION . . . . . . . . . . . . . . Ill — 121

General remarks . . . . . . . . . . . . Ill

Earlier Observations of Schwann, de Seynes, Reess, de

Bary, Engel, and Brefeld 111—112

Pasteur and his predecessors . . . . . . . . 113 — 115

His dispute with Liebig . . . . . . . . . . 114

Pasteur's views on the fermentation organisms, his theory

of fermentation, and his controversy with Brefeld and

Traube * 114—118

Aeration experiments of Pedersen, Hansen, and Buchner 118
Nageli's, Brown's and Hueppe's objections to Pasteur's

theory of fermentation ; Nageli's theory . . . . 118 — 120

TABLE OF CONTENTS. Vll

PAGE

Observations on budding-fungi by Bail, Reess, Zopf, and

Brefeld .. : 120

Chemical investigations by Rayman and Kruis . . . . 120 — 121

HANSEN'S INVESTIGATIONS . . . . . . . . . . 122 — 159

General remarks . . . . . . . . . . . . 122

1. Preparation of the Pure Culture 122—123

2. The Analysis 124—158

(a) The Microscopic Appearance of the Sedimentary

Yeast 124—125

(6) Formation of Ascospores . . . . . . . . 125 — 137

(c) The Formation of Films 137—142

(d) The Temperature Limits for the Saccharomycetes 142 — 143

(e) Cultivation on a Solid Nutritive Medium . . 143 — 144
(/) The Behaviour of the Saccharomycetes and

Similar Fungi towards the Carbohydrates and
other Constituents of the Nutritive Liquid —

Diseases in Beer . . . . . . . . 145 — 151

(y} Variations in the species of Saccharomycetes . . 151 — 156
(A) Gelatinous Formation secreted by the" Budding-
fungi 156—158

The Microscopic Appearance of a Yeast-cell . . . . . . 158 — 159

Classification of the Genus Saccharomyces . . . . . . 159 — 188

Saccharomyces Cerevisiae I. Hansen . . . . . . . . 159 — 161

Saccharomyces Pastorianus I. Hansen . . . . . . 161 — 165

Saccharomyces Pastorianus II. Hansen . . . . . . 165 — 167

Saccharomyces Pastorianus III. Hansen . . . . . . 167 — 170

Saccharomyces ellipsoideus I. Hansen . . . . . . . . 170 — 172

Sacchuromyces ellipsoideus II. Hansen . . . , . . 173 — 174

Two ellipsoid yeasts described by Will . . . . . . . . 174 — 175

Saccharomyces Ilicis. Gronlund . . . . . . . . 175

Saccharomyces Aquifolii. Gronlund . . . . . . . . 175

Saccharomyces pyriforrnis. Marshall Ward . . . . . . 175

Saccharomyces Marxianus. Hansen . . . . . . . . 176

Saccharomyces exiguus (Reess). Hansen . . . . . . 176 — 177

Saccharomyces membransefaciens. Hansen . . . . . . 177 — 178

Saccharomyces Hansenii. Zopf . . . . ... . . . . 178

Saccharomyces Ludwigii. Hansen . . . . . . . . 179 — 180

Saccharomyces acidi lactici. Grotenfelt . . . . . . 180

Saccharomyces minor. Engel . . . . . . . . . . 180

Saccharomyces anomalus. Hansen . , . . . . . . 181 — 182

Saccharomyces conglomerates. Reess . . . . . . . . 182 — 183

Carlsberg bottom-yeast No. 1 and No. 2 . . . . . . 184 — 186

Grouping of cultivated yeasts for practical purposes . . . . 186 — 187

Observations made by Irmisch . . . . . . . . . . 187

Viii TABLE OF CONTENTS.

Distillers' yeasts, Pressed yeasts (Delbriick, Lindner, Stenglein,

Belohoubek, Schumacher, Wiesner) . . . . . . 187 — 188

Other Budding-fungi 188—203

Torula 189—194

Saccharomyces apiculatus . . . . . . . . . . 195 — 200

Mycoderma cerevisise and vini . . . . . . . . . . 200 — 203

CHAPTER VI.

THE APPLICATION OF THE RESULTS OP SCIENTIFIC RESEARCH
IN PKACTICE.

The Technology of Sterilisation brought about by the earlier
investigations on spontaneous generation. Spallanzani,
Scheele, Appert, Schulze, Schwann, Schroder, Dusch;
Pasteur's results 204—206

Hansen's System and the Reform brought about by it : Diseases
in beer ; Different Species of Saccharomyces cerevisiae ;
Pure Cultivation of brewers' yeast ; Constancy of the
Characters of yeast-species under the conditions of the
brewery ; Method for the Practical Analysis of yeast ;
Yeast-propagating Apparatus ; Flasks for the despatch of
yeast ; Preservation of Pure Cultures . . . . . . 206—214

Accounts of the Results obtained by Hansen's investigations
(Lintner, Aubry, Will, Reinke, Belohoubek, Bungener,
Lintner, jun., Frankland, MacCartie, de Bavay, Wilson,
Kokosinski, Van Laer, and others). , .. .. .. 214 — 226

Resumd . 226—227

Bibliography . . .... 228—251

Index . 2-32—257

MICRO-ORGANISMS

AND

FERMENTATION.

CHAPTEE I.
Microscopical and Physiological Examination.

1. MICROSCOPICAL PREPARATIONS, STAINING, AND
MICRO-CHEMICAL EXAMINATION.

THE Microscope will be for all time of paramount aid in the
investigation of micro-organisms, since these, as individuals,
are almost always invisible to the naked eye. The earliest
important observations in the physiology of fermentation we
owe to purely microscopical investigations, and it was not
until the last decades that biological and physiological
investigations were undertaken. After a certain probability
had arisen that the same species of micro-organism did not
always occur in the same form, work was eagerly commenced
in different laboratories with so-called " culture experiments,"
in which attempts were made, by conditions of growth
artificially brought about, to observe the different phases of
development in one and the same spot, in order to thus
determine the entire process of development. The idea was
correct, but the way in which it was worked out was at that
time so faulty that " culture experiments " threatened in
consequence to fall into utter disrepute. The work was carried
out without any proper precautions, as the following example
shows. Beer yeast was sown on a moist slice of bread ; the

2 MICRO-ORGANISMS AND FERMENTATION.

culture was carefully covered with a glass shade, and all
manner of precautions were observed in order to protect the
growth from external contamination. After some days a
growth of mould appeared, as is always the case with moist
bread ; and the conclusion was therefore drawn that the beer
yeast was the origin of the mould, and that, consequently,
yeast and mould-fungi were different phases of development
of one and the same species.

A number of years elapsed before what are now universally
acknowledged to be self-evident requirements of such in-
vestigations were put in practice, namely, that the first thing
to be ascertained, before drawing definite conclusions, must be
the point from which to start. This requirement was
gradually defined with greater precision, and at last, as we
shall see later, a point was reached which satisfies this
demand in a higher degree than has hitherto been the case
in the allied branches of science.

A microscope capable of magnifying to the extent of
1.000 diameters is, as a rule, necessary for the investigation
of micro-organisms. For the yeast and mould-fungi the only
preparation generally required consists in placing a drop of
the liquid containing the organisms on an object-glass, and
spreading it out in a thin layer by means of a cover-glass.
When cultivated on solid substances, a very small portion of
the growth is first mixed with a drop of water. At any rate,
the preliminary examination of bacteria must always be
performed in this manner. In modem bacteriological
research, and especially in the case of pathogenic forms, a
number of different methods of drying and staining are
employed, partly in order to facilitate observation, and
partly with a view to bring out characteristics which would
otherwise be observed only with difficulty or not at all. An
objection to these methods, urged with unquestionable
correctness, is that the violent treatment often alters the
proportions of length and thickness, etc., of the bacteria.
On the other hand, it must be alleged that certain pathogenic

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 3

forms — for instance, the tubercle-bacillus, investigated by R.
Koch, — could not be determined with certainty until such a
preparation had been made : and, indeed, staining is often
necessary in order to detect such bacilli. As an example of
the methods of staining, we will enter somewhat more closely
into the examination of the tubercle-bacillus^ which led to
one of the most important observations made in modern
science. Koch gave the following method for its examination :
The section of the tissue which contains the bacilli is
immersed for 24 hours in a mixture of 200 parts of distilled
water, 1 part of concentrated alcoholic solution of methylene
blue, and 0-2 part of a 10 per cent, potash solution. By
this treatment the section is stained dark blue, and is then
immersed, for a quarter of an hour, in a concentrated aqueous
solution of vesuvin. The section is now rinsed in distilled
water until the blue colour disappears, and a more or less
strong brown stain remains ; finally, the section is treated
with alcohol, mounted in clove oil, and examined. The cell-
nuclei and most species of micrococci are stained brown by
this treatment, whereas the tubercle-bacilli assume an intense
blue colour. (Of the known species of bacilli, only the
bacilli of leprosy behave in the same way; they differ,
however, in other respects from those of tuberclosis.)
According to Koch, this result depends on the alkaline reaction
of the staining solution, since these bacilli never take the
stain in acid or neutral solutions ; the neutral solution of
another colouring matter entirely removes the first stain,
except in the case of the tubercle-bacilli, which retain the
original staining. Subsequently, various other methods
were proposed for the identification of this micro-organism,
the most preferable of which is that of Ehrlich, who used
aniline instead of potash. Aniline is a faintly yellow, oily
liquid, the saturated aqueous solution of which has the
power of taking up more colouring matter than the solution
of potash. Ehrlich has also employed mineral acids for
decolourising, proceeding on the supposition that the tubercle-

4 MICRO-ORGANISMS AND FERMENTATION.

bacilli are surrounded by a cell- wall which is only permeable
by alkaline liquids. Therefore, when the bacilli, cell-nuclei,
plasma, etc., are stained by the alkaline solution, and the
first -named are consequently practically indistinguishable in
the mixture, treatment with an acid removes the stain from
all the other parts of the section and from all foreign
organisms ; but, as the presumed envelope of the tubercle-
bacilli cannot be penetrated by the acid, these bacilli will
remain as the only stained bodies in the otherwise entirely
decolourised material. Ehrlich carries out the staining in the
following manner : — Finely-powdered gentiana-violet is
dissolved in a saturated aqueous solution of aniline ; 10 to 20
drops of this solution are filtered into a watch-glass, in
which the section to be examined is allowed to remain for
about 24 hours. It is then rinsed with distilled water and
again placed in the watch-glass with a solution of 3 parts
of nitric acid in 100 parts of alcohol. After three to five
minutes the section is decolourised ; it is then transferred to
pure alcohol, and finally examined in clove oil.

As is known, photographic illustrations of bacteria have
recently come into general use, having been first introduced
by R. Koch. In order to obtain these, staining and
decoloration are quite necessary, partly in order to render
the contours of the bacteria sharper, and partly in order to
remove all bodies detrimental to the picture.

Staining and decoloration are not generally required in
investigations connected with the physiology of fermentation,
where the organisms are almost always free, and only seldom
mixed with disturbing elements, and only in a few cases has
staining led to the discovery of specific characters (Bacterium
aceti and B. Pasteurianuin, see Chapter III.).

On the other hand, however, it is sometimes necessary
in the examination of the organisms of fermentation, and
especially of bacteria, to adopt another method of preparation.
The particles of organic and inorganic matter which separate
from the solutions frequently have a deceptive similarity to

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 5

various bacterial forms ; and, indeed, it is often a matter of
the greatest difficulty, if not altogether impossible, even for
the most experienced observer to determine with certainty
whether the small spherical bodies in the field of the micro-
scope are micrococci or particles deposited by the solution.
In such doubtful cases it is advisable, before entering on the
physiological examination described later on, to have recourse
to micro-chemical reagents, which often give good pre-
liminary indications. In beer and in nutritive liquids generally
which contain albuminoids, these often separate in spherical
and thread-like forms; the starch granules, the dextrins formed
from starch, and even some of the hop constituents may also
appear as small spherical bodies. The addition of a small
quantity of alcohol, ether, chloroform, acetic acid, soda, potash,
etc., is often able to throw some light on the nature of these
bodies, the resinous substances being dissolved by the former
liquids, whilst the albuminoid matter is acted on more or less
by the latter solutions ; the addition of iodine will impart a
blue colour to the starch granules which are present, whilst
certain dextrins are coloured red by the same reagent.

In the case of the higher organisms of fermentation — yeast
and mould-fungi, — staining is employed for a different purpose,
namely, in order to obtain information concerning the sub-
stances which are present in the cell-wall or cell-contents at
different stages of their development. On the addition, for
instance, of a solution of iron chloride, or any other salt of iron,
to cells which contain tannic acid, a bluish-black or green
coloration appears in the cells ; in this way it was observed
that the cells of Saccharomyces cerevisise contain a fairly con-
siderable quantity of tannic acid during the earlier stages of
fermentation. If yeast cells are treated with a solution of
hsematoxylin or osmic acid, small, sharply-defined, dark-
coloured bodies can be seen, which may be regarded as cell-
nuclei of the same nature as those generally observed in the
cells of the majority of plants without the aid of this treat-
ment.

6 MICRO-ORGANISMS AND FERMENTATION.

2. BIOLOGICAL RESEARCH BY MEANS OF THE MICROSCOPE ;
MOIST CHAMBERS.

A true and thorough insight into the nature of the organ-
isms of fermentation is not attainable until the method of
physiological investigation is resorted to. As stated above,
endeavours were made long ago to devise methods of this
nature ; the entire neglect of precautions in carrying out the
experiments resulted, however, in complete failure, and a re-
action then set in, which found expression, e.g., in the work of
Reess on the Saecharomycetes (1870), in which he expressly
stated that he had taken no precautions to obtain pure
cultures, — to such a degree had these cultures fallen into
discredit. In the course of the following years, however, the
matter took a different turn, and it is, perhaps, an almost
unique fact in the history of science, that, in so short a time,
a new method of investigation not only made its way, but also
yielded practical results, both in pathological science and in
our own special branch, results which have brought about a
revolution in many previously-accepted doctrines.

The aim of physiological investigations of micro-organisms
is to gain an insight into their development and vital functions.
The means to be employed in order to attain such an insight
is naturally to determine such conditions for their growth and
propagation which will make it possible to observe the changes
gradually taking place in the organism itself and in the sub-
stances influenced by it. When the object aimed at is solely
to obtain a knowledge of the various forms which the organ-
ism assumes during its development, the conditions are much
more easily attained than when a culture on a large scale of
individuals originating from one cell of the species is required
for the purpose of gaining an insight, through physiological^
chemical, or purely practical experiments with larger quan-
tities of these organisms, into the relations between their
forms and external influences and into all their biological
functions. In the former case all that is required is a culture

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 7

in which the organism is able to develop itself undisturbed,
apart from the question whether foreign individuals or species
are present in the same preparation. In the latter case, on
the contrary, an absolutely pure culture is required.

Cultures of the first-mentioned kind may in certain cases
be of use in affording information when the case previously
mentioned occurs : a nutritive solution in which deposits
of various kinds have assumed a more or less deceptive
similarity to different bacteria, in consequence of which it is
impossible to obtain any certain information by means of an
ordinary microscopical examination ; the question to be
answered by the experiment is accordingly, whether these
small bodies are capable of multiplying.

, FIG. 1.
Ranvier's Moist Chamber : a, seen in plan ; 5, in section.

A drop of the liquid is transferred to the so-called " moist
chamber" as, for instance, Ranvier's (Fig. 1). This apparatus
is made by grinding a slight hollow in the middle of a common
object-glass ; around this hollow a groove is made of greater
depth to receive the water ; the drop of the nutritive solution,
which must be very small, is placed in the middle of the
hollow and covered with a cover-glass, which extends beyond
the groove ; when the cover-glass is in place, it is cemented
by means of vaseline, and the drop is thus spread out between
the cover-glass and the hollow of the object-glass, and is at the
same time protected by the water in the groove from
evaporating.

8 MICRO-ORGANISMS AND FERMENTATION.

Another kind of moist chamber, invented by Bottcher,
consists of a glass ring cemented to a common object-glass,
upon which, within the ring, some drops of water are placed.
A cover-glass, on the under side of which a small drop of
nutritive liquid containing the organisms has been placed, is
fastened to the edge of the glass ring by means of vaseline.

This apparatus is brought under the microscope, and the
changes of the organisms are observed from time to time ; or
it may be placed in an incubator, maintained at a suitable,
constant temperature, and withdrawn at intervals for a
thorough microscopical examination.

These forms of apparatus are adapted to morphological or
botanical examinations under the microscope. If, on the other
hand, a physiological examination is to be carried out, it is

FIG. 2.

Bottcher's Moist Chamber : a, thin cover-glass ; b, layer of nutritive material ;
c, glass-ring ; d, liquid.

necessary that the pure cultures should be developed on an
extensive scale. Among the investigators who have developed
the methods in this direction, Pasteur, Lister, Koch, and
Hansen, deserve special mention. (See " Preparation of the
Pure Culture," p. 22.)

Every fermentation, no matter whether the product be
beer, wine, spirit, vinegar, or other liquid, is caused by a
vegetation of living organisms, " organised ferments," and, in
practice, it is endeavoured to obtain, as far as actual circum-
stances will permit, a pure culture of the forms best suited
to the manufacture. Although, in our time, with a better
understanding of aims and means, great progress has been
made in this direction, yet there must always be limits which,
from purely practical reasons, cannot be overstepped; the
cultures in the factories will never reach such a perfection

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 9

as to keep continually in a state of absolute purity. It is,
however, one of the most salient features in the present
development of the industry of fermentation, that efforts
based upon the right understanding of the paramount im-
portance of the fermentation organisms are being made to
emancipate the chief useful species from the action of injurious
forms. The very great importance of this was not, however,
appreciated until Hansen, through methodical selection of
certain types of yeast, showed that such a pure growth insures
far greater certainty and uniformity than the impure and
unknown yeast mixtures hitherto used. We shall come back
to this point later on (Chapter VI.).

In experiments in the laboratory, where the object is
likewise to prepare cultures of fermentation organisms,
greater demands may naturally be made than on a practical
scale. In this case it is necessary to work with absolutely
pure cultures, partly in small quantities, partly in masses
so large that they may be transferred at a given point of
time from the laboratory to the brewery. Conditions which
are wanting in practice are sought to be realised in the
laboratory, which is specially arranged for such investigations.
We will now briefly mention these requirements and the way
in which they are met, and, for purely historical reasons,
we will begin with the last link, viz., with the vessels and
liquids which receive the originally-prepared small pure
cultures, and the expedients to be employed in their cultiva-
tion. It is necessary that these vessels and liquids be sterile
before the inoculating substance is introduced, i.e., they must
be freed from all living germs ; also that the various utensils
and the air in the place where the work is performed should
contain as few living germs as possible. The same applies
of course to the clothing and hands of the experimenter.

3. STERILISATION.

The principles of the technology of sterilisation as well
as the models of the various apparatus appertaining thereto

10 MICRO-ORGANISMS AND FERMENTATION.

had been given in the old experiments on spontaneous
generation.

As early as the year 1765, Spallanzani argued against the
doctrine of Needham and Buffon, that living beings came
into existence through spontaneous generation in putrefying
liquids or other substances. SpaUanzani warmed extract of
meat in closed flasks, and demonstrated that the contents
of the flasks remained unaltered until air is allowed to
gain admittance. From this he concluded that the germs
which developed in the opened bottles had come in from the
air. Later (1782) Scheele demonstrated that vinegar may
be made to keep sound by means of warming. But his
discovery was not heeded. In 1810 Appert published his
book on a means of preserving various foods and liquids
through warming. In the 4th edition of his book, which
appeared in 1831, he gives directions for the treatment of
wine, beer, and other liquids, his method being essentially
the same as that employed in our day (the so-called " Pas-
teurisation " ).

To the following period, which was of such great impor-
tance for micro-biology, belong the highly meritorious
researches of F. Schulze and Th. Schwann, in which it was
shown, that perishable liquids which had been vigorously
boiled in flasks would remain sterile if the air subsequently
entering were made to pass through sulphuric acid or through
red-hot tubes. At the same time Cagniard-Latour and
Schwann described the yeast-cells, and Kutzing the acetic
acid bacteria. Turpin started (1838) that most important
doctrine : " No decomposition of sugar, no fermentation
without the physiological action of vegetation."

Finally, the objection against the experiments of Schulze
and Schwann, that the air entering the flasks had been
affected in some manner by the violent treatment to which
it had been submitted, that it was no longer able to furnish
the conditions of growth required by the germs existing in
the liquid, was overcome by the beautiful experiments of

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 11

Schroder and Dusch (1854), who caused the air to pass
through cotton-wool filters, and by this means still obtained
the same result.

The principles of the whole technology of sterilisation
being thus established, the matter under consideration reached
a high state of development and great importance both for
science and for industry, more particularly through Pasteur
and, subsequently, several other eminent scientists, who
devoted their energies to these investigations.

1. Sterilisation of glass and metal articles. — Sterilisa-
tion properly so-called must always be preceded by a thorough
mechanical and, in many cases, also a chemical cleansing.
Articles of daily use in the laboratory, as, for instance, spatulas,
pins, wire, etc., are heated directly in a flame and allowed
to cool in a germ-free space. Many pieces of apparatus,
however, do not admit of this treatment, and must be sterilised
either by heating in steam or in a water-bath, or else in dry
air by means of a sterilising oven, in which the objects are
heated for one or two hours at a temperature of about 150° C.
According to the nature of the objects, some may be put
directly into the sterilising oven, whilst others must be pre-
viously wrapped in paper. The necks of the flasks are closed
by cotton-wool, which is, in addition, often covered by several
layers of filter-paper.

2. Sterilisation of nutritive liquids and solid nutritive
substrata.— Nutritive liquids can be sterilised by filtration
or by heat. The former method presents the advantage that
the liquids treated undergo less change than when heat is
employed, and are, consequently, better suited for the develop-
ment of many species of micro-organisms. The necessary
condition for sterilisation is, that the pores of the filter must
be smaller than even the smallest micro-organisms. Gypsum,
asbestos, charcoal, and porcelain have been employed for this
purpose, the liquids being forced by pressure and suction
through thick layers of these substances. The form most
generally used is the Chamberland porcelain filter, which,

12 MICRO-ORGANISMS AND FERMENTATION.

however, requires frequent cleansing, and must also be
frequently sterilised by ignition, it having been proved
that the bacteria are able in time to grow through the
pores1.

Liquids and solid nutritive substrata are in most cases
sterilised by heat. The way in which this must be done,
as well as the duration of the heating process, are dependent
on the nature of the substratum in question. Direct boiling
on the sand-bath may be employed for the purpose of steril-
ising, for example, brewery-wort in Pasteur-flasks, otherwise
the water-bath may be used. An excellent means of sterili-
sation is afforded by steam either at 100° C. or under pressure
(110— 120° C.) by means of Papirfs digestor (autoclave).
During cooling, care must be taken that only absolutely pure
air comes in contact with the sterilised substance, the air
entering the vessel being filtered through cotton-wool or
passed through tubes bent several times, if it is drawn in
slowly and with no great force2.

1 In breweries the filtration of beer has been resorted to during the
last few years, the filtering media commonly used being paper, cellulose,
asbestos, etc. By such filtration brewers sometimes succeed, it is true, in
freeing a beer originally sound from deposits of various kinds, and in
rendering it bright ; but, on the other hand, the fact must be emphasised,
that an indiscriminate employment of this method may occasion great
dangers, as has been directly proved by the experiments made by Thausiny,
Wichmann, Reinke, and others. If, namely, the filters are not sufficiently
effective, it may happen that only the yeast cells are retained, but not the
bacteria, which are then enabled to act with much greater energy upon
the liquid. Another great danger lies in the fact that a filter, owing to
deficient cleansing, may become a seat for the development of different
kinds of germs, contaminating all the beer passing through it. If a single
cask of a store-room has become infected, and the filter is not effectually
sterilised after the filtration of this beer, the disease will be communicated
to all the other beer.

2 In the so-called Pasteurisation of beer, a merely relative sterilisation
is all that is generally aimed at ; that is to say, by a cautious treatment
of the beer at elevated temperatures, it is sought to check the yeast cells
to such a degree that they are capable only to a very limited extent of
multiplying and producing fermentation. It is only for transportation
to a great distance that it is attempted to kill all living germs contained

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 13

Nutritive gelatines must be treated with particular care,
as they often lose their power of gelatinising if the heat is
too great or if it be applied too long.

If the substance cannot be boiled without suffering great
changes or entirely losing its original nature, fractional
sterilisation must be resorted to.

This, for instance, is the case with blood-serum, which is
employed in a gelatinous condition in bacteriological studies.
This substance, when heated to 100° C., becomes fluid, and
does not again solidify, and it is, therefore, necessary to pro-
ceed in a different way in order to get it sterilised in the
gelatinous state. It was observed that a temperature of 58°
to 62° C. was sufficient to kill the vegetative bacteria which
develop in blood-serum. By this treatment of the substance
only the spores of bacteria remain unkilled. If the gela-
tinised serum is placed for two or three days in an incubator
at a temperature favourable to the development of the spores
(30° to 40° C.), the greater number of these germinate, and
the new vegetative rods can then be killed by again heating
to about 60° C. If this process is repeated several times,
the gelatinous mass will remain sterile for an unlimited
time. This process, which is also used for the sterilisation of
milk, and which was discovered by Tyndall, has been further
established by R. Koch.

A similar method is employed in zymotechnical laboratories
for the treatment of nutritive liquids, which, when boiled, are
apt to deposit a considerable amount of albuminoid matter,
and would thus become comparatively bad nutritive liquids
for the alcoholic yeast.1

in beer. No general rules can be laid down for a treatment of that kind.
The correct procedure depends on the nature of the liquid as well as on
the properties of the particular species of yeast, and preliminary experi-
ments must accordingly always be made with regard to temperature as
well as to the duration of the action of the particular temperature.

1 Sterilisation is also attempted in practice, namely, for the purpose
of introducing wort in a sterile condition by means of closed cooling and
aerating apparatus into the fermenting-tuns. It is true that the wort

14 MICRO-ORGANISMS AND FERMENTATION.

3. Sterilisation of the Air is best attained, as stated
above, by means of cotton-wool filters ; sulphuric acid, salt
water baths, cloth filters, etc., are less efficient. In labora-
tories, where work must often be performed in germ-free air,
a glass chamber is employed, the front of which can be raised
sufficiently for the operator to introduce his hands. Some
time before using the chamber, the whole of its inner surface
must be washed, and the chamber then closed. The particles
and germs suspended in the air will then settle to the moist
bottom and remain there.

4. DISINFECTION.

Another method of killing disturbing germs is by the use
of disinfectants, which act as poisons on the micro-organisms.
Not a few of these substances have found application in
practice. The limit for the employment of such poisonous
substances must be determined for each individual case. As
manipulations with such poisons may be deleterious to the
operator, it is important to ascertain their proper degree of
dilution.

Investigations having for their object the determination of
the poiver of resistance of the various species of micro-
organisms to poisons have proved that it varies greatly
in different cultures of one and the same species, not only
for the spores, but also for vegetative forms. A young
culture behaves differently from an old one, and the same
applies to individuals belonging to one and the same culture.

cannot keep absolutely free from germs when the fermentation takes
place in open tuns, but a great deal can be effected in this direction by
acquiring a true and thorough understanding of the matter. ' The expert
brewer will always take care that the air in the fermenting room is
kept as free from germs as possible, and also that the tuns as well as all
utensils that are immersed into the fermenting liquid, e.g.. thermometers,
sample glasses, etc., are always perfectly clean. As a matter of course,
these precautionary measures could not acquire any real practical import-
ance until, through Hanseris reform, absolutely pure yeast had been
introduced into the fermenting room.

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 15

In all experiments of this kind, organisms which have been
tested with some disinfectant should afterwards be brought
under the most favourable conditions of growth, otherwise
they will not develop, even though they be alive and capable
of development. In such cases, the ordinary temperature of
the room and solid nutrient substrata are not sufficiently
favourable ; it is also necessary to allow ample time for the
observation of such growths before definitely deciding whether
they are dead or not ; in fact, it often happens that they have
merely been somewhat checked in their development, and
that they may develop again, after some time, with their full
vigour. Furthermore, the temperature and the medium
in which the organisms are present when the disinfectant is
employed may be of some importance. Before testing a
culture thus treated, great care must be taken to previously
free it from all remains of the disinfectant by washing and
dilution.

The first information on this subject we owe to R. Koch.
Subsequently, these researches were continued by Gruber and
others.

Koch examined several poisons not only with reference to
the degree of concentration requisite for destroying bacteria
and spores of bacteria, but also with a view to ascertain the
particular quantity necessary for checking the micro-organ-
isms in their power of development in suitable nutritive
solutions.

I will here briefly state the results obtained by Koch.
Carbolic acid was found to be a less efficient disinfectant than
it is commonly held to be. A solution containing 5 per cent,
destroyed the power of development of anthrax spores only
after 48 hours, whilst the anthrax bacilli were killed in two
minutes by a 1 per cent, solution. A solution of 1 part in
850 proved sufficient to check the growth of the bacillus,
and when anthrax spores were moistened 5 to 7 times with a
5 per cent, solution, their development was somewhat retarded.
A 5 per cent, solution of carbolic acid in oil or alcohol had

16 MICRO-ORGANISMS AND FERMENTATION.

absolutely no effect on the anthrax cells and spores. In the
form of vapour, carbolic acid acts more strongly, although
even two hours' action of its vapour at 75° C. proved unable
to destroy the vitality of these spores. Sulphurous acid is not
able to destroy all germs, even under very favourable con-
ditions. On the other hand, chlorine, bromine, and corrosive
sublimate are efficient disinfectants. According to Koch,
corrosive sublimate in the proportion of 1 in 1000 acts fatally
on all germs. According to experiments made by Johan-Olsen,
however, mould-fungi are only destroyed by more concentrated
solutions ; e.g., Penicillium glaucum only by a solution contain-
ing 1 in 400. Several bacteria, the organisms of puerperal fever,
abscesses, and putrefaction, likewise germinate and grow,
although more slowly than usual, on slices of potato saturated
with a solution of sublimate containing 1 part in 500, and
their growth is only checked by a concentration of 1 in 300.
Gruber found from recent investigations, carried out with all
precaution, that anthrax spores, for instance, were only killed
by 5 parts of sublimate in 1000, 1 part of sublimate hydro-
chloric acid in 1000, 1 part of sublimate tartaric acid in
1000.

For cleansing pipes, coolers, etc., which often contain very
considerable deposits of organic matter liable to decomposition
through the agency of micro-organisms, a solution of soda is to
be recommended ; it acts by dissolving and loosening resinous
and albuminoid matters, which can then be removed by means
of water. Experiments made by Aubry and Will have proved
that chloride of lime, even when strongly diluted (2 — 5 per
cent, of chlorine) is a very efficient disinfectant, owing to its
deadly action on fungi. This substance being very cheap is
therefore specially suited for cleansing walls, pavements,
gutters, etc. Bisulphite of lime is also very efficient (used in
solutions containing 2 — 4 per cent, of sulphurous acid). The
filter-bags — which, according to the investigations of Will,
often contain very considerable accumulations of wild yeasts
and bacteria in the very texture of the material, and which

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 17

in many breweries are never sufficiently cleansed — should be
disinfected by treatment with a solution of chloride of lime.
Will, who experimented with material obtained from a brewery,
recommends a solution containing 1 per cent, of active
chlorine (corresponding to about 3 — 3^ kg. of good commercial
chloride of lime to 100 liters of water). The filter-bags must
be washed with pure water after this treatment In physio-
logical laboratories, where it is of especial importance to guard
against any invasion of foreign germs, an alcoholic solution of
salicylic acid will prove of service (it is often used by Hansen

FIG. 3.

Pasteur's Flask.

in the Carlsberg laboratory for cleansing tables). The action
of this substance, even in a diluted state, in checking fer-
mentation is generally known.1 Eecently, hydrofluoric acid
has also been employed as a disinfectant (see p. 25).

5. FLASKS : PASTEUR, CHAMBERLAND, FREUDENREICH,
HANSEN, AND CARLSBERG FLASKS.

All vessels in which cultures are made must satisfy the

1 Biernacki and others have proved that substances otherwise
possessed of antiseptic power can, when very much diluted, act as
stimulants on yeast-fungi. Thus, alcoholic fermentation is promoted by
the addition of corrosive sublimate in a dilution of 1 in 300,000, by
salicjlic acid solution of the strength 1 in 6000, and by boracic acid
solution of the strength 1 in 8000.

c

18 MICRO-ORGANISMS AND FERMENTATION.

condition that they are proof to every contamination from
without. Pasteur's flasks satisfy this demand in the highest
degree.1 The illustration shows this flask in the slightly
modified form employed in the Carlsberg Physiological
Laboratory directed by Hansen. When the nutritive liquid is
boiled, the steam first escapes through the wide straight tube,
at the end of which is a piece of India-rubber tubing ; when
this is closed the only outlet for the steam is through the
bent tube. After some time the flask is taken from the
sand-bath, and the bent tube may be closed with a plug of
asbestos. The sterilisation is then complete, and the contents
of the flask can remain for years without suffering alteration.
During the cooling and the indraw the air is partially filtered
through the asbestos-plug ; any germs that are carried
further are deposited in the lowest part of the bend, or,
at the most, do not pass the enlargement of the thin tube,
and therefore do not come into contact with the liquid.
Hence, it is evident that the lower part of the bent tube
must be heated whenever the flask is to be agitated or
emptied through the straight tube, without exposing it to
contamination. If the flask is to be opened and placed in
connection with another flask, this must be effected either in
some small germ-free space, or the opening and connecting
must be done in a flame. A Bunsen burner is placed
directly in front of the operator, the flask to be emptied to
the left, and the one that is to receive the liquid or culture
to the right, close to the burner. Then the tube of the
left-hand flask is opened in the flame by quickly removing
the India-rubber tube with its glass stopper ; while the open
tube is in the flame, the glass stopper of the flask to the

1 Chevreul and Hoffmann had previously found that when vessels
employed in sterilising liquids are provided with open but bent tubes
their contents will remain sterile. Although Chevreul was thus the
first to indicate the principle of these flasks, I will not mention them
by any other name than that of Pasteur, through whom, indeed, they
have obtained a wide application.

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 19

right is quickly withdrawn, and the hot tube of the first
flask is introduced into the India-rubber tube of the second
flask. The liquid is now poured into the latter flask, the
bent tube of the former flask being at the same time
heated. Then the side tube of the left flask is again
introduced into the flame, while the stopper of the right
flask is heated and put back into its place ; finally, the left
flask is closed in, the flame with its tube and stopper.
When the operation is quickly performed, there is seldom
any danger of contamination.

Pasteur flasks will be found indispensable in certain

FIG. 4.
Chamberland Flask.

operations ; as, for instance, in physiological researches where
one has to deal with large quantities of liquids.

In recent years various other flasks and vessels have
been brought into use, notably the Chamberland flask
(Fig. 4), the neck of which is closed with a ground cap,
which terminates above in a short, open tube ; this tube
is filled with tightly-packed sterilised cotton-wool.

The Frewdenreich flask is constructed on exactly the
same principle ; it has, however, a cylindrical shape.

For certain special purposes Hansen's flask (Fig. 5) is
employed. The ground cap is provided with a cotton-

20 MICROORGANISMS AND FERMENTATION.

wool filter (a), and the flask has a small side-tube closed
with an asbestos stopper (d). This flask is used partly for
the preservation of pure cultures, partly for sending small
cultures or samples from the propagating apparatus.1
For the first-named purpose the flask is half filled with a
10 per cent, solution of cane-sugar, to which a trace of
the yeast-culture is then added. The asbestos stopper and
lower edge of the cap is coated with sealing-wax (c). For
the last-named purpose the lower part of the flask is
filled with cotton-wool (e\ and some cotton-wool (6) is
also put into the cap, below the filter. For the mode of
employment, see Chapter VI.

FIG. 5.
Hansen Flask.

For the development of very large cultures the Carlsberg
vessels (Fig. 6) are employed. They have a capacity of
10 liters, are made of tinned copper, are cylindrical in
shape and conical at the top; at the apex of the cone a
twice-bent tube (c d) with an enlargement (e) is either
soldered or screwed. At one side of the cooe is the
inoculating tube and glass stopper (a), and at the bottom
of the vessel is another tube (6) for drawing off the
fermented liquid and the yeast. This tube is provided
with a pinch-cock. When the liquid is sterilised, the

1 For the description of this apparatus, see Chapter VI.

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 21

bent tube is closed with an asbestos or cotton-wool filter,
which is either screwed on or placed over the end (d).
For further particulars respecting the treatment of these
vessels, see Hansen's " Untersuchungen aus der Praxis der
Garungsindustrie."

6. NUTRITIVE SUBSTRATA.

With regard to the nutritive substrata, the problem
naturally always consists in finding those which are best
suited to the respective organisms. If they also possess the

a

FIG. 6.
Carlsberg Vessel.

advantage of being per se less favourable for the development
of competing forms, it is a great point gained. The rule
must of course be borne in mind, when comparative investi-
gations are made in different directions, that the nutritive
liquid must always remain the same. For the investigation
of alcoholic ferments Hansen generally uses hopped wort from
the filter-bags ; in special cases of investigations of this kind
yeast- water with an addition of glucose, or a solution of cane-
sugar or some other sugar, is employed. If it is desired to
use a solid nutritive material, the liquid may be mixed with

22 MICRO-ORGANISMS AND FERMENTATION.

5 to 10 per cent, of gelatine. Similar liquids, or more
frequently, meat-extract with an addition of peptone, are
employed for bacteriological investigations ; this mixture is
neutralised with sodium carbonate. Either gelatine or agar-
agar is used for rendering the medium solid. Solid nutritive
substrata are the best for the study of mould-fungi, in most
cases preferably sterilised black bread. Where liquids are
employed, the most suitable are beer-wort, fruit decoctions, or
mixtures of sugar with an addition of tartaric acid or tartrates.
Pasteur used exclusively liquids as substrata in his investi-
gations on the organisms of fermentation. Later, solid
substrata were very extensively employed, and in this respect
Koch has given many practical illustrations.

We have now briefly explained how our micro-organisms
are cultivated, and guarded against contamination from the
liquid itself, from the vessels and apparatus, from the air,
and from the experimenter. We have now before us the
first and most important question: How are we to obtain
the first absolutely pure culture to be introduced into the
flask? I have, on purely historical grounds, first sketched the
conditions for the preservation of the pure culture, because
these were known long before a certain method for preparing
the pure culture itself had been discovered.

In this respect it will be instructive to see how we have
advanced step by step, and we will again take up the subject
historically, from the moment when really rational endeavours
were made for the attainment of this object.

7. PREPARATION OF THE PURE CULTURE.

It is only by starting with one, individual cell that we
can be certain of obtaining a really pure culture, and such
a culture is the indispensable condition for exact scientific
investigations of the micro-organisms. These investigations
may, as stated above, be carried out for different purposes,
namely, with a view to observe the individual, the isolated
cell through its successive phases of development, — morpho-

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 23

logical investigation; or the object may be to study the
vital functions of a growth developed from a single cell, —
biological and physiological investigation. As these two
methods of investigation are of different natures, the means
to be employed must likewise differ.

(a) Pure Cultures for Morphological investigations. —
After the discovery had been made, by means of the microscope,
that yeast consists of cells, it was not long before the attempt
was made to determine, by closely observing one of these cells,
the way in which they multiply, and in what forms the new
generations occur. In other words, a morphological examina-
tion of a pure culture was made. For this purpose it became
necessary to guard against such disturbances as would arise
from other cells hindering the selected one from multiplying
or withdrawing it from the observer's view. On the other
hand, it would not matter if foreign cells occurred in other
portions of the preparation.

Ehrenberg^ as early as 1821, observed the germination of
the spores of some fungi by means of investigations of this
kind. Later, the propagation of yeast-cells was observed by
Mitscherlich, Kutzing (1851), and F. Schulze (1860), in the
same way. A small quantity of high-fermentation yeast was
diluted with beer-wort until it contained only one or two
yeast-cells ; from a drop of this an ordinary preparation was
made, the cover-glass was cemented fast on the glass slide,
and the development of the cell was watched under the
microscope. The same method was employed, in its main
features, by Tulasne (1861) and De Bary (1866) in their
famous researches on the germination of the spores of the
fungi. The investigation was carried further by Brefeld, who
followed the development of the mycelium until it in its turn
again formed spores. He sowed the spores on the object-glass.
When his investigation was to extend over a longer space of
time, during which an ordinary drop of liquid would evaporate,
he added gelatine to the liquid, and placed a small shade of
paper over the apparatus; this shade was attached to the

24 MICRO-ORGANISMS AND FERMENTATION.

tube of the microscope in order to keep out foreign germs
as much as possible. When the development took place in
ordinary fluid drops, the preparation was placed, in the interval
between two observations, under a moist glass-shade ; thus,
an unbroken observation was not attempted, and was not even
possible for the larger fungi. Accordingly, in consequence
of the whole arrangement of the experiment, absolutely pure
cultures are quite out of the question. As stated above,
however, such an investigation may very well be carried on
with an impure material.

(6) Pure Cultures for Physiological experiments with
mass-cultures. — When the object of the pure culture is to
employ it for biological or physiological researches, so
that a mass-culture of the growth becomes necessary, a
direct microscopical control is impossible, and the methods
described above cannot be employed. The methods made
use of for this purpose may be divided into two groups,
namely, the physiological methods and the dilution methods.
In the former, liquids are used, in the latter liquids or
gelatines.

(«) Physiological methods. — The physiological methods
employed by Pasteur, Colin, and others, start with the
fundamental idea, that the various species occurring in a
mixture will multiply unequally according to their different
natures, when they are cultivated in one and the same nutritive
liquid and at the same temperature, so that those species for
which the conditions are unfavourable will be gradually
suppressed by the one or more species for which the condi-
tions are favourable. Different liquids have been employed for
such cultures in the course of time ; as, for instance, alkaline
liquids for growths of bacteria, acid liquids for the purpose of
freeing yeast-growths from bacteria (lactic, tartaric, hydro-
fluoric acids, etc.). The weak point of all such methods is,
that they start from an unknown material, namely, the
impure mixture. Hence, it is impossible to know what
results a treatment of this kind will lead to, because it is

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 25

evident that any agency exerted will be hap-hazard, and
this does not, properly speaking, constitute a method ; in
fact, there is always the possibility that the weaker species
are not destroyed at all, but merely checked and retarded,
so that when the stronger species, after having reached the
height of their development, enter into a condition of weak-
ness, other species will get a chance of multiplying. Likewise,
there is always the possibility that not one but two or more
species thrive equally well in the liquid, and, consequently,
develop to the same extent. If we examine, for instance,
common brewers' yeast, we may often separate several
typically different species of " culture-yeast," as they are
termed, from the same yeast-mass by means of Hansen's
method. The method given by Pasteur for the purification
of a brewers' yeast may be mentioned as a marked illustration
of the dangers connected with the physiological method of
treatment. The impure yeast-mass is introduced into a cane-
sugar solution to which a small amount of tartaric acid has
been added. The object of the method is to free the yeast from
any disease germs with which it may be infected. Hanserfs
investigations have, however, proved that, even if the bacteria
are suppressed or checked by this treatment, the so-called
wild yeasts, and among them those productive of diseases,
will develop abundantly, and in many cases the culture-yeast
becomes totally suppressed instead of being purified. Even
if there is primarily only a trace of the wild yeasts, or yeasts
of disease, these are apt to develop to such an extent through
this treatment that finally they may form the chief portion
of the yeast-mass. Thus, this unmethodical treatment of the
unknown material has led to an exactly opposite result to
that intended. Even when the yeast-mass consists entirely
of the so-called wild yeasts, it is not possible by this process
of Pasteur's to prepare with certainty a pure culture of a
definite species.1

1 According to Effronfs method, hydrofluoric acid is used as an anti-
septic in distilleries. But the use of this material for the purification of

26 MICRO-ORGANISMS AND FERMENTATION.

If, now, we ask, whether it is advisable to employ any of
the various methods mentioned above for the purification
of an unknown impure yeast-mass, the answer must accord-
ingly be in the negative ; and this will be the case whether
the culture is intended for purely scientific or for industrial
purposes, for the danger will always remain of furthering the
growth of species other than the desired one. And, the
starting-point being uncertain, it necessarily follows that the
result must be so too. In fact, all such methods must now be
regarded as antiquated, and will, whenever resorted to, prove
utter failures. Yet in certain cases they may have some
value when employed preparatory to the preparation of a
pure culture. In the different branches of the fermentation
industry there is only one way that will lead to the goal,
namely, the application of the same principles which have
for many years been followed in agriculture and horticulture
— the selection, by means of methodical experiments, of
the particular species or type which gives the best results
under the circumstances, and which is therefore to be sown
alone, without any admixture of other types. The only
possible way of effecting this is, however, by the adoption
of the methods discovered by Hansen, which will come
under consideration later on.

(/3) Dilution methods. —The second group of methods
employed for physiological purposes embraces the dilution
methods, or the so-called "fractional cultivation," the

an impure yeast-mass, whether brewers' or distillers' yeast, as proposed by
Effront, will give rise to the same dangers as were mentioned above in
the case of tartaric acid. In fact, a long series of methodical experi-
ments made in the laboratory of the author of this book have shown that
by the treatment of impure yeast according to Effront's method the wild-
yeasts and Mycoderma species will develop far more actively than culti-
vated yeast ; and our experiments have also shown that in many cases
even such a dangerous species as Bacterium aceti cannot be suppressed at
all by this treatment of the ye<ist-mass ; on the contrary, it was found to
multiply much more actively when treated with hydrofluoric acid or
fluorides.

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 27

principle of which is to dilute the material to such a
degree that it is ultimately possible to isolate a single
cell. In most of these cultures we can only reckon on their
probable purity, whereas for the alcoholic ferments Hansen
has developed the process into an exact method.

Lister was the first (1878) who brought methods of this
kind into use. In order to prepare pure cultures of lactic
acid bacteria he first determined microscopically the number
of bacteria in a very small drop of sour milk, counting them
in several fields of the preparation, and thus calculating their
number in the whole preparation. He then calculated the
amount of sterilised water required to be added in order that
after dilution there would be on an average less than one
bacterium in each drop. With five of these drops he
inoculated in one case five glasses containing boiled milk.
The result was that the milk in one of these coagulated,
showing that it contained Bacterium lactis, whilst the four
other glasses remained unaltered and did not show the
presence of bacteria. The same method was subsequently
employed by N'dgeli and Fitz.

Air has also been made use of for such a dilution
(Pasteur). A small portion of yeast is dried and ground
with powdered gypsum. The resulting fine powder is thrown
into the air from a height, a series of vacuum flasks (p. 37)
being opened while the particles are falling. Isolated yeast-
cells which are distributed in the resulting dust-cloud may
then perhaps enter some of the flasks.

In comparison with the physiological methods the dilution
method now described is a distinct advance ; indeed, we have
here approached much nearer to the goal. On the other
hand, it is clear that, even if the dilution is carried as far as
in the case mentioned, in which only one of several flasks
shows development, it is not yet proved that this one flask
has received only one germ. Thus, there is still great
uncertainty, even in such cases where the individuals with
which we are working can be counted. Moreover, such

28 MICRO-ORGANISMS AND FERMENTATION.

countings are very difficult in the case of the bacteria, and
often, indeed, quite impossible. In all cases the accuracy of
such calculations is very questionable. Thus, the question
remains to be solved : How are we to distinguish the flasks
which have only received one cell from those which, in spite
of the counting, have been infected with several cells f For
the bacteria, no means has as yet been found of solving this
difficulty.

In the case of the yeast, this problem was solved by
Hansen, who developed the method to such a degree of
perfection as to create, in fact, an exact method (1881). He
employed dilution with water, in the following manner : —
The yeast developed in the flask is diluted with an arbitrary
amount of sterilised water, and the number of cells in a
small drop of the vigorously-shaken liquid is found. The
counting, in this case, is effected in a very simple manner
by transferring a drop to a cover-glass, in the centre of
which some small squares are engraved, and this is then
connected with a moist chamber (Fig. 2) ; the drop must not
be allowed to extend beyond the limits of the squares. The
cells present in the drop are then counted. Suppose, for
instance, that 10 cells are found ; a drop of similar size is
transferred from the liquid, which must first be again
vigorously shaken, to a flask containing a known volume,
e.g., 20 ccm. of sterilised water. This flask, then, will in all
likelihood contain about 10 cells. If it is now vigorously
shaken for some length of time, and then 1 ccm. of the
liquid introduced into each of 20 flasks containing nutritive
liquid, it is probable that half of these 20 flasks have received
one cell each. But, here again, as in Lister's experiments,
it is entirely a calculation of probabilities. If the flasks are
left in repose for further development of micro-organisms,
there will be a chance of getting a pure culture in some of
them. But no certain inferences can be drawn. Hansen
succeeded, however, in adding a new link, which first gave
certainty to this experiment. If, namely, the freshly

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 29

inoculated flasks are vigorously shaken, and then left in repose,
the individual cells will sink to the bottom, and become
deposited on the wall of the flask. It is self-evident that if
the flask contains, for instance, three cells, these cells will
always, or at least in the majority of cases, be deposited
separate from each other and apart, on the bottom. After
some days, if the flask is raised carefully, it will be observed
that one or more white specks have formed on the bottom of
the flask. If only one such speck be found, we have obtained
a pure culture.

It is evident that by means of this method we are also
able to introduce a single cell into the flask with nutritive
solution.

It was by this method that Hansen prepared all his first
pure cultures, with which he carried out his fundamental
researches on alcoholic ferments.

Solid nutrient media have also been employed for the
preparation of pure cultures for use in physiological investi-
gations. The foundation of such methods was laid by Schroeter
(1872), who, in his researches on pigment-bacteria, employed
slices of potatoes among other nutrients. He had observed
that when such slices had been exposed for some time to the air,
specks or drops of different form and colour made their appear-
ance. Each of these specks contained most frequently one
species of micro-organism.

R. Koch subsequently considerably improved this method.
He at first prepared his pure cultures by means of streak
growths in nutritive gelatine. Afterwards he devised a far
better method, the so-called plate-culture method (1883).
He proceeds in the following manner. A trace of the crude
culture is transferred to a large proportion of sterilised water.
From this a small quantity is transferred to a flask containing,
for instance, a mixture of meat-broth and gelatine warmed to
30° C. The flask is shaken in order to distribute the germs,
and the contents poured on to a large glass plate, which is then
covered with a bell-glass. The gelatine quickly sets and the

30 MICRO-ORGANISMS AND FERMENTATION.

germs remain enclosed in the solid mass. In a few days
they develop to colonies — points or specks which are visible to
the naked eye. The purity of the specks of bacteria in the
gelatine is ascertained, according to Koch, partly by their
appearance, colour, form, etc.

When regarded more closely it will be seen, however, that
there is no essential difference between this distribution of
the germs in the liquid gelatine, and the former dilution by
means of liquids. The same uncertainty is always present :
neither the macroscopical observation of the appearance of the
colony nor the microscopical examination of its contents
gives any surety of its only containing one species.

The only possibility of securing a really pure culture in
the gelatine consists in the direct observation of one indivi-
dual germ and its development.

Hansen has done this in the case of yeast-cells, and the
method which he contrived for the purpose is as follows. The
layer of gelatine formed by the solidified nutritive liquid is
arranged in such a way that the position of the isolated
germs can be observed under the microscope. The position
of these germs, then, is accurately marked, and the cell can
be seen to develop and propagate step by step.

For the glass-plate is substituted a round cover-glass of
about 30 mm. diameter. This is fastened to a glass-ring,
which again is cemented to a thicker glass, thus forming the
moist chamber previously described (Fig. 2), and which is
adapted to the purpose, and carries a layer of solid gelatine on
its inner upper surface. The essential point in Hanserts
method is, that the leading principle — " the starting-point of
a pure culture must be a single cell " — is consistently carried
out, which is not the case in Kochs method. The germs
must be so sparsely distributed that comparatively few are
present in the gelatine layer ; the chamber is then either
allowed to remain under the microscope, in order that the
multiplication of the germs may be directly followed, or the
positions of the well-isolated germs are marked, either by

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 31

dividing the glass-cover into small squares or by means of the
object marker, and the apparatus is placed in the incubator
until the colonies are completely grown. On one cover-glass
there may be 50 to 60 well-isolated germs. When the colonies
are completely developed, they are transferred to flasks by
means of a small piece of platinum wire, which has been pre-
viously ignited and cooled. During this transference the
culture is for an instant in the air, and is here exposed
to contamination. But the danger of contamination at
this, the single weak point, is reduced to an insignificant
minimum, and disappears if the above-mentioned opera-
tion, be performed in a small enclosed germ-free space ;
as, for instance, in a small chamber with glass sides
which is sufficiently large to admit the apparatus and the
hands of the experimenter (see p. 14). In this way the
transference of the colonies is effected with all possible
security. From the first flask the culture can be transferred
without contamination to a continually increasing number of
larger flasks. Thus, Hansen's method approaches the desired
end as nearly as is possible, and is consequently employed
everywhere in exact experiments of this kind.

As early as the year 1883 Koch's method of plate-culture
was tested by Hansen. He prepared a mixture of two species
of yeast which can be distinguished from each other micro-
scopically, namely, Saccharomyces apiculatus and a species
of the group Sacch. cerevisise. This mixture was introduced
into wort-gelatine, and after shaking was poured on to a glass-
plate. Of the specks formed, about one half contained one
species exclusively, the other half the other species, and in
one of the specks both species were found.

Later (1888) a similar control was carried out for the
bacteria by Miq^lel, who introduced 100 colonies from a
plate-culture obtained in an air-analysis into 100 flasks
containing meai>broth with peptone. The examination of
the growths developed in the flasks showed that they con-
tained 134 different species of micro-organisms. The cause

32 MICRO-ORGANISMS AND FERMENTATION.

of this evidently depends upon the fact that it is very
difficult, and often quite impossible, to separate all germs
of bacteria and other organisms from each other by simply
shaking the gelatine mixture. This test proves therefore
that the plate-culture involves very material errors.

Holm has subjected the method to a thorough analysis
(1891), and has experimented with a considerable number
of yeast-species, absolutely pure cultures of which were
prepared by the above-mentioned method of HanserCs.
The result of 23 series of experiments with different
mixtures was, that only in a single case were 100 colonies
developed from 100 cells; that is to say, all the colonies
were pure cultures. In all the other series the method
proved faulty. In the most unfavourable case 100 colonies
were yielded by 135 cells, and the average number obtained
was 100 colonies from 108 cells. This proves the plate
method to be faulty also in the case of yeast.

Thus, the advantage of Han-sen's method over Koctis
for the pure cultivation of yeast is, that it has a certain
starting-point. Even if the plate-cultures are repeated
several times, one can never be certain whether the desired
result has been attained or not. With regard to the
bacteria, however, it is generally impossible to secure a
starting-point from one individual cell. In such cases
Koch? 8 plate-culture is still the best method we have.

8. COUNTING THE YEAST-CELLS.

In the yeast and spirit manufactures it is of impor-
tance to determine the multiplying capacity of the yeast-
cells during the growth of the yeast. This must naturally be
effected by a direct counting of the number of cells which
occur in a determinate volume of the liquid at different
stages of the fermentation. Experiments having this object
in view have been undertaken especially by Delbruck,
Durst, Hansen, Hay duck, and Pedersen, whilst Fitz has
applied the method of counting to bacteria.

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 33

The counting is performed by means of an apparatus
constructed by Hayem and Nachet (Fig. 7), which was first
employed for counting the corpuscles of blood (hence termed
hcematimeter). The late Prof. Panum, of Copenhagen, was
the first to employ this apparatus for counting micro-
organisms, in order to determine their multiplying capacity.
The hsematimeter consists, as shown in Fig. 7, of an object-
glass on which a cover-glass of knoivn thickness (0*2 mm.,
for instance) is cemented, and from the centre of which a
disc has been cut out. A small drop of the liquid containing
the cells is brought into the cavity thus formed, a cover-glass
is placed over the opening, and thus rests on the cemented
and perforated cover-glass. The drop of liquid must not be

FIG. 7.

Hsematimeter : a, object-glass ; b, cemented cover-glass with circular opening ;

c, cover-^

so large that the pressure of the cover-glass causes it to flow
out from the enclosed space, yet it must be high enough to

be in contact with the cover-glass. The thickness of the

o

layer of liquid is then known. In order to determine the
other two dimensions, and thus be able to work with a given
volume of liquid, one of the generally known micrometers,
e.g., a thin piece of glass on which 16 small squares are
engraved, is introduced into the eye-piece of the microscope.
The actual value of each of these squares is known when a
given system of lenses is employed, and thus, when the
square is projected on the object, a small prism of known
volume is defined. In certain cases it may be more expedient
to make use of an appliance constructed by Zeiss, of Jena,
from the instructions of Thoma, and which consists of a fine

34 MICRO-ORGANISMS AND FERMENTATION.

system of squares of known size, engraved on the object-
glass itself at the bottom of the cavity. This also improves
the microscopic definition of the cells which are on the
bottom of the chamber.

When it is merely desired to determine the rapidity with
which the cells multiply, or to make repeated observations of
the number of cells in the same volume, it is quite super-
fluous to determine the size of this volume ; it is then only
necessary to work always with the same volume.

It is always necessary that the sample taken should be a
fair average one. In most cases it must be diluted and
thoroughly agitated for a long time, in order to obtain an
equal distribution of the cells ; the specific gravity of the
liquid must also be such that it will allow the cells to remain
suspended in it for a short time. A small drop is then
withdrawn in a capillary tube, transferred to the counting
apparatus, and covered with the cover-glass. The apparatus
is now allowed to remain at rest for some time, in order that
the cells may settle to the bottom of the enclosed space, and
on this account the specific gravity of the liquid must not
be greater than will allow this to take place in a convenient
time. Both these requirements are generally satisfied by
the wort employed in breweries.

If it is found that the determinate volume contains too
many cells to be counted with certainty, the liquid must be
diluted. This may also be advisable for other reasons, partly
to prevent the formation of froth, which otherwise will
generally form abundantly from the violent agitation, and
partly to isolate the single cells which are frequently clustered
in colonies or large masses in the wort, and are not always
separated by shaking, and, finally, in order to bring about a
discontinuation of the fermentation and multiplication of
the yeast-cells at the beginning of the experiment.

Hansen found that dilute sulphuric acid (1 : 10) on the
whole answers these requirements ; hydrochloric acid,
ammonia and caustic soda may also be used, but they are not

MICROSCOPICAL AND PHYSIOLOGICAL EXAMINATION. 35

so good. If a very great dilution is required, distilled water
can be added, after the addition of one to two volumes of
dilute sulphuric acid.

When the different volumes of liquid are measured with
accuracy, and particular care taken that the cells are
thoroughly distributed by vigorous and prolonged shaking,
the determination can be made with great accuracy. Two
similar dilutions must always be made, and samples taken
from each for counting. As a matter of course, experiments
must also be made in order to determine the number of the
small squares whose cell contents must be counted in order
to arrive at a true average. Such a counting and determina-
tion of the average numbers is continued until the number
finally obtained is found to have no further influence on the
average value. The number of countings necessary, and the
accuracy generally, depend on the experience and care of the
observer. Hcinsen found that, as a general rule, it was
sufficient to count the cells in 48 to 64 small squares.

CHAPTER II.
Examination of Air and Water.

As the water was hitherto regarded as one of the obscure
factors in the fermentation industries, and had often to
bear the blame of irregularities which could not be explained
in any other way, so also many peculiarities in the results
obtained at a certain point have at all times been considered
to originate from the air. In this was involved a vague
misgiving that this invisible air contained substances which
act prejudicially to our operations — the nature of these
substances, and how it was possible to obtain a closer know-
ledge of them, was, until the most recent times, involved in
obscurity. Chemical investigations of the air, which have
been carried out for more than a century, gave no information
on this point.

In the course of time a new factor was added ; it was
incontestably proved that the air is not everywhere equally
favourable to the human system ; there might possibly be
something present which attacked our organism; this
unknown matter was called "Miasma" (mixture), the word
being taken in a purely chemical sense. Since, however,
these miasmata were not traced further, science was thereby
not advanced one step.

The discoveries of Spallanzani (mentioned in the last
chapter), and of later investigators, opened up an entirely
new path, namely, the study of micro-organisms. Pasteur
especially showed that these micro-organisms are of essential
importance to the fermentation industries, when he proved
that the air contained both bacteria and alcoholic ferments.

EXAMINATION OF AIR AND WATER. 37

The questions then arose : What is the nature of these
germs floating in the air ? To what degree and extent do
they occur in space ? Do their number and nature vary
with the different seasons of the year? And, finally, are
they really able to effectually interfere with technical
operations ?

It will be of interest to glance at the different methods
by which the analyses of air with regard to its germs have
been attempted.

The majority of the analyses of air have been under-
taken with the view to obtain some light on the mysterious
obscurity which envelops most contagious diseases, nearly all
of which are, as is well-known, attributed to the agency of
micro-organisms. With regard to the organisms of fermen-
tation, these have been investigated chiefly by Pasteur, and
later by Hansen. The French savant stated that these
germs are always floating about in the air, but that they are
present in much larger quantities in the dust which settles
on the vessels and apparatus employed. The true alcoholic
ferments are present in comparatively small numbers in the
air, whilst the germs of mould-fungi are more frequent ; he
further showed, as was subsequently done by Tyndall, that
the germ-contents of the air vary both with regard to the
quantity and the species. These results were obtained by
exposing in open, shallow dishes, in different places, beer-
wort, wine-must, or yeast-water containing sugar ; after some
time their contents were examined for microscopical germs.
Pasteur also employed for this purpose the so-called vacuum-
flasks, containing nutritive liquids and rarefied air. On
opening the flask the air with its germs entered.

The most important air-analyses undertaken in recent
years are, without doubt, those undertaken by Miquel, the
director of the laboratory specially arranged for this purpose
at Montsouris, near Paris. His fellow-worker, Freudenreich,
has also added very valuable contributions to our knowledge
in this direction.

38

MICRO-ORGANISMS AND FERMENTATION.

Miguel performed his first experiments with a so-called
aeroscope (Fig. 8), which is constructed in the following
manner. From the top of a bell. A, proceeds a tube, C, through
which air is aspirated, thus causing it to pass through the bell.
To the latter is screwed a hollow cone, the mouth, B, of which
points downwards ; in the apex, D, of this cone there is a
very fine opening through which the aspirated air is drawn,
and immediately over this opening is fixed a thin glass-
plate covered with a mixture of glycerine and glucose. The
particles carried in by the air settle to a great extent on
the viscous mixture. The micro-organisms here intercepted
are distributed as equally as possible on the glass-plate,
and counted under the microscope. This method is so

FIG.
Aeroscope.

far defective in that it gives no information on the most
important point, namely, which and how many of the inter-
cepted germs are actually capable of development.

In order to determine the number of germs capable
of development, and also their nature, Miquel employs the
following apparatus (Fig. 9). The flask A has fused into it
a tube, K, tapering below and nearly reaching to the bottom ;
the upper end of this is fitted with a ground cap, H, provided
with a narrow filter-tube containing sterilised cotton-wool,
asbestos, or glass-wool, as. On one side of the flask is a
tube, Asp, which is constricted in the middle and is provided
with two cotton-wool plugs, wf and w. On the other side is
another glass tube, which is connected by rubber, fc, with the
tube B, which is drawn out to a point, and closed by fusing the

EXAMINATION OF AIR AND WATER. 39

end. The flask is charged with distilled water, and the whole
apparatus sterilised. When the apparatus is to be employed,
the tube Asp is connected with an aspirator ; for instance, a
bottle filled with water and provided with a cock below ; the
cap H is taken off, and the air then passes, bubble by bubble,
through the opening o, through the water g, and out through
the cotton-wool plugs of the tube Asp. Since all the germs
of the air are not retained by the water when the air-bubbles
ascend through the latter, the cotton-wool plug w is intended
to catch those which get past the water. When the experiment
is finished, the cap H is replaced over the tube K. By blowing
through Asp, the liquid is made to ascend in R in order that

FIG. 9.
Miquel's Apparatus for Air-Analyses.

any germs which may have settled on the walls of the tube
may be washed down into the liquid. Then, by blowing with
greater force, the inner cotton-wool plug w is driven down
into the liquid, and its germs shaken off into the latter. After
sterilising the thin tube B in a flame, the point is nipped off,
and the liquid is now — by blowing through Asp — transferred,
drop by drop, into a large number of flasks containing
sterilised broth.

The main point here is, by means of preparatory experi-
ments, to obtain such a dilution of the air-infected water
that a considerable proportion of the small flasks (one-half
for example) remain sterile after inoculation; or several
samples of the water may be diluted to different degrees,

40 MICRO-ORGANISMS AND FERMENTATION.

and a series of flasks inoculated from each dilution. When
a large number of the flasks do not show any development
of organisms, there is a certain probability that in each of
the remaining flasks in which growths have developed,
only one germ has been sown. A simple calculation will then
show how many germs capable of development in the medium
employed were present in the volume of air aspirated through
the original flask ("fractional cultivation").

By these methods of investigation Miquel found that
similar volumes of air in the same locality contained at
different times a different number of bacteria. A prolonged
rain greatly purifies the air from bacteria, and their number
continually diminishes as long as the earth is moist ; but when
the ground dries, they again gradually increase. In the dry
seasons of the year the number of bacteria is thus usually
the greatest, whilst the mould-fungi, which thrive best in
moisture, and whose organs of reproduction project upwards,
are most abundant in the air during the wTet seasons. The
purest air is found in the winter time ; the air of towns is less
pure than that outside the towns ; germ-free, or nearly germ-
free air is found at sea and on high mountains. In certain
localities — hospitals, for instance — the air has been found to
be very rich in bacteria ; in one case even 50 times richer
than the air in the garden at Montsouris.

An entirely different method for the examination of the
organisms contained in air is that employed in Koch's
laboratory, and more completely developed by Hesse. A
glass tube, about 1 meter long and 4 to 5 cm. wide, is closed
at one end with a perforated india-rubber membrane, over
which another non-perforated cap is bound. A little liquid
nutritive gelatine is then poured into the tube, after which
the other end of the tube is closed with an india-rubber
stopper, through which passes a glass tube plugged with
cotton-wool. The whole apparatus is then heated sufficiently
to render it sterile, after which the tube is placed in a
horizontal position, so that the gelatine sets in a layer in the

EXAMINATION OF AIR AND WATER. 41

lower part of the tube. When the air is to be examined, the
outer india-rubber cap is removed, and air slowly drawn
through the tube. The germs contained in the air then
settle down on the gelatine, and after the aspiration is con-
cluded the tube is again closed and placed in the incubator,
where some of the germs then produce visible colonies,
which are easily counted. The results show that with a
sufficiently slow current of air the bacteria, which are often
floating about in the air in larger or smaller aggregations,
frequently clinging to dust-particles, settle sooner than the
mould-spores ; so that in consequence the gelatine in the front
part, of the tube generally contains the majority of the
bacteria colonies, whilst the mould-spores develop further
along the tube.

Hueppe, v. Schlen, and others, employ liquid gelatine for
air-analyses, the air being aspirated through the gelatine, after
which the latter is poured on to glass-plates.

Frankland, Miquel, and Petri, use porous solid substances
for the nitration of air for analytical purposes ; as, for example,
powdered glass, glass-wool, sand, sugar, etc. The sand-filter
employed by Petri is 3 cm. long and 1*8 cm. wide. It is
filled with sand which has been heated, the size of the grains
being O25 to 0*5 mm. Two such sand-filters are placed one
behind the other, in a glass-tube. The first filter should re-
tain all the dust- particles containing germs, whilst the other
filter should remain sterile, and thus serves as a control. The
sand charged with germs is distributed in shallow glass-dishes
and covered with liquid gelatine. The germs contained in
the dust-particles will then develop colonies in the gelatine.

When samples of air are to be sent from one place to
another, these air-filters will answer the purpose. On receipt,
the sand may be washed into gelatine or, preferably, into
sterilised water. After vigorously agitating the water, it is
added in drops to flasks containing nutritive liquid, or it may
be used in plate-cultures.

Against the employment of gelatine plates for these

42 MICRO-ORGANISMS AND FERMENTATION.

purposes, an objection based upon numerous experiments
has been raised by Miquel, who asserts that many bacteria,
when exposed to a temperature of 20° to 22° C., require an
incubation of a fortnight before developing distinct colonies
in gelatine ; on the other hand, however, there are species
which will very soon liquefy the gelatine, thus rendering
further observation impossible for the next fortnight. The
same is the case with the mould-fungi, which will often
spread over the whole plate in a few days. Thus, it becomes
necessary to count the colonies at such an early stage when
many are not to be seen. An additional drawback to the
gelatine plates is, that the development cannot take place at a
temperature higher than 23° to 24° C., otherwise the gelatine
will become liquid ; but many species of bacteria give a fair
development only at considerably higher temperatures. Other
species, moreover, do not develop in gelatine at all, but only
in liquids. Finally, it is urged as a very material objection
to the gelatine-plates, that many of the colonies consist of
several species (see p. 31) ; Miquel proved this by intro-
ducing the colonies, one by one, into meat-decoction with
peptone, and then again preparing plates from these growths.
This is in part due to the fact that bacteria, as shown by
Petri, often occur in aggregates in the air, and these will
either fall directly on to the gelatine-plate or become mixed
in the liquid gelatine, where it will always be very difficult to
separate the individuals from each other by agitating.

Hansen's investigations of the air were made between
1878 and 1882. The main object of his investigations was
to throw light on questions affecting the fermentation indus-
tries. As is known, his researches on Saccharomyces apicula-
tus (1880) were partly based on work of this nature. Since
the question concerned the organisms which occur in brewing
operations, the choice of a nutritive liquid was easily made,
namely, ordinary wort as employed in breweries. The appara-
tus employed consisted either of ordinary boiling flasks closed
with several layers of sterilised filter-paper, the contents of

EXAMINATION OF AIR AND WATER. 43

which were boiled for a certain time, or of flasks of the same
kind as Pasteur's vacuum flasks, the necks of which were
drawn out to a fine point, and were closed with sealing-wax
whilst boiling. A little below the point a notch was made
with a file, in order that the point might be easily broken off
when it was desired to admit the air.

When these flasks had become filled with the air of the
locality to be examined, they were again closed with sealing-
wax and thoroughly shaken in order to mix the contents of
the infiltrated air with the liquid The flasks were then put
aside for a shorter or longer time, up to six weeks, and their
contents examined under the microscope.

In these investigations Hansen often found that the wort
remained bright and apparently unchanged, even although
a growth had taken place. Hence, the examination with
the naked eye alone cannot be relied on. He names the
following forms which, when present in a feeble state of
growth, cannot be detected macroscopically : — Aspergillus,
Mucor, Penicillium, Cladosporium, Bacterium aceti and
Pasteurianum, and Mycoderma cerevisise. Even when these
micro-organisms have formed vigorous growths, the above-
mentioned nutritive liquid has remained bright.

It was further shown that pure cultures may often be
obtained by the use of these flasks, when only one species was
drawn into the flask with the air. It very seldom happened that
three or four species were found in the same flask. This arises
from the fact that only a very small volume of air enters each
flask. The advantages of this are evident: — a true knowledge
of these germs can only be obtained when they have developed;
in cases where several germs penetrate into the same flask,
the strongest germ would by its growth, in all probability,
prevent the development of the others, so that these would
not be detected in a subsequent examination. At the same
time, however, this method necessitates the opening of a large
number of flasks, which makes the operation cumbersome
and costly. As the flasks only show what was present in the

44 MICRO-ORGANISMS AND FERMENTATION.

air at the moment of opening, Erlenmeyer flasks were also
used to give supplementary information, for which purpose
they were allowed to remain in the same locality for some
length of time, in some cases as long as 48 hours.

After these preliminary remarks we will give a brief
summary of the results obtained by Hansen.

He confirms the statement first made by Pasteur, that
the air at neighbouring points, and at the same time, may
contain different numbers and different varieties of organ-
isms ; and he found that this rule also holds good for places
lying close together in the same garden. Hansen states,
as other characteristics of the distribution of micro-organisms,
that those forms, for instance, which in the first half of July
commonly occurred under the cherry trees in the garden,
were in the latter half of the same month entirely absent
from the same place; further, that organisms which atone
time were found under the cherry trees, but not under the
grape-vines, were to be found later only under the latter ; as
a proof of the inequality of distribution of the organisms, it
is shown that the flasks opened in the same place in the
same series of experiments often had the most diverse
contents.

The experiments with the vacuum flasks have further
taught us that the micro-organisms of the air often occur
in groups or clouds, with intermediate spaces, which are
either germ-free or only contain quite isolated germs. As
the organisms are not generated in the air, but have their
place of growth on the earth, it follows that their presence in
the air must be dependent on the condition of the surface of
the ground, which again depends, in certain respects, on the
weather.

ffansen's numerous analyses have further proved that the
Saccharomycetes occur comparatively seldom in the dust of
the air. Their number in the air increases from June to
August in such a way that the flasks at the end of August
and the beginning of September are frequently infected

EXAMINATION OF AIR AND WATER. 45

with these organisms, after which a decrease takes place.
Those organisms from the air which at other times of the
year are found to enter the flasks, must be regarded as un-
important and accidental, and therefore falling outside the
principal rule. As most species of the Saccharomycetes have
in all probability — like Saccharomyces apiculatus — their
winter quarters in the earth and their places of growth on
sweet succulent fruits, these latter must apparently be
considered as the most important source of contamination.
At the same times of the year bacteria are also found in the
largest numbers. This constitutes an important danger in
technical operations, since the wort, which is spread in a
thin layer on the open coolers, is exposed at the above-named
season of the year to a great source of contamination from
the germs of the air.

Bacteria are found in the flasks in somewhat greater
number than the Saccharomycetes, whilst the mould-fungi
occur in still greater numbers. Amongst the latter Clado-
sporium and Dematium are especially prevalent in gardens,
and after these Penicillium; whilst Botrytis, Mucor, and
Oidium are less frequent.

After Hansen has thus stated which of the micro-
organisms existing in the open air are capable of develop-
ing in flasks with sterilised wort, he proceeds to communi-
cate the results of his examination of different localities in
the brewery.

When grains (draff) are allowed to stand in the open air,
they evolve, as is known, acid vapours, and since they always
contain a rich growth of bacteria when they remain exposed
for a short time, the following question suggests itself:—
What is the condition of the air in the neighbourhood of the
heaps of grains ? It was found that only 30 per cent, of the
flasks opened in these vapours became contaminated, and of
these 3-6 per cent, with Saccharomycetes and 2'4 per cent,
with bacteria, whilst parallel experiments in the garden gave
a contamination of about 44 per cent., of which 8*5 per cent.

46 MICRO-ORGANISMS AND FERMENTATION.

were bacteria. The air near the grains thus contained fewer
bacteria than the air of the garden. The most abundant
contamination here was that of mould-fungi, as in all the
other localities. After a thorough examination Hansen
came to the conclusion that, without any doubt, scarcely a
single organism which entered the flasks proceeded from the
grains. At all events the great abundance of bacteria in the
grains does not bear any correct relation to the above-stated
result, which, with far greater probability, admits of the
explanation that the air in this, as in other cases, does not
take up any contingent of organisms from moist surfaces.

This, however, must not be misunderstood to mean that
grains may be accumulated, without risk, in any chosen
place, and the remains after removal exposed to the weather.
It is clear that this would constitute a great danger. When
the remains become dry and are blown about in the air as
dust, masses of bacterial germs will be carried up at the
same time, and will, without doubt, constitute a source of
constant bacterial contamination. For this reason, places
where grains have remained for any length of time must
be washed with lime-water or, preferably, with chloride of
lime.1

In a corridor which led to the room where the barley was
turned, the flasks always received a greater contamination
than anywhere else ; bacteria especially were found in great
abundance.

On the malt floors the condition of the air was also
characteristic ; it always contained a very strong growth of
mould. In the case in question this growth consisted of
Eurotium Aspergillus, which was otherwise rare. On the
malt itself, as always, Penicillium glaucum occurred the most
frequently.

1 The germs are not killed during the treatment of the grains in the
drying machines. Such apparatus, therefore, constitute a very great
danger in the brewery, in cases where the bacteria can become transported
from the dried grains to the open coolers.

EXAMINATION OF AIR AND WATER. 47

The greatest interest, however, attaches to the examina-
tion of the different fermenting-rooms, partly in u Old
Carlsberg " and partly in the brewery " N." In the first-
mentioned rooms the air contained fewer organisms than in
any of the localities examined in the whole research ; in the
fermen ting-cellars of the brewery " N," on the contrary, a
large number of the flasks (55-75 to 100 per cent.) became
infected. The organisms which occurred in the air of these
cellars were : Saccharomyces cerevisise, Mycoderma cerevisise,
Sacch. Pastorianus, Sacch. ellipsoid eus, Torula Pasteur, and
other yeast-like forms ; further, Penicillium, Dematium,
Cladosporium, and rod bacteria. Hansen was thus enabled
by a favourable chance, to show the following contrast in the
state of the air in the most important place in the two
above-named breweries: on the one hand an almost germ-
free air, on the other hand an atmosphere teeming with
germs. That the product of the latter place at this time
must have borne the stamp of this condition admits of no
doubt, and we find here one of the most important of all
facts connected with the practice of the fermentation
industries. The air in the fermenting room itself may
contain a world of those germs which are productive of the
most calamitous results ; it is, however, possible to keep the
air free from these invisible germs, and it admits of no doubt
that, on the one hand, the purification of the air entering
the fermen ting-room by passing it through a salt-water bath,
and, on the other hand, the very rigidly maintained order and
cleanliness in the cellars of the Old Carlsberg brewery stand
in direct relation to the above-mentioned result. Hansen's
investigations, therefore, here again contain a warning which
cannot be repeated too frequently.

Based upon a long series of comparative investigations,
Hansen gave the following method for the zymotechnical
analysis of air and water.

The principle of this method of air- and water-analysis is
as follows: — For brewing purposes it is only necessary to

48 MICRO-ORGANISMS AND FERMENTATION.

know whether the water and the air contain such germs as
are capable of developing in wort and beer. This cannot,
as was formerly assumed, be ascertained by means of the
meat-decoction peptone gelatine employed in hygienic air-
and water-analysis. The zymotechnologist has this great
advantage over the hygienist, that he is in a position to make
direct experiments with the same kind of liquid as that
employed in practice, namely wort. All disease germs that
have hitherto been shown with certainty to occur in beer are
also capable of developing in wort. Hansen's comparative
investigations have proved beyond dispute that the use of
gelatines introduces great sources of error. Thus, for
instance, in a series of comparative experiments with corres-
ponding samples of water, the following numbers were
obtained:— In Koch's nutritive gelatine: 100, 222, 1000,
750, and 1,500 growths were obtained from 1 ccm. of water ;
in wort 0, 0, 6*6, 3. and 9 growths ; whereas, in beer, none of
these water-samples gave any growth. In another series,
Koch's gelatine gave for 1 ccm. of water 222 growths, wort-
gelatine 30 ; but none of the flasks containing wort and beer,
and infected with the water, showed any development of
organisms. Thus, only very few, or none at all, of the great
number of living germs in the water developed in wort or
beer.

Hansen has further shown, that in zymotechnical analyses
of water and air, it is a mistake to employ gelatine at the
outset, and then to transfer the colonies that have been
formed into wort-flasks. Thus, he demonstrated by experi-
ments that several of the bacterial germs existing in atmo-
spheric dust and in water are capable of developing in
nutritive gelatine, but not in wort; but several of these
species become invigorated to such a degree after having
formed a new growth in the gelatine, that they are then
enabled to develop in the less favourable medium, wort.
In such cases the experimenter is therefore deceived.
Another, and a still greater, objection to the gelatine

EXAMINATION OF AIR AND WATER. 49

method is, that several important organisms do not develop
at all when transferred directly to the gelatine in the en-
feebled condition in which they generally occur in atmospheric
dust and in water.

Based upon these observations, Hansen devised the
following method : Small quantities of the water, either
in its original state or diluted, are added to a series of
Freudenreich flasks containing sterilised wort and beer.1
After incubation at 25° C. for fourteen days the contents
of the culture-flasks are submitted to an examination. If
only a part of them show any development, the rest re-
maining sterile, it may be assumed with approximate
certainty that each of the flasks belonging to the former
set has received only one germ. Information is thus gained
concerning the number of germs capable of development
existing in a determinate volume, and the different germs
are also under more favourable conditions for their free
development. An exact examination will show to what
species these germs belong.

Although, in this method, the wort-cultures give a very
small number of growths in comparison to the plate-cultures,
yet in many cases the numbers of wort-growths will be too
high, since these growths are able to develop in the flasks
undisturbed and without hindrance from other organisms;
when wort is mixed with good culture-yeast in the ferment-
ing vessel, many of these germs will be checked. Further,
the flasks which show a formation of mould will have no
importance for the brewery, but only for the malt-house.
By way of a nearer approach to practical requirements,
Hansen proposes the following method of procedure. The
flasks containing a development of yeasts and bacteria are
divided into two groups : (1) those in which the growths soon
appeared, and (2) the remainder, in which they made their

1 In the analyses of air the germs are introduced directly, by means
of an aspirator, into water, or first into cotton-wool and then into
water.

50 MICRO-ORGANISMS AND FERMENTATION.

appearance later ; as, for instance, after five days. Among
the latter growths are those species which develop less
readily in wort ; and in the brewery these will therefore
be generally suppressed by the yeast, and are consequently
of less importance in the examination of water or air.
Analyses according to this method have been executed by
Holm, Wichmann, and several others.

For the control of air- and water-filters KoclCs gelatine
method is the best.

CHAPTER III.
Bacteria.

THE more our knowledge of these micro-organisms becomes
enlarged, the more difficult it is to give a general defini-
tion -of them. They are known in all forms, from the
smallest specks or spheres to green, alga-like filaments ;
and they occur very nearly in all possible localities, under
the most various conditions, as the cause of putrefaction or
decay (Saprophytes), of diseases (pathogenic forms), and of
fermentation (zymogenic forms).

The first knowledge of these forms was obtained by placing
small quantities of the different substances under the micro-
scope and examining them with high powers. In putrefying
meat very small spherical bodies were found, which clearly
multiplied by successive divisions ; in sour milk short, rod-
like bodies occurred : and yi putrefying vegetable matter
larger spherical bodies and long, fine, thread-like forms ; in
saliva, on the contrary, very fine, spirally-twisted threads
were found, etc. On this account it was convenient to
provisionally retain these /orms, and to describe them as
so many distinct species. Cohn especially has earned credit
in this respect, since to him is due the first systematic
classification of bacteria.

We will first consider the various forms and individuals
somewhat more closely. As before stated, the bacteria in
their simplest form occur as spherical bodies of different sizes,
often so small that they can only just be seen even with the
strongest powers, and only give evidence of their existence
as organisms by their multiplication by division. They are

52

MICRO-ORGANISMS AND FERMENTATION.

accordingly divided into macrococci and micrococci (Fig. 10 a).
When the spheres occur in pairs, they are called diplococci (6);
they also appear in groups consisting of four individuals,
sarcina (6); or of a greater number, arranged irregularly,
or in chains, streptococci (c). From the coccus forms there is a
gradual transition to the rod forms — bacterium, bacillus (e\

./ t' "• A

FIG. 10.

Growth-forms of Bacteria : a. Cocci ; b, Diplococci and Sarcina ; c, Strepto-
cocci ; d, Zooglcea ; e, Bacteria and Bacilli ; /, Clostridium ; g, Pseudo-
filament, Leptothrix, Cladothrix ; h, Vibrio, Spirillum, Spirochsete, and
Spirulina ; i, Involution-forms ; k, Bacilli and Spirilla with cilia or
llagella ; I, Spore-forming bacteria ; m, Germination of the Spore.

which vary greatly both in length and thickness. When the
rods are enlarged in the middle and taper towards the ends,
i.e., spindle-shaped, we have the clostridium form (/). If
the rods are elongated so as to become more or less thread-
like, they are called leptothrix (g), which may also occur as
pseudo-filaments (g), when several rods are arranged length-
wise, or as cladothriX) when they lie so close to one another
and in such a way that they become seemingly ramified ; a

BACTERIA. 53

true ramification, like that of the mould-fungi, does not occur
in bacteria. Eods and filaments frequently assume wavy or
spiral forms (h) ; when they are only slightly curved, we have
the vibrio form ; when the spirals are more prominent, the
spirillum, and spirochcete forms ; when they intertwine like
a plait of hair, the form called spirulina is produced. To
these must be added the remarkable irregular, swollen, or
curved forms which many bacteria can assume ; the cause of
this alteration is, however, not sufficiently known — involution
forms (i\

We will now select one of these forms and submit it to a
thorough examination with a magnifying power of about
1 ,000 diameters. Like every other cell, it contains protoplasm,
a homogeneous, feebly refractive mass, in which infinitesimal
particles can be detected here and there, especially if the
cell is not in its most active growth. Sometimes a bright
spot is found in the middle of the cell, which, from analogy
to the higher plants, is considered to be a sap-cavity or
vacuole. In some bacteria certain solid substances have been
detected, as, for instance, sulphur grains in bacteria which live
in water containing sulphur ; in some species the plasma can,
under certain conditions, be coloured blue by iodine, which
indicates the presence of substances resembling starch.

Surrounding this protoplasmic body we find a cell-wall
or membrane. An examination of this by means of staining
will generally show that this membrane in its outer layers is
swollen up into a gelatinous mass, which becomes especially
distinct when masses of bacteria are aggregated together.
From a chemical standpoint it must be provisionally assumed
that this cell-wall is of a different nature in different species.
In some it reminds us of the cellulose of the higher plants,
whilst in others it appears rather to resemble the albuminoids
in its properties.

Many bacteria contain blue, red, yellow, or green colouring
matters, which sometimes cause very intense coloration.
Under the microscope, however, the individual bacteria

54 MICRO-ORGANISMS AND FERMENTATION.

appear only very faintly coloured. It has not yet been
determined with certainty in what part of the organism the
colouring-matter is situated. Some species of bacteria are
phosphorescent under certain nutritive conditions.

A remarkable property of many bacteria is their — at least
apparent — free locomotion. This is either quick or slow, the
bacteria rotating about their longitudinal axes, assuming the
forms of open or contracted spirals. In some of these motile
forms we can observe, under high magnifying power, very fine
cilia or flagella (Fig. 10 &) ; whether these are to be considered
as organs of locomotion is not yet determined, nor has it been
decided whether they issue from the membrane or from the
cell contents.

The multiplication of bacteria takes place in different
ways. In the main, multiplication by division and by spore-
formation in the interior of the cell may be distinguished.
The first mode of multiplication has been observed in detail
in the larger forms. Fine transverse lines appear, which
gradually increase in thickness and become gelatinous ; after
this the organism separates at these transverse walls into
smaller rods (Fig. 10 <gr). Long before a trace of these
transverse walls can be observed, a staining of the organism
will show that it consists of a series of segments, each of
which corresponds to a subsequently-formed member. The
newly-formed segment-cells are all in the same plane. A
division in two or three directions of space has only been
observed in certain micrococci (Sarcina).

It was proved by the investigation of the shapes of
bacteria in the above-mentioned manner (especially by Zopf),
that the same species of bacterium can occur in very different
forms, e.g., as spirillum, leptothrix, bacillus, bacterium,
and coccus ; and we thus obtained the important addition to
our knowledge of the history of these plants that the names
quoted very often only express growth forms of the same
species, and not distinct species. The following question,
however, remains to be answered : — Under what conditions

BACTERIA. 55

does a species occur in this or that particular form ? Upon
this point we know very little at present.

In the case of many bacteria multiplication by spores
takes place in the following manner. The plasma in the
cell becomes darker, and often distinctly granular ; after that
a small dark body appears, which quickly increases in size,
and at the same time becomes strongly refractive ; mean-
while by far the greater portion of the plasma of the ceil
disappears, becoming used up in the formation of the spore ;
this is seen enclosed in a clear liquid, which gradually
disappears ; finally, the cell-wall shrivels up, and only remains
as a withered appendage to the ripe spore. This organ is
often termed a resting-spore (Dauerspore), for two reasons,
namely, first, because it actually possesses far greater
durability and resistance to external influences than the
vegetative rods ; and, secondly, because the spore formation
generally takes place when the nutriment of the organism is
either exhausted or unfavourable to the further vegetative
growth of the latter ; the spores, then, serve to preserve the
life of the organism during this critical period.

As soon as favourable conditions of nutriment and tem-
perature again occur, the spores germinate. They first
increase in size, and the contents lose their strong refractive
power. A bacterium then grows out from the spore, the wall
of which is sometimes seen to burst or divide into two shells
(Figs. 10, 13). The full-grown rod then multiplies in the
usual manner.

Bacteria are now sometimes divided into endosporous and
arthrosporous bacteria, of which the first-named form their
spores in the interior of the vegetative rods, whilst in the
latter group no such interior spore-formation has hitherto
been observed ; in these forms, members of a series of united
generations of vegetative cells separate from the rest and
assume the character of spores immediately without previous
endogenous rejuvenescence, and become the origin of new
vegetative generations (for instance, Bact. aceti). Perhaps

56 MICRO-ORGANISMS AND FERMENTATION.

by continued investigation endogenous spores may also be
found in all species of tbe last-mentioned group. It is only
a supposition that the above-mentioned separated members
must be considered as analogous to the spores.

Finally, in the morphology of bacteria, we must mention
the so-called zooglcea formation (Fig. 10 d). It is known that
in all branches of the fermentation industries, in places where
the cleaning is not strictly attended to, slimy, fatty masses
may occur, which gradually increase in thickness. The cause
of this is commonly a growth of bacteria occurring in such a
manner that the single cells lie very close to each other, whilst
at the same time the outer gelatinous layers of the cell-wall
greatly swell up. During the continued growth of the
bacteria the slimy layer increases in thickness, and often
assumes certain characteristic forms. Such slimy masses —
known in the sugar manufacture as "frog-spawn" — occur
both on solid and in liquid media.

Pasteur made the important discovery, that there are
certain bacteria and other micro-organisms which do not
require free oxygen, and even produce very active decomposi-
tions of the fermenting material when oxygen is excluded.
He, therefore, distinguished two classes of micro-organisms,
naming the last-mentioned anaerobic and the others aerobic.
More recently Duclaux has stated that there are intermediate
forms between the two extremes. As an example of anaerobic
bacteria, Pasteur's bacterium of the butyric-acid fermenta-
tion may be mentioned.

We will now pass in review the more important species
which are of special interest in the fermentation industries.

1. ACETIC ACID BACTERIA.

The acetic acid bacteria w^ere first thoroughly described
from a morphological standpoint by Hansen. The correct-
ness of his investigations was afterwards confirmed by Zopf,
deBary, and A. J. Brown.

As early as in the year 1838 the view was expressed by

BACTERIA. 57

Turpin and Kutzing that the acetic acid fermentation is
caused by a micro-organism, which Kutzing described and
delineated under the name of Ulvina aceti. Starting from
this, Pasteur, first in his treatise (1864) and subsequently in
his work "Etudes sur le vinaigre" (1868), furnished experi-
mental proof of the correctness of this view, and also gave a
method, based on the results, for the manufacture of vinegar.
He assumed that the acetic acid fermentation was caused by a
species of micro-organism which he called Mycoderma aceti.
Subsequent research has, however, shown that there are
different species of acetic acid bacteria. With Pasteur,
therefore, it was not at all a question of the employment of
one definite, selected species. His method consists in giving
a large surface to the liquid employed — two parts of bright
wine to one part of wine-vinegar — and then sowing on the
surface of the mixture a young film consisting of " mother
of vinegar." When the temperature, the composition of the
liquid, and all other conditions are favourable, the formation
of acetic acid will proceed more quickly than in the older
Orleans process. The installation is claimed to be cheaper,
and the loss of alcohol not greater — at all events not to any
appreciable extent — than in the last-named process. Yet,
as far as I have been able to learn, Pasteur's process is never
employed. The cause of the uncertainty of the results may
be sought in the fact that the composition of the nutritive
liquid varies, and especially in the fact that the bacterial
culture was not a pure culture, and might, therefore, also
contain varieties of bacteria which possessed different pro-
perties, required different conditions for their growth, and, con-
sequently, would give different products in varying quantities.
This will hold good even in those cases in which the composite
culture consists only of such varieties which can produce
vinegar. As early as 1879 Hansen discovered that at least
two distinct species are hidden under the name of Mycoderma
aceti, namely, Bacterium aceti and Bact. Pasteurianum ;
and he has shown that also in this branch of industry it is

58

MICRO-ORGANISMS AND FERMENTATION.

necessary to start with an absolutely pure culture of a
methodically-selected species. — The old Orleans process still
prevails in France. In this method the wine which is to be
converted into vinegar is placed in tuns, to which atmo-
spheric air has moderately free access. The formation of
acetic acid, as in Pasteur's process, takes place in consequence
of the liquid becoming covered with a film consisting of
"mother of vinegar." — In other countries the German
" quick vinegar process " is employed, in which the growth of
bacteria, through free access of air and by dividing the liquid

FIG. 11.
Bacterium aceti and Bact. Pasteurianum, after Hansen.

into small drops, and distributing these over very large
surfaces (shavings), comes into intimate contact with the
air. The nature of the micro-organisms taking part in this
manufacturing process has not yet been investigated.

Whilst Pasteur, in the above-named work, does not
explicitly maintain the theory that the oxidation of alcohol
to acetic acid is a purely physiological process, yet Adolf
Mayer expresses this opinion, and Hansen emphasises as a
certainty the fact that the formation of acetic acid is
commonly effected by the action of bacteria. Hanserfs

BACTERIA. 59

researches are, moreover, among the first which proved that a
definite fermentation is not induced by one species of
bacterium only, but by several ; more recently a large number
of instances have been discovered. By placing lager-beer in
an incubator at 30° to 34° C., he obtained a vigorous film-
growth of the acetic-acid bacterium. This consists of long
chains of hour-glass-like members, partly as bacterium and
bacillus, partly as wavy or curved forms. As a peculiarity
of Bad. aceti it may be mentioned that this species often
yields at a very early stage the different, irregularly swollen
involution forms above described, whilst other bacteria do not
produce these forms until a very advanced stage, and even
then, possibly, only as a consequence of a deficient supply of
nutriment; this, however, cannot be the case with the
organism under discussion. We have here also one of the
first cases, in which it has been shown that the same species
can occur in very different forms.

By means of his staining experiments with Bact. aceti,
Hansen discovered, as already stated, that two distinct species
are hidden under this name, of which the one. like most
other bacteria, is stained yellow by iodine, whilst the other
assumes a blue coloration with the same reagent. For the
former he retains the old name Bact. aceti, whilst the one
stained blue he names after Pasteur — B. Pasteurianum. In
a lecture he communicated the following new observations : —
The film formations on wort and beer, and likewise the
growths on wort-gelatine, give a fine blue colour with tincture
of iodine, or iodine dissolved in a solution of iodide of potassium,
whilst the growths which develop on yeast-water and meat-
decoction with peptone and gelatine are coloured yellow ;
even very old films on beer show a yellow reaction. It is the
gelatinous formation secreted from the cell-wall that is
coloured blue ; it has hitherto not been possible to determine
whether the contents of the cells are coloured or not. In
wort-gelatine, Bact. Pasteurianum develops round specks or
colonies with a smooth or wavy border, whilst the corresponding

60 MICRO-ORGANISMS AND FERMENTATION.

specks of Bact. aceti have a tendency to assume stellated
forms. From a morphological standpoint the two species
behave alike. Spores have not been observed. A fact of
practical interest is the observation that pure cultures of the
two species of acetic acid bacteria in beer do not exercise
any influence on the colour or brightness of the liquid.
It is therefore possible, to a certain extent, to ascertain
whether or not these organisms are alone present, since other
bacteria, when present in beer, produce turbidity in the liquid.
In order to develop vigorously, Bact. aceti not only requires
a very plentiful supply of oxygen, but also a fairly high
temperature. Hansen found that a temperature of about
33° C. was the most favourable when Carlsberg lager beer
was used. In a well-conducted store-cellar ( 1 ° to 3° C.) there
is therefore nothing to fear from Bact. aceti. But as soon as
the beer leaves the cellar, and is exposed to higher temperatures,
there is always a danger.

In leaven, especially when it has become old and extremely
sour, Peters recently discovered an acetic acid bacterium
which distinctly differs from Bact. aceti and B. Pasteurianum.
The colonies in ordinary plate-cultures are circular in shape,
of a homogeneous appearance, and, when seen in transmitted
light, of a strong brown colour ; the surface colonies are
largely expanded. The single individuals are 1*6 /* long,
0-8 /* broad, truncated at one end, tapering at the other;
they occur singly or in pairs, rarely in groups of four. The
bacterium does not exhibit any motion. In yeast-water
containing 5 per cent, of alcohol it first produces turbidity,
then a thin film forms on the surface, and gradually becomes
viscous. This bacterium is perhaps identical with the species
described by Duclaux.

The Bacillus ethaceticus discovered by Percy Franldand
induces a vigorous fermentation in various substances (e.g.
mannite), the chief products of which are ethyl-alcohol and
acetic acid.

Pasteur has shown that, by the oxidation of alcoholic

BACTERIA. 6.1

liquids, ethyl-alcohol is converted into acetic acid, and by
farther oxidation the latter is converted into carbonic acid
and water. This has been recently confirmed by A . J. Brmvn.
to whom we are indebted for the most complete researches on
the chemical action of acetic acid bacteria.

2. LACTIC ACID BACTERIA.

When milk is exposed at a temperature of 35° to 42° C.
it will soon become sour, and a considerable portion of the
acid produced is lactic acid, which is formed by the agency
of various species of bacteria. When a certain quantity of
lactic acid has been formed, the fermentation ceases. It will
recommence, however, if the liquid be neutralised with car-
bonate of lime, or on the addition of a small quantity of
pepsine or pancreatine, which causes the casei'ne of the milk
to be dissolved.

A method commonly employed for inducing a spontaneous
lactic acid fermentation is the following : — To a liter of water
are added 100 grams of sugar, 10 grams of casei'ne or old
cheese, and an abundant quantity of powdered carbonate
of lime. This mixture is exposed in an open vessel to a
temperature of 35C to 40° C. The liquid is occasionally
agitated, or a current of air is passed through it. After
completion of the fermentation the liquid is evaporated,
when calcium lactate crystallises out, and from this the
lactic acid is liberated by treatment with sulphuric acid.

In addition to milk-sugar, lactic acid bacteria are also
capable of fermenting cane-sugar, glucose, maltose, and
various other substances. According to Bourquelofs investi-
gations, a species of lactic acid bacterium, which makes its
appearance in the spontaneous acid fermentation of milk,
is capable of fermenting cane-sugar without previously
inverting it.

In milk-sugar solutions which were free from casein e,
Fokker could only obtain feeble lactic acid fermentations,

62 MICRO-ORGANISMS AND FERMENTATION.

whilst, on the addition of this substance, the lactic acid
was proportionately increased.

In breweries the lactic acid fermentation takes place even
in the malting, also in the wort and in the after-fermentation ;
in the Belgian beers, obtained by spontaneous fermentation,
lactic acid is formed in large quantity, and cousequently
imparts a sharp taste to the beer. In modern low-fermenta-
tion breweries it is endeavoured to exclude the lactic acid
ferment, and bacteria in general, from the fermentations.
" In distilleries," according to Maercker, " these are still
provisionally regarded as a necessary evil. The production
of lactic acid takes place during the yeast-dressing, and its
importance appears to be confined to preventing the develop-
ment of bacteria, and in this way rendering possible the pure
fermentation of the alcoholic yeast."

FIG. 12.

Lactic acid bacteria, after Pasteur. In order to give an idea of the size of
the bacteria, some yeast-cells are figured amongst them.

We are indebted to Pasteur for the first important work
on the subject of lactic acid bacteria. In 1858 he described
the species which appears when milk spontaneously ferments.
In his " Etudes sur la biere " he figures some bacteria which
develop in wort or beer in which lactic fermentation has set
in (Fig. 12) ; he describes them as short rods slightly narrowed
in the middle, and commonly occurring singly, rarely united
in chains.

Later, Hueppe found a bacterium in a spontaneous lactic
acid fermentation which converts milk-sugar and other sugars
into lactic acid with the simultaneous formation of carbonic acid.
It consists of short, plump, motionless cells, the length of which
exceeds their breadth by at least one half; they are united in
pairs or in groups of four. In sugar solutions and less distinctly

BACTERIA. 63

in milk they form spores, which appear as lustrous spheres
attached to the end of the rods. In gelatine-plates they
form whitish colonies which, as long as they are submerged,
are circular, uniformly dark, and have sharp contours ; those
on the surface have lighter borders. Atmospheric oxygen is
necessary for fermentation with this species. It coagulates
the casei'ne of milk.

In recent publications descriptions are found of a large
number of lactic acid bacteria ; thus, two species of micro-
cocci have been found in saliva and the mucus of teeth ;
amongst the pigment-forming bacteria, species are also found
which, in addition to their pigment-fermentation, are able to
produce so much lactic acid from the sugar of milk that the
casei'ne of the milk coagulates ; to these belong, according to
Hueppe, the famous blood-portent (Micrococcus prodigiosus)
and, according to Krause, a pathogenic form, the micrococcus
of osteo-myelitis.

According to statements made by Delbriick, Zopf has
obtained a lactic acid bacterium by preparing a mash from
200 grams of dry malt and 1000 grams of water, and
keeping it for some time at 50° C. The material was then
sown in a solution of milk-sugar, on the surface of which the
organism formed a film. The filaments consist at first of
small rods, later of both rods and cocci.

Peters found a bacterium in leaven, which produces a typical
lactic acid fermentation. In plate-cultures it forms circular
colonies with concentric stratification. The rods have a rapid
sinuous motion ; in a neutral solution of sugar in yeast-water
at 30° C., this species forms after some time a slimy film ;
the rods have here developed into long filaments. Spore-
formation has not been observed.

The so-called Pediococcus acidi lactici examined by
Lindner gives, when cultivated in a neutral malt-extract
solution at 41° C., a strong acid reaction ; both in a solution
of this kind and in a hay-decoction, which have not been
sterilised, this bacterium develops so vigorously that, accord-

64 MICRO-ORGANISMS AND FERMENTATION.

ing to Lindner, all other organisms are suppressed. It has
been proved chemically that the acid, which is abundantly
produced, consists for the most part of lactic acid. When a
malt-mash or malt-rye mash is maintained at 41° C., the
Pediococcits develops vigorously, and the rod-shaped lactic
acid bacteria are suppressed. In a neutral malt-extract
solution, the Pediococcus is killed after five minutes' exposure
to 62° C. In gelatine it does not thrive well; it is only in
puncture-cultivations in neutral malt-extract gelatine, that
very vigorous white colonies are formed below the surface.
This species appears, on the whole, to thrive better when the
air is excluded.

The Saccharobacillus Pastorianus described by Van
Laer, which occurs in the form of rods of different lengths,
produces a characteristic disease (" tourne ") in beer, which
manifests itself as follows : the liquid gradually loses its
brightness, and when it is agitated filaments of a silky lustre
rise from the bottom, and the beer assumes a disagreeable
odour and taste. The bacillus, in cultures, develops both in
the presence of free oxygen and when this is excluded. In
nutrient liquids it ferments the carbohydrates, and amongst
them the saccharoses, without previously inverting them.
Amongst the fermentation-products lactic acid, acetic acid,
and alcohol, are especially mentioned. The acids produced
cause the precipitation of nitrogenous compounds in the
liquid, and these, mixed with the bacilli, produce the
above-mentioned clouds, consisting of lustrous filaments.

Besides the investigators mentioned, several others have
likewise carried out researches in this field ; as, for instance,
Pasteur's co-operator Duclaux. Grotenfelt has recently
described some species which must doubtless be regarded as
new ones ; at any rate he could not identify them with those
described by Hueppe and Marpmann* Some species were
observed to yield alcohol in addition to lactic acid by the
decomposition of sugar ; he expresses the belief that these
species take part in the formation of the aroma of butter.

BACTERIA. 65

Recently a methodical pure cultivation of certain lactic
acid bacteria has been introduced into practice, the principles
being the same as those carried out by Hansen in the case of
alcoholic ferments in breweries, the object being to attain a
more rational acidification of the cream employed for making
butter. Wcigmann, Storch, and Qvist have isolated a series of
lactic acid bacteria which, when employed for the acidification
of the cream, have imparted to the butter a more or less
pure sour taste, and also a more or less aromatic odour, whilst
the durability of the butter likewise varies with the different
species.

Storch particularly mentions one species which, in experi-
ments on the acidification of cream for the use of dairies, not
only gives it a pure and mild slightly sour taste, but also
imparts a markedly pure aroma both to the cream and to the
butter made from it. In gelatine this species forms very
small colonies of a pure white colour and with a smooth
border. In milk and whey it occurs as plump, oval, or
spherical bacteria, which form flexible chains. It bears
some resemblance to Pasteur's " ferment lactique." At
28° C. it develops a marked fermentative activity.

Qvist has cultivated another species, which has been
employed with still greater success in practice. It occurs
both as micrococcus and in other forms, according to the
different nutrient media in which it is cultivated. In
gelatine it forms small, circular, slowly-growing colonies of
a whitish-yellow colour. In puncture-cultivations spherical
colonies arise throughout the puncture-channel, and in streak-
cultures this organism forms a continuous streak with wavy
borders. It was prepared from a sample of butter of remark-
able aroma and durability.

On the other hand, several species of bacteria have been
discovered in recent years, which cause diseases in milk.
Thus Schmidt-Mulheim found a micrococcus which occurs in
the form of moniliform chains, and causes the milk to become
viscous; another species, discovered by Ratz, possesses the

r

66 MICRO-ORGANISMS AND FERMENTATION.

same property and also produces a vigorous lactic acid
fermentation ; other slime-forming species have been described
by Adametz, Duclaux, Loffler, and Guillebeau ; Weigmann
isolated a species which imparts a bitter taste to milk and
secretes a ferment which dissolves caseine. Jensen likewise
found a species which causes marked abnormal changes in the
taste of milk and butter ; it has the form of thick, motile
rods, of varying length, partly resembling micrococci. Storch
proved that the disagreeable taste of tallow which butter
sometimes has, is caused by a certain species of bacterium,
which acidifies and coagulates the milk.

3. BUTYRIC ACID BACTERIA.

When milk which has stood for some time, and in which
lactic acid bacteria have developed, is neutralised by the
addition of lime ( chalk), so that calcium lactate is formed, it
will, as a rule, enter into a butyric fermentation, which is
caused by different species of butyric acid bacteria. This
spontaneous butyric acid fermentation takes place most
vigorously at 35° to 40° C. Starch, dextrin, cane-sugar, and
dextrose, are likewise fermentible by the butyric acid ferments,
and these fermentations set in very readily, as the different
bacteria belonging to this group are very widely distributed
in nature. It is doubtless also such species which take part
in the ripening of cheese, and which help to impart the
characteristic taste and aroma to the different kinds of
cheese. In order to induce a butyric acid fermentation, Fitz
recommends the employment of a mixture of 2 liters of
water, 100 grams of potato-starch or dextrin, 1 gram of
ammonium chloride, the ordinary nutrient salts, and 50 grams
of chalk ; this mixture is to be maintained at 40° C.
Bourquelot recommends exposing water containing slices of
raw potatoes for two or three days at a temperature of 25°
to 30° C.

The most important products of the butyric acid fermen-
tation are butyric acid, carbonic acid, and hydrogen.

BACTERIA. 67

In the saccharine mashes of breweries, distilleries, and
pressed-yeast factories, some species of butyric acid bacteria
always occur, and if the mashes are maintained for a
lengthened period at certain temperatures, these bacteria
develop very rapidly and exercise a retarding influence on
the alcoholic ferments. If butyric acid occurs to any extent
in beer, it will acquire a very unpleasant taste.

According to Pasteur's experiments, the butyric acid
ferment can perform its functions without access to the free
oxygen of the air. More recent investigations have shown, how-
ever, that many butyric acid bacteria exist which not only yield
different fermentation products, but also behave differently
with regard to free oxygen, in that some are not capable of
developing when the latter is present, — so-called anaerobic
species, — whilst others multiply and induce butyric acid fer-
mentation when they have access to oxygen, — aerobic species.

One of the first species which were minutely described is
Prazmowski's Clostridium butyricum (Bacillus butyricus)
(Fig. 13). It occurs in the form of short and long rods,
which may be either straight or somewhat curved. Before
the formation of spores in the rods, the latter swell and form,
as shown in the figure, peculiar spindle- and lemon-shaped,
elliptical, or club-like forms ; at the same time there is the
important fact that they are coloured blue by iodine. On
germination the spores burst their outer envelope, and the
germ filament grows in the same direction as the longitudinal
axis of the spore. Clostridium butyricum grows most readily
at a temperature of about 40° C., and is especially able to
predominate in a solution of sugar if the lactic acid ferment
has previously converted a portion of the sugar into lactic
acid. This species is decidedly anaerobic.

Fitz has described a species belonging to the aerobic
forms. It is a bacillus of a short cylindrical form, which is
not coloured blue by iodine, is motile in a moderate degree,
and forms no spores. It ferments all carbohydrates, with the
exception of starch and cellulose.

68

MICRO-ORGANISMS AND FERMENTATION.

Hueppe has likewise described a species, which was found
in milk, and occurred in the same forms as the species
discovered by Prazmowski, but was much less sensitive
towards oxygen, and must therefore be classed with the

A

FIG. 13.

Clostridiunf butyricum Prazm., after Prazmowski : A, vegetative state ; c, short
rods ; d, long rods ; at a and b, rods and filaments curved like vibriones ;
£, formation of " resting-spores " (Dauersporen) ; b, d, rods, previous to,
c, e, during, /, g, h, after the formation of resting-spores ; c, of an
elliptical ; d and h, of a lemon-like ; e, g, of a spindle-like ; /, of a
tadpole-like shape ; at a, rods still in their vegetative state ; (7, germina-
tion of resting-spores : the spore a expands into b ; c shows the
differentiation of the membrane into exo- and endosporium. The con-
tents surrounded by the endosporium issue from the polar fissure of
the spore in the form of a short rod (d), which appears prolonged at e.

BACTERIA. 69

aerobic species. Gruber found associated under the name of
Clostridium butyricum three well-defined species, two of
which are exclusively anaerobic. One of these last-mentioned
species consists of straight or slightly-curved rods, which
become spindle- or barrel-shaped during the formation of
spores. In nutrient gelatine it forms colonies which, when
seen in transmitted light, appear blackish-brown or black.
The second species consists of strongly-curved vegetative
rods, in which the spores are formed at the end ; it forms
yellowish or yellowish-brown colonies. The third specie's is
also capable of growth and of causing fermentation in the
absence of oxygen ; its development is, however, decidedly
promoted by the presence of oxygen, and it is only then
able to produce spores. The vegetative rods are cylin-
drical ; with the formation of spores the rods become
spindle-shaped, and in the centre of the spindle the large
spore is formed. The colonies in nutritive gelatine are
of a yellow colour. All three species form butyric acid
and butyl- alcohol.

According to Fitz the spores of butyric acid bacteria can
withstand a boiling temperature for a period of time which
is naturally dependent, as in all cases, on their condition and
on the nature of the substratum ; Fitz gives three to twenty
minutes as the limits. They can, however, also be killed by
a lower temperature, if continued long enough ; thus they
are killed by being heated for six hours at 90° C. in a solution
of grape-sugar ; but in glycerine, at the same temperature,
only after six to eleven hours.

Thus the same holds good for butyric acid fermentation as
for lactic acid fermentation, namely, that it is not produced
exclusively by one species. When butyric acid fermentation
occurs in distilleries, breweries, and pressed-yeast factories,
bacteria are frequently found which are entirely different from
those described above.

Clostridium butyricum, and various other species, are
capable of dissolving cellulose, and therefore play an im-

70 MICRO-ORGANISMS AND FERMENTATION.

portant part in the cellulose fermentation, which is employed
in various branches of industry.

4. KEPHIR-ORGANISMS.

The so-called "Kephir" on which the investigations of

Kern have thrown some light, is an effervescent alcoholic,

and sour milk, which is prepared by the inhabitants of the

Caucasus from cows', goats', or sheep's milk. It is prepared

by adding a peculiar ferment, " kephir-grains," to milk.

These are white or yellowish, irregularly-shaped, uneven

grains, about the size of a walnut and of a tough gelatinous

consistency, and when dried become cartilaginous and brittle.

The most essential part of these grains consists of rod-like

bacteria, which are connected in threads and have developed

gelatinous membranes. Kern calls this bacterium Dispora

Caucasica. Yeast-like fungi are also found in kephir-grains,

and among these different varieties of true Saccharomycetes.

In the preparation of kephir a little milk is first poured on the

grains and allowed to stand for twenty-four hours ; the milk is

then poured off, and the grains preserved for future use. This

milk is now mixed with fresh milk, and poured into bottles

which are corked, or into leather sacks which are tied ; after

some days a fermentation has taken place. It now contains

about two per cent, of alcohol. This result is probably brought

about by the simultaneous action of the above-mentioned

Dispora and the yeast cells in combination with the lactic

acid ferments which are probably always present in milk.

These ferments convert a portion of the milk-sugar into

lactic acid ; the alcohol and a part of the carbonic acid

probably result from the action of the yeast cells. Then,

as the fermented milk contains considerably less coagulated

caserne than ordinary sour milk, it may further be assumed

that the above-mentioned Dispora is also able to partly

liquefy (peptonise) the coagulated caserne, perhaps with the

help of the gelatinous mass secreted by the bacterium and

which is found in the kephir-grains, but is not present in the

BACTERIA. 71

fermenting milk. If one of the above-mentioned kephir-
grains is allowed to remain in milk, it will grow very slowly
and only attain, according to the researches of de Bary, a
double size after the lapse of several weeks. This author
considers it probable that under such conditions single
Dispora cells separate themselves and give rise to new
kephir-grains. According to the mode of preparation pub-
lished by A. Levy, kephir can also be obtained without the
addition of Kern's ferments. When milk which is becoming
sour is repeatedly and violently shaken, an effervescent alcoholic
kephir-like drink is obtained, which, as regards taste, etc.,
does not perceptibly differ from kephir prepared with kephir-
grains. According to de Bary the kephir obtained by shaking
contained about one per cent, by volume of alcohol, whilst a
sample of the ordinary kephir contained only 0'4 per cent, by
volume (Schmiedeberg). According to the recent investiga-
tions of Duclaux, Grotenfelt, Adametz, and others, there are
also certain yeast-fungi which are capable of fermenting
milk-sugar by themselves, without the aid of bacteria (see
Chapter V.).

The Ginger-beer Plant, which presents morphological
resemblances to the Kephir ferment, has been examined
from a botanical and biological point of view by Professor
Marshall Ward. If this ferment is introduced into
saccharine solutions to which ginger has been added, it
transforms them into an acid effervescing beverage, ginger-
beer. When fresh, it occurs as solid, white, semi-transparent,
irregular, lumpy masses, brittle like firm jelly, their size
varying from that of a pin's head to that of a large plum.
It induces an alcoholic fermentation in the saccharine solution,
which at the same time becomes viscous. Marshall Ward
isolated the numerous micro-organisms existing in the masses
described above, and gave accurate descriptions of a series
of yeast-fungi, bacteria and moulds, among which two
organisms proved to be essentially concerned in the fermen-
tation of ginger-beer. One of these is a Saccharomyces,

72 MICRO-ORGANISMS AND FERMENTATION.

belonging to the ellipsoid group of this genus, and probably
originating from the ginger and brown sugar employed in
ordinary practice; the author has named it Saccharomyces
pyriformis. It inverts cane-sugar, actively ferments the
products, and forms a white pasty deposit at the bottom of
the flasks. It yields spores on gypsum blocks at 25° C.
in 40 to 50 hours ; it also forms spores on gelatine.

In hopped wort it induces a not very vigorous fermen-
tation, and it forms a film on the surface ; the cells in this
film are usually pyriform or sausage-shaped.

The other constant and essential form is a Schizomycete,
Bacterium vermiforme, which, according to Professor Ward,
originates from the ginger. It is a peculiarly vermiform
organism, enclosed in hyaline, swollen, gelatinous sheaths,
and imprisoning the yeast cells in brain-like masses formed
by its convolutions. It is the swollen sheaths of this
organism which constitute the jelly-like matrix of the
"plant." It also appears without the sheaths, and with
all the various growth-forms which we meet with among
the bacteria. It is a markedly anaerobic bacterium. The
gelatinous sheaths are only developed when the saccharine
liquid is acid, and free from oxygen.

Of the other organisms which occur in the ginger-beer
plant, a Mycoderma species and Bacterium aceti were
found in all the specimens examined, and a variety of other
bacteria and fungi also occurred as casual intruders.

The author has proved experimentally that Saccharo-
myces pyriformis and Bacterium vermiforme are the only
two essential species in the ginger-beer fermentation, since
it was only by inducing a fermentation with these two species
that he was able to produce an effect of the same kind as
that obtained when the ordinary ginger-beer plant is em-
ployed. But it is only when both species develop together
in the liquid that they bring about this result, and the
author's experiments point to the view that the relations
between the yeast and the bacterium are those of true

BACTERIA. 73

symbiosis, so that the two species form a lichen-like
compound organism, which induces a " symbiotic fermen-
tation."

5. SLIME-FORMING BACTERIA.

Among the various species of slime-forming bacteria there
are several which are of peculiar interest in the fermentation
industries, as they occur in wine and fermenting wort, in which
they cause morbid changes. According to all analogy, this
slime formation may be regarded as a phenomenon closely
related to the commonly-occurring zooglcea formation of
certain bacteria (see p. 56). In the case of certain species
the slime is, however, also regarded as a product of the
decomposition of sugar, and not as a substance separated
from the organism itself.

In the viscous fermentations examined by Bechamp a kind
of gum termed viscose was formed together with carbonic
acid, and frequently also mannite.

In his "Etudes stir la biere" (Plate 1, Fig. 4) Pasteur
describes bead-like chains of spherical organisms, which
render wine, beer, and wort so viscous that they can be drawn
out in threads.

In Berlin " Weissbier " (white beer), which had become
ropy, Lindner found a strong development of a certain
Pediococcus. The disease could be produced by adding
pure cultures to sterilised white-beer wort. On the other
hand, this organism had no action on hopped beer-wort or
low-fermentation beers.

In ropy Belgian beer Van Laer found the cause of this
disease to be small, very thin rods (1*6 to 2*4 micro-milli-
meters long), which were partly isolated and partly united in
pairs by means of a zooglcea-like substance. When added to
beer-wort, this first became turbid, and afterwards ropy. On
meat decoction with gelatine these rods gave colonies with
concentric rings of different colours and with a hollow in
the middle ; streak cultures give broad, white bands, with a

74

MICRO-ORGANISMS AND FERMENTATION.

sinuous border; puncture-cultivations give a white stripe,
which soon extends to the bottom of the glass ; the gelatine
forms fissures which become filled with the growth, while at
the same time a speck is formed on the surface. Experiments
carried out with pure cultures of this bacterium in beer-wort
have shown that one and the same form includes several
species, which have a somewhat different action on wort.

•i!

s!\

«ee^ ^^

FIG. 14.

Leuconostoc mesenterioid.es Cienkowski, after Zopf. A, cell cluster of the
variety with no envelopes, taken from a potato cultivation ; B, series
showing the development of a cultivation grown in gelatine, free from
sugar ; B a, has no envelopes ; B b, the same after 24 hours' growth in a
solution of molasses, the envelopes are already seen but are not strongly
developed ; B c, after 48 hours' growth in molasses, the envelopes are
more strongly developed and partly encased in each other ; O, a small
gelatinous mass from which the cells have been expelled.

They are all included under the name Bacillus viscosus.
If sterilised wort is infected with this bacterium and alcoholic
yeast added after the lapse of some hours, the liquid becomes

BACTERIA. 75

viscous. If the wort be infected with a mixture of absolutely
pure yeast and bacteria, the disease will develop in a varying
degree, according to the proportion of bacteria. If, however,
these are only added after the completion of the primary
fermentation, the disease will not appear at all. The greater
the proportion of nitrogenous matter in the liquid, the
sooner it will become viscous ; even liquids which do not
contain sugar can be made ropy by these species ; on the
other hand, the phenomenon does not occur in pure sugar
solutions.

The so-called frog-spawn fungus (Leuconostoc mesen-
terioides) was investigated by Cienkowski and van Tieghem,
and more recently by Zopf and Liesenberg. Both the
European form and the variety found by Winter in Java
occur spontaneously in beet-root sap, and in the molasses
from the manufacture of sugar, in which they form large
slimy masses (frog-spawn) and multiply vigorously. The
fungus forms chains of cocci, two of which are always more
closely united ; in opposition to earlier observations, Zopf
found that these cocci present no differences with reference
either to their morphology or physiology ; spore formation
could in no case be proved. Consequently, the analogy which
was formerly assumed to exist between this fungus and the
algal genus Nostoc (implied in the name Leuconostoc) falls
through.

Under certain conditions the cells become enclosed in a
strong gelatinous envelope, which consists of a mucilaginous
carbohydrate, the so-called dextran. This formation — a
product of assimilation — only occurs in the presence of grape
sugar, and not in solutions of milk-sugar, maltose, and dextrin,
because these carbohydrates and likewise glycerine cannot be
assimilated. Under certain conditions of cultivation, e.g., in
potato-cultures, the species develops quite a different form, in
which the gelatinous envelope is completely absent.

The Leuconostoc ferments grape-sugar, cane-sugar (after
previous inversion), milk-sugar, maltose, and dextrin, with

76 MICRO-ORGANISMS AND FERMENTATION.

production of gas and acid. The fungus secretes an enzyme
which inverts cane-sugar; but no other enzymes could be
detected.

Especially characteristic of this fungus is its power of
resisting elevated temperatures, the younger growths pos-
sessing this power in a higher degree than older cultures.

It is also remarkable that the growth and fermentative
action of the fungus are very favourably affected by the
presence of considerable quantities of calcium chloride.

6. BACTERIA EXERCISING AN INVERTING, DIASTATIG, OR
PEPTONISING ACTION.

Bacteria play a very great part in the formation of soluble
chemical ferments. This constitutes one of the chief means
by which these organisms exercise such an important activity
in the economy of nature.

According to statements made by Hansen, many species
of the bacteria which generally occur in beer secrete invertive
ferments. Amongst these species are a number of bacteria
which exhibit an invertive action in a pure cane-sugar
solution but lose this property when yeast-water is added.
Similar properties were observed by Wortmann in the case
of bacteria which develop diastatic ferments. He found these
on putrefying beans and potatoes, and grew the cultures in
mixtures of nutritive salts and wheat-starch. Marcano also
found a species which exercises diastatic action and which
frequently occurs in the outer envelope of maize. Peters
found a bacillus in leaven which brought about the solution
of starch. In ordinary gelatine-plate cultures this fungus
forms peculiar curved colonies, consisting of long filaments
about 0-5 p- thick ; in young colonies the filaments are shorter
and motile. In beer-wort the bacillus forms rods which
exhibit very active movements, and which gradually produce
a film on the surface. The spores are rod-like, and their
highly refractive contents are for the most part situated at
the ends.

BACTERIA. 77

Peters described another bacillus, which he found among
the organisms occurring in leaven, and which possesses a
peptonising power. Small rods grow out of the spores, and
these rods increase to long filaments, which in their turn
divide into rods. In ordinary nutritive gelatine this species
does not thrive well, if at all ; whilst, on the other hand,
it thrives readily and vigorously when " soluble starch " is
added ; the gelatine rapidly becomes liquefied. Spores appear
abundantly in cultures in neutralised yeast-water. In sus-
pended drop-cultures it was found that small pieces of boiled
white of egg were much acted on or completely dissolved by
this- species.

7. SARCINA FORMS.

In addition to the above-mentioned Pediococcus acidi
lactici there also occur in fermenting liquids a number of

FIG. 15 : Sarcina.

other spherical bacteria, the life-histories of which are only
very imperfectly known. Both in bottom-fermentation and
in top-fermentation (especially in distilleries and pressed-
yeast factories) different varieties of Micrococci occur, the
injurious action of which is strongly emphasised in the
journals relating to these industries. This has, however, only
been satisfactorily demonstrated by direct experiment in a
single case (see Section on " Slime-forming bacteria "). In
bottom-fermentation lager-beer these forms appear as small,
more or less spherical, water-grey bodies, sometimes isolated,
sometimes arranged in groups, generally in groups of four.
They were described by Hansen under the name of Sarcina
(Fig. 15). Organisms belonging to this group are found in
very different localities. The true places of growth of the
individual species are, however, not yet known.

Reincke often observed such forms, both in bottom- and

78 MICRO-ORGANISMS AND FERMENTATION.

top-fermentation beers. He found that lager-beer, when so
attacked, soon yielded a considerable sediment, and developed
a bad odour and taste. The Berlin white beer often assumed
a red colour, and it was then always found to be infected with
many Sarcina forms, the growth of which increased
considerably after a few days at a somewhat higher tempera-
ture; temperatures between 10° and 14° C. are stated by
Reincke to be particularly favourable in this case. However,
he correctly lays stress on the fact that it is not certain
whether Sarcina or the rod bacteria, which are also present,
are the actual cause of the disease ; it is only known that in
red beer the presence of Sarcina is a symptom of abnormal
conditions ; whether it is the cause or the result can only be
determined by exact investigations.

In the fresh residues from the distillation of spirit, which
are employed as fodder, Brdutigam found a sarcina-like
micrococcus, which possesses pathogenic properties. It has
not yet been determined by direct experiments whether the
so-called " malanders " or " greasy heels " of domestic animals
is caused by this organism.

Lindner examined a series of sarcina-like organisms, and
contributed largely to our knowledge of the life-histories of
these organisms. The so-called Pediococcus cerevisice appears
in cultures in the form of cocci, diplococci, or tetrads.
Cultures made on meat broth with peptone gelatine, and
partially covered with thin plates of gypsum, showed that
the access of air is favourable to the growth of the colonies
of this bacterium ; during the first days all the colonies were
found to be colourless ; subsequently a yellowish, or yellowish-
brown, tinge began to appear. The gelatine was not liquefied.
On meat-broth gelatine this organism gave, in streak cultures,
a greyish white, moist streak, with nearly smooth borders,
and which, in thin layers, was strongly iridescent. In
puncture-cultivations it developed throughout the length of
the puncture, forming a white tuft on the surface of the
gelatine, which spread out like a leaf. On boiled slices of

BACTERIA.

79

ale

FIG. 16.

Crenothrix Kiihniana, after Zopf : a—e (600 : 1), cocci in different stages of
division; / (600: 1), small, round cocci-zoogloea ; g (natural size),
zoogloea ; h (600 : 1), colony of short filaments composed of rod -like
cells, originating from the germination of a small collection of cocci :
i — r, filaments, partly straight, partly spirally curved (I, m}, of very
varying thickness, with more or less pronounced contrast between the
the base and apex, and different stages of the division of their members
and sheaths ; the sheathed filament r shows short rods at the base, which
higher up are divided into small cylindrical pieces ; at the apex the
cocci are seen arising from the longitudinal divisions of the cylindrical
discs.

80 MICRO-ORGANISMS AND FERMENTATION.

potato this species thrives but poorly ; in older cultures of this
kind peculiar involution forms appear. In meat-broth
gelatine the organism was killed after eight minutes' heating
at 60° C., but not at 50° to 55° C. after the lapse of 12
minutes. In hopped beer-wort it yields a sediment, and
subsequently forms a film. The formation of acid in the
liquid after the action of this Pediococcus is very slight, and
the author assumes that traces of lactic acid are formed.
Lindner states that in no case was he able to produce any
real disease in wort or beer by inoculating these liquids with
a vigorous growth of this bacterium ; he therefore remarks
that the change in the flavour of the beer may not be caused
by this species, but by other bacteria co-existing in the
infected beer ; on the other hand, he states that Pediococcus
cerevisice causes a turbidity. The slime-forming species
described by Lindner has been mentioned above.

A. Petersen observed that an abundant development of a
Sarcina could take place in bottom-fermentation lager-beer
without causing any disease ; on the contrary, the beer was
bright and stable, and had an agreeable taste and odour.

Thus, Sarcina species exist which are not productive of
any disturbance in the brewery. The investigation of this
problem has not, however, as yet been carried further.

8. CRENOTHRIX.

In microscopical examinations of water we often meet the
very typical forms of Crenothrix Kuhniana (Fig. 16).

This ferment (frequently associated with Beggiatoa alba)
occurs in every water which contains organic matter ; some-
times it multiplies to such an extent that it may make the
water unfit for use. Thus, according to Zopf, great
calamities have been caused by this fungus in the water
supplies of Berlin, Lille, and certain Russian towns. In
consequence of its power of storing iron compounds in its
walls, it forms red or brown flocks in water. Its forms are
very beatitiful ; it occurs in the form of cocci (a — -/), which

BACTERIA. 81

by partition and formation of viscous matter form zoogloea ;
these cocci frequently grow to articulate filaments, which are
provided with distinct sheaths (h, i — r) ; they increase in
thickness towards the apex ; when they have arrived at a certain
age, they divide within the sheath into smaller pieces, which
become round and issue forth as rods, macro- or micrococci ;
these are sometimes seen floating about in water. We do
not yet possess a more exact knowledge of the life-history
of this beautiful bacterium.

CHAPTEE IV.
The Mould-Fungi.

THE mould-fungi ordinarily affect the fermentation industries
in a somewhat different manner from the bacteria. Whilst
the latter — in distilleries as a rule, in breweries only
exceptionally — make their appearance in great force during
the fermentation, and are therefore able to bring about
important changes in the course of the fermentation, and in
the resulting products, the mould-fungi, on the contrary,
usually occur outside the true field of the fermentation in
that they select as places of growth the vessels, tools, rooms,
the green malt, and the quiescent masses of yeast, especially
top-fermentation yeast. Accordingly the mould-fungi have a
more subordinate, but nevertheless very real, importance. If
we only sufficiently examine a growth of mould which has
developed on the ceiling or walls of a fermenting room, or
on the sides of a vessel, it will very soon be found that we
have practically never to do with a mould growth alone ; in
nearly every case bacteria and yeast-like cells are found
amongst the filaments of mould. These filaments extend
upwards, and thereby raise the foreign bodies which in this
exposed position are more readily carried away, partly by the
workmen, and partly by the air.

During malting, all sorts of microscopic organisms are
present on the raw materials containing starch. The mould-
fungi are usually regarded as the most dangerous enemies,
and this is certainly due to the fact that they are visible to
the naked eye during development, and thus obtrude them-
selves upon our notice in an unmistakable manner. If,

THE MOULD-FUNGI. 83

however, numerical superiority be taken into account, the
bacteria, which are always present in large numbers on green
malt, must certainly be placed in the front rank. Judged
from this side, it may even be considered doubtful whether
the greatest influence on the product must be attributed
to the mould-fungi (Penictttium, Aspergillus, etc.,) when
these are met with in a state of vigorous development
on the malt, or whether it is not far more probable that it is
the numerous other organisms accompanying them which
here play the most important part.

I have often found on the surface of pieces of pressed
yeast a fine white parasitic growth, which most frequently
consists of a mould mycelium, belonging principally to forms
resembling Chalara and Dematium. It is very possible that
when these plants form a thick layer on the surface of the
yeast-mass, they retain by their respiration a portion of the
free oxygen which is necessary to enable the quiescent yeast
to remain alive for a longer time. Even here I always, with-
out exception, found bacterial growths.

The truth is, that from observations made in breweries
and elsewhere, a growth of mould nearly always serves to
indicate that other organisms of a doubtless more injurious
and more active character are present in the growth. It is,
therefore, of great importance that the walls of fermenting-
rooms should be smooth ; this is effected with the greatest
certainty by employing the enamel paint now so much in
use.

The following is a review of the most important mould
forms which are of interest for the fermentation industries.

1. BOTRYTIS CINEREA

forms small greyish -yellow patches on moist, decaying
vegetable matter, and can also occur on wort. From the
greyish-brown mycelium the conidiophores are thrown up ;
these are perpendicular, articulated filaments, generally
arranged in tufts. They grow to the height of 1 mm.,

84

MICRO-ORGANISMS AND FERMENTATION.

after which the apical cell throws out near its point, and
almost at right angles, two to six small branches (C"). The

y/v

FIG. 17.

Botrytis Cinerea, after de Bary : a, b (natural size), Sclerotia, from which
at a the conidiophores, at b the apothecia (fruits with asci), are thrown out ;
c, 0, conidiophores (C", with conidia just ripe), springing from the
mycelium filament m ; C" , end of a conidiophore with the first .com-
mencement of formation of conidia from the ends of the branches ; k,
germinating conidium ( x 300) ; p, s (slightly magnified), section through
a sclerotium s, from which a very small apothecium (p, p) is thrown up ;
7i,' single ascus. with eight ripe spores ( X 300).

THE MOULD-FUNGI. 85

lowest of these branchings are the longest ; these again
develop below their ends one or more short side branches.
The topmost branches are almost as wide as they are long.
Thus a system of branchings is formed which is shaped like
a raceme or a bunch of grapes. When the longitudinal
growth is at an end, the inner space of the branches becomes
separated from the main stem by the formation of a transverse
wall close to the latter. At the same time the ends of the
branches and of the main stem swell, and on the upper
half of each swelling several small papilla? now appear near
together ; these quickly increase to oval blisters, filled with
plasma, and become narrowed, stalk-like, at their base.
When these conidia (C') are completely developed, the walls
of the branches carrying them are shrivelled up, and the
conidia are consequently brought so closely together that
they form a loose, irregular aggregation which readily falls
off. If these clusters are placed in water, the conidia become
detached from their stalks, and the envelopes of the branches,
devoid of plasma, shrivel up or are only to be found in traces;
their former place of attachment to the main filament appears
only as a slightly raised scar. The member next below can
now throw on one side the shrivelled apex, grow upwards,
and form a new cluster ; this can be repeated several times,
whereby the conidiophores attain a considerable length.

Under certain conditions this mould can assume a peculiar
state of rest, the so-called sclerotium (skleros = hard) (a, 6, ss).
The hyphal threads branch extremely freely, and the branches
intertwine themselves into a continuous body of diverse shape,
circular to narrow spindle-shape, and of varying size up to
a few lines ; the extreme ends of the filaments are brown to
black, and the ripe, solid sclerotium thus consists of an outer
black rind and an inner colourless tissue. Such bodies are
capable, after a long period of rest — at least one year — of
forming a new growth, and may in so far be compared with
the bulbs and roots of the higher plants. If the sclerotium
is brought into a moist place soon after it comes to maturity,

86 MICRO-ORGANISMS AND FERMENTATION.

the inner colourless branches break through the black outer
rind and throw up the conidiophores (a). If, however, the
sclerotium is not brought into a moist place until after it has
been in rest for some time, a large tuft of filaments develops
from the inner tissue, and these shoot up perpendicularly and
finally spread out to a flat, plate-shaped disc (6 and_ps) ; the
ends of the filaments appear parallel on the free upper surface
of the disc ; some of them remain thin, others swell up to
club-shaped asci, and each of these asci forms in its interior
eight oval spores (n). The mould has now entered upon the
stage in which the formation of apothecia takes place. The
spores germinate when they are set free, and the germ tubes
grow into conidiophores.

According to Bersch, Fitz, and Reess, this organism is
the cause of one of the diseases of wine, which manifests
itself as an unpleasant smoky taste and smell. Similar cases
of disease have been occasionally observed in breweries ; it has,
however, not yet been determined with certainty whether
they are caused by this mould.

2. PENICILLIUM GLAUCUM.

A mould which is far more widely distributed in the
fermentation industries, especially in green malt, is Peni-
cillium glaucum. It forms a felt-like mass on the sub-
stratum, is at first white, then greenish or bluish-grey,
and spreads with great rapidity. The mycelium consists of
transparent branched and divided filaments, which, wThen
immersed in liquids, are able to swell somewhat irregularly.
From these filaments the conidiophores (A) are thrown
up perpendicularly. They consist of elongated cylindrical
cells, the terminal cell of which soon stops in its longi-
tudinal growth and becomes tapering and pointed; the cell
next below throws out one or more opposite branches, which
rise up close to the terminal cell and, like this, consist
of one pointed cell. In more vigorous individuals the
branches may again ramify (compare Fig. 18J., above),

THE MOULD-FUNGI.

87

FIG. 18.

Penicillium glaucum, after Brefeld and Zopf: A, conidiophore ; B, organs of
generation ; G, first development of the sclerotium (a, ascus-formirig
hyphse ; b, sterile filaments) ; D, very young sclerotium in section (a,
ascus-forming hyphae ; b, sterile portion of the sclerotinm ; m, myce-
lium) ; E and F, ascus-forming hyphse (a) with young asci (s) and
sterile mycelium threads (m) from a more developed sclerotium) ; G,
group of asci with spores ; H, spore ; /, germinating spores ; K, young
mycelium (with spore at x) ; A — E (below), germination of a conidium,
after Zopf (more highly magnified) ; A, conidium before germination ;
B, it has thrown out a germ tube ; C, three germ tubes have been formed ;

D, each germ tube shows towards the spore a transverse septum (.s) ;

E, each germ tube has become divided by another septum (s'} into
a terminal cell («) and an inner cell (6).

88 MICRO-ORGANISMS AND FERMENTATION.

or similar branches may also spring from the next cells,
and these again ramify and become pointed as described
above. In this tuft of branches each pointed cell (sterigma)
breaks up into a series of spherical conidia, and finally the
tuft carries a large number of conidia, arranged in series,
which, when ripe, are readily scattered. These round, smooth
conidia give to the patches of mould their greyish-blue
colour ; when they fall upon moist surfaces, they are able
to germinate at once.

In culture experiments with this fungus, Brefeld made the
interesting observation that PeniciUium can occur under
certain conditions with an entirely different form of growth.
He enclosed cultures of this mould-fungus on slices of coarse,
non-acidified bread, between glass-plates, and allowed the
culture to further develop with the greatest possible exclusion
of atmospheric air. There then appear in pairs on the
mycelium short, thick branchings, which become entwined
(5, above) ; one part of this spiral throws out short, thick
filaments ((7), whilst the hyphal thread carrying the spiral
develops numerous fine branchings, which envelop the spiral
and form a covering (D), consisting of an inner solid and an
outer felt-like layer ; the inner cells gradually become coloured
yellow, and the outer loose cells are cast off. In this small
yellow ball — sclerotium — a formation of swollen cells (E, F,G}
gradually takes place by the continued branching of the
above-mentioned spiral filaments, and in each of these new
cells eight large and lenticular spores are produced, which
have a circular furrow on the margin, and three or four slight
ridges on the outer membrane (Exosporium). After the
collapse and absorption of all the remaining interior elements
the spores are at last set free, and the small yellow ball is
then filled with the spore-dust. The entire development
requires six to eight weeks. The sclerotia may be preserved
in a dry state for several years without losing their power of
germination. When the spores (H) are sown, the exosporium
bursts open like a shell at the circular furrow, and the endo-

THE MOULD-FUNGI. 89

sporium swells and emerges (/), and elongates itself to a
germ tube, which quickly develops conidiophores.

Penitillium possesses the power of secreting an invertive
ferment, which is able to convert cane-sugar into other sugars.

3. EUROTIUM ASPERGILLUS GLAUCUS.

The development of this fungus was first thoroughly
described by the celebrated de Bary. It forms a fine felty,
greyish or greyish-green covering on various materials, and is
able to grow with the greatest luxuriance on green malt.

The mycelium consists, as in the case of Penicillium, of
fine transparent and branched threads, provided with trans-
verse septa. Some of the hyphal threads are thrown up
perpendicularly, are thicker than the rest, and very rarely
branched or divided by septa. Their upper ends swell to
spherical flask-shaped heads (c); and these throw out from
their entire upper portion radially divergent papillae of an
oblong form ; these sterigmata then .throw out at their apex
small round protuberances, which are attached to the sterig-
mata by greatly contracted bases, and after some time are
defined from the former as independent cells (spores, or
conidia). Below the base of the first spore, a second begins
to form from the crown of the sterigma, and pushes the first
upwards ; a third then forms, and so on. Each sterigma thus
carries a chain of spores, the youngest of which is closest to
the sterigma. This occurs at the same time over the whole
surface of the enlarged ends of the conidiophore, which is
thus finally covered with a thick head of radially-arranged
chains of spores. These masses of spores form the greyish -
green dust which covers the mycelium.

Finally, the conidia separate from one another ; they have
then a warty appearance on their outer surface. These
small bodies are able to germinate (p) directly after they
have become detached, and quickly develop a new mould-
fungus; on this fact depends the rapidity with which the
plant spreads. Under certain conditions, which are not yet

M

Fro. 19.

THE MOULD-FUNGI. 91

sufficiently known, but which in every case appear to be
connected with a free supply of nutriment, the mould develops
perithecia. These appear at first as tender branches, which,
at the termination of their longitudinal growth, begin to
twine their free ends in the form of a spiral of four to six
turns (/) ; the threads of the spiral gradually approach
nearer together, until finally they are brought into contact,
so that the entire end of the filament takes the form of a helix
(the ascogonium). There then grow from the lowest turn
of the helix two or more small branches, which cling closely to
the spiral. One of these small branchings ($, T, p) quickly
outstrips the others in growth, and its upper extremity reaches
the uppermost turn of the helix, and becomes fused with it.
The other brancli or branches likewise grow upwards along
the spirals, shoot out into new branches, and gradually become
so interlaced that finally the spiral becomes surrounded by
an unbroken envelope (W). These branches become divided
by septa perpendicular to the surface, and the envelope
consequently consists of short, angular cells, in which new
septa appear parallel to the surface, so that the envelope
becomes thicker and composed of many layers (F, X, F).
The small sphere now formed is about one-quarter mm. in
diameter ; the outermost layer is yellow, whilst the inner

FIG. 19.

Eurotium Aspergillns Glaucus de Bary : m, m, hyphal thread, carrying a
conidiophore c (from which the conidia have fallen), a perithecium F,
and the first rudiments of an ascogoninm, / (X 190) ; .<?, three sterig-
mata from the crown of a conidiophore, showing the conidia-constric-
tions ; p, germinating conidium (x 250 — 300); A, ASCIIS; r,
germinating ascospore ; k, germ tubes ; S, spiral ascogonium ; at p
the commencement of the growth of one of the enveloping hyphse ;
T, older stage ; Jf , ascogonium, already surrounded by the envelope ;
V, longitudinal section of an older stage ; in the centre the ascogonium,
surrounded by the envelope, which now consists of several layers ;
X, longitudinal section of a later stage of development ; the ascogonium
is enveloped in a sheath of many layers, and it has loosened its convo-
lutions, and commences to throw out the ascus-forming branches ;
My portion of an older ascus-bearing branch ; a, a young ascus ; a', an
older ascus which has burst.

92 MICRO-ORGANISMS AND FERMENTATION.

layers remain soft, and later are dissolved. The spiral after
a time extends and throws out on all sides branched filaments
which dislodge the interior layers of the envelope. These
branchings finally take the form of an ascus (If, and J.), and
in each eight spores are formed. After the breaking np of
the asci the spores lie loose in the interior of the perithecium,
and are liberated by the rupture of the now fragile wall of
the latter. The spores, as in the case of Penicillium, are
bi-convex, warty, and possess an outer stout membrane and an
inner one, which, on germination, bursts the outer membrane
into two valves (r).

This mould-fungus contains a diastatic ferment, which
converts starch into dextrin and maltose.

In addition to this species, several others, closely related,
occur in nature, and also find their way to the places
mentioned here. In the greater number only the conidia
stage is known.

4. ASPERGILLUS ORYZJE.

In the preparation of the strong fermented Japanese
rice wine (" sake "), the so-called Aspergillus Oryzce is
systematically employed.1 The rice grains, freed from the
hulls, are steamed, but the aggregation and gelatinisation of
the grains are avoided. In order to prepare a malt serviceable
for the brewer from these grains, which are not capable of
germination, and from which the ordinary diastatic action is
consequently excluded, the mass of grains is mixed with the
so-called " Tane kosi " — rice grains, which are coated over
with the mycelium and conidia of Aspergillus Oryzce ; or
the yellowish-brown spores of the fungus are mixed with the
steamed rice grains. In the moist and warm air there
develops on the rice at the end of about three days a white
velvety mycelium, which gives to the whole mass an agreeable
odour, resembling apples or pine-apples. Before the fructifi-

1 Researches on this ferment were made by Ahlburg, Atkinson,
Biisgen, Cohn, Ikuta, Kellner, Mori, and Nagaoka.

THE MOULD-FUNGI. 93

cation of the fungus takes place, a fresh quantity of steamed
rice is introduced, and this also becomes coated over with
mycelium ; this process is repeated several times. In the
koji-rnass thus produced a part of the starch has been converted,
and some of the albuminoids, which before were insoluble in
water, have become soluble. The koji-mass is mashed, 21
parts of koji being mixed with 68 parts of rice boiled by
steam, and with 72 parts of water. This pasty mass is
allowed to remain at about 20° C. ; after some days it becomes
clear, the saccharification of the starch and dextrin continually
progresses, and at the same time a spontaneous and very violent
fermentation sets in, being caused by a yeast-like fungus,
which does not stand in any genetic relation to the Aapergillus,
and about which nothing is known. At the end of two or
three weeks the fermentation is finished, and the product,
after being filtered, is a clear, yellow, sherry-like liquid,
containing 13 to 14 per cent, of alcohol. It is then
pasteurised at 44° C. in iron vessels.

Atkinson found a ferment in koji which is soluble in water,
and which inverts cane-sugar and converts maltose, dextrin,
and starch-paste into dextrose. The researches of Kellner
Mori, and Nagaoka, likewise showed that the koji-mass
possesses a strongly invertive ferment, which converts cane-
sugar into dextrose and levulose, maltose into dextrose,
starch into dextrin, maltose, and dextrose. The various
micro-organisms which occur in the koji-mass in all like-
lihood possess different invertive ferments. The presence
of such different invertive ferments has before been pointed
out by Bourquelot.

5. MUCOK.

The genus Mucor belongs to the most interesting of the
groups of mould-fungi with which we have to deal, since it
embraces species with very" marked fermentative action.
These generally occur as a grey or brown felt-like mass,
sometimes of very considerable height — even several inches

FIG. 20.

THE MOULD-FUNGI. 95

— in which small yellow, brown, or black spherules can be
distinguished by the naked eye.

We give a description of the most frequently occurring
species.

Mucor Mucedo (Fig. 20), one of the most beautiful
mould-fungi, and one which occurs very generally on the
excreta of phytophagous animals, has a transparent white
mycelium, which develops numerous and delicate ramifi-
cations on the surface of and within the substratum, and
which, in its earliest stages of development, and until the
sporangia begin to form, is without transverse septa, and
therefore unicellular. From the mycelium are thrown up
single vigorous branches, the sporangium-carriers ; the
points of these branches which, according to Zopf, contain
a reddish-yellow fatty colouring matter, swell greatly, and
below the swelling a transverse septlim is finally formed,
whereby the sporangium is marked off from the sporangium-
carrier. The transverse wall becomes arched upwards, and
forms a short column — termed the columella — in the interior
of the spherical head, whereby an inner space of peculiar
form (1) results. The protoplasm of this space breaks up
into a number of small portions, which become surrounded
with a membrane and are rounded off; these are the spores.
At the same time the sporangium becomes coated on its
outer surface with small needle-shaped crystals of calcium
oxalate. As soon as the ripe black sporangium takes up
moisture, the wall is dissolved, and the spores with their
yellowish contents are scattered on all sides along with the
swelling contents of the sporangium. The columella, which
projected upwards in the sporangium still remains at the

FIG. 20.

Mucor Mucedo, after Brefeld and Kny : A, tree-like ramified mycelium
with isolated thicker upright branches (a, b, c). 1, Sporangium ; 2.
columella and spores ; 3, 4, germinating spores ; 5, 6, development ot
the zygospore ; 7, germinating zygospore with sporangium.

96 MICRO-ORGANISMS AND FERMENTATION.

end of the sporangium-carrier ; this is now surrounded at
its base by a collar (2), the remains of the outer wall of
the sporangium. When the refractive spores fall on a
favourable substratum, they swell very considerably and
send out one or two germ tubes (3, 4), which quickly
develop to a vigorous mycelium.1

In addition to this mode of reproduction, Mucor Mucedo
and the other species possess also a sexual method of repro-
duction, which takes place by means of a conjugation of two
branches of the same mycelium. Two such short branches,
filled with plasma, and growing towards each other, form club-
like swellings and come in contact at their free ends, which
become flattened (5). Each of the branches is then divided
into two cells by a septum, and the end cells, which are
in contact (the conjugating cells), coalesce by the dissolu-
tion of the originally double wall which separated them.
The two conjugated cells are either equal in size, as in Mucor
Mucedo, or unequal, as in Mucor stolonifer. The new
cell thus formed — zygospore (6) — quickly increases in size
and expands to the shape of a ball (in Mucor stolonifer
to the shape of a barrel), after which the wall becomes
thickened and stratified ; externally it is coloured dark and
covered with wart-like excrescences. These outer layers are
very resistant to the action of acids. The contents possess
an abundance of reserve substances (fat). The zygospores
are generally able to germinate only after a long period
of rest ; the germ tube, after bursting the outer layers,
quickly develops the above-mentioned sporangia (7). In
the zygospore we thus find a resting-stage of the plant, an
organ which by its structure enables the mould to preserve
its life during periods which are unfavourable for its growth.

Mucor racemosus, which occurs especially on bread and
decaying vegetable matter, has a branched, many-celled
sporangium-carrier, which can also attain to a consider-

1 Many of the above-stated botanical characters do not apply to
M. Mucedo alone, but must rather be considered as generic characters.

THE MOULD-FUNGI. 97

able height. The brownish sporangia are developed at the
ends of the branches. The spores are colourless. When
this fungus is cultivated in wort, the submerged mycelium
swells irregularly, and a large number of transverse septa
appear, which divide it into large barrel-shaped or irregular
cells filled with highly refractive plasma. These cells — gemmce
— are readily separated, and then assume a spherical shape
(compare Fig. 21,7), as was first observed by Bail, and multi-
ply by budding like the true yeast-fungi ; the same takes
place with the submerged spores (Mucor-yeast, spherical
yeast). The mycelium produces a similar characteristic for-
mation of gemmae when cultivated on solid substrata. The
plasma of the filaments collects in certain places in a
compact mass, and is then enclosed at both ends by a
transverse wall. At the same time the cell swells, the, walls
become thickened, and fatty substances are stored in the
interior. The intermediate portions of the hyphae gradually
lose their contents.

Mucor erectus occurs, for example, on decaying pota-
toes and has the same microscopic appearance as Mucor
racemosus ; physiologically, however, it differs from this.

Mucor circinelloides (Fig. 21) has a very characteristic
appearance. The mycelium (1) shows the remarkable
branching which occurs in some of the species of Mucor : —
the main branches (6) send out short, root-like, repeatedly-
forked branches (c) ; at the base of these grow new mycelial
branches (r), which become erect, and are able to form
sporangia (2 to 5) ; the sporangium-carrier is branched.
During its development it becomes considerably curved, and
to this the species owes its name of circinelloides. In
this form, as in Mucor spinosus, whose chocolate-brown
sporangia are distinguished by the columella being studded
on its uppermost part with pointed, thorn-like protuberances,
the mycelium, when submerged in a saccharine liquid,
produces a similar formation of gemmae, as Mucor racemosus
and Mucor erectus.

H

98

MICRO-ORGANISMS AND FERMENTATION.

Mucor stolonifer (Khizopus nigricans) attains a very
considerable size, and occurs very commonly, for instance, on
succulent fruits. This mould is easily recognised, since the
brownish-yellow mycelium sends aslant into the air thick
hyphse without septa. These attain a length of about 1 cm.,
then sink their points to the surface of the substratum, and
send out fine, greatly ramified hyphae, resembling rootlets,

FIG. 21.

Mucor Circinelloides, after van Tieghem and Gay on : 1, Mycelium ; 6, main
branch ; c, root-like branches ; r, axillary branches ; 2—4, develop-
ment of sporangia ; 5, opened sporangia ; 6, spores ; 7, submerged
mycelium and budding cells.

into the latter, whilst other hyphse rise perpendicularly and
develop sporangia ; other branches again form new " runners."
The black sporangium possesses a high, dome-shaped
columella, and develops a number of dark-brown round or
angular spores. When these become free by the absorption
of the sporangium wall, the columella is turned over on the

THE MOULD-FUNGI. 99

sporangium-carrier like an umbrella, the line of junction
of the external wall remaining in evidence in the form of a
collar.

The species of Mucor have, considered from one point of
view, very considerable interest, since they are able to act,
in different degrees, as true alcoholic ferments. As previously
mentioned, some of the species of Mucor,, when immersed in
a fermentable saccharine liquid, very quickly change their
appearance ; and whilst the mould thus approaches the yeast-
like fungi in its appearance, it at the same time causes an
actual alcoholic fermentation, yielding alcohol and carbonic
acid- as the chief products. If then the above-mentioned
free cells of the mould-fungus are brought to the surface of
the liquid by the bubbles of carbonic acid, they are able to
again develop the mould form. The power of bringing about
an alcoholic fermentation is possessed by the majority of the
species of Mucor, but in a different degree ; still the fermen-
tative power is not exclusively connected with the formation
of the above-mentioned budding gemmae, since these have
not been observed in Mucor Mucedo and stolonifer.

According to the recent investigations of Hansen, the
various species, as far as they really are alcoholic ferments,
induce fermentation not only in solutions of dextrose and
invert-sugar, but also in solutions of maltose. Of all the
species which he investigated, Mucor racemosus is the only
one thai is capable of inverting a cane-sugar solution ; the
others are consequently unable to bring about fermentation
in a solution of this sugar.

The most active fermentative power is possessed by Mucor
erectus. In beer-wort of ordinary concentration — 14 to 15°
Balling — it yields up to 8 per cent, by volume of alcohol. It
also induces alcoholic fermentation in dextrin solutions, and
converts starch into reducing sugar. Mucor spinosus yielded
up to 5*5 per cent, by volume of alcohol in beer- wort. In
maltose solutions distinct fermentation phenomena were
observed, and at the end of eight months the liquid con-

100 MICRO-ORGANISMS AND FERMENTATION.

tained 3 -4 per cent, by volume of alcohol. Mucor Mucedo has
only a comparatively feeble fermentative power both in wort
(up to 3 per cent, by volume of alcohol) and in maltose and
dextrose solutions. Mucor racemosus produces in wort as
much as 7 per cent, by volume of alcohol, develops invertase,
and ferments the inverted cane-sugar ; thus, as mentioned
above, it stands quite alone.

Mucor circinelloides is, according to Gayon, without
action on cane-sugar, whilst it exercises a very powerful
action on invert-sugar (yielding 5' 5 per cent, by volume of
alcohol). "Oayon concluded from this that this mould might
with advantage be employed to extract the cane-sugar from
the molasses in the manufacture of sugar. So far, however,
as I have been able to learn, this observation has not yet
received any practical application.

6. MONILIA.

Under this name are found described in works on
mycology a large number of different fungi of comparatively
simple structure ; from a mycelium, the colour of which
varies according to the species, branches are thrown up, which
give rise to series of egg-shaped or elliptical spores. The
genus has lately attracted interest on account of one of its
species, which Hansen has provisionally named Monilia
Candida, from Bonorden's description, and which shows very
remarkable physiological properties. It occurs in nature in
the form of a white layer covering fresh cow-dung, and on
sweet, succulent fruits. When introduced into wort, it
develops a copious growth of yeast-like cells, which resemble
Saccharomyces ellipsoidetis, or cerevisice. At the same time
it excites a vigorous alcoholic fermentation, and whilst this is
progressing forms a mycoderma-like film on the liquid ; the
cells of this film extend more and more, and finally form a
complete mycelium. In the first period the fungus produced
only 1-1 per cent, by volume of alcohol, whilst Sacch. cerevisice
gave 6 per cent. ; but the Monilia continued the fermentation,

THE MOULD-FUNGI.

101

FIG. 22.

Monilia Candida, after Hansen : A, growth in beer-wort or other saccharine
nutritive liquids ; B, cells of a young film-formation ; G (p. 102), growth
of mould : forms like a are frequent ; they consist of chains of elongated
more or less thread-like cells, rather loosely united ; at each joint there
is generally a verticil of oval cells, which readily fall off ; b represents
another form, also very frequently occurring, but distinguished from the
former by having no verticillate cells ; instead of these there generally
issues from every joint a branch of the same form as the mother cell, but
shorter ; the links of these chains are not seldom closely united together,
the constrictions in many cases disappear, and a very typical mycelium,
with distinct transverse septa (c), is produced ; the forms b and c occur in
the nutritive medium, a commonly on the surface. Forms like d have
much resemblance to Oi'dium lactis. e shows a chain of pear-shaped cells
with verticils of yeast-cells resembling Sacch. exiguus ; the chain of
lemon -shaped cells represented at /closely resembles Ehrenberg's figures
of Oidium fructigenum. Between the principal forms here described
there are numerous yeast cells of different forms, and differently arranged
in colonies ; as is usually the case, there also appear forms like Sacch.
conglomerate Reess.

FIG. 22 c.
Monilia Candida, after Hansen. (For description see preceding page.

[p. 102.]

THE MOULD-FUNGI. 103

and produced, at the end of six months, 5 per cent, by
volume of alcohol, whilst the culture-yeast did not give more
than the above-mentioned quantity.

Further experiments with this fungus led to the remark-
able discovery that it does not possess the poiver of secreting
the soluble chemical ferment invertase, and yet ferments cane-
sugar as cane-sugar. As is known, cane-sugar has heretofore
been considered to be not directly fermentable ; Hansen has
thus proved that this statement is not universally applicable.

His investigations have likewise proved that this species
also ferments maltose. Since Monilia does not form invertase
and is yet able to excite a fermentation in maltose solutions,
it follows that a previous conversion of maltose into dextrose
is not necessary in order to bring about a fermentation of
this sugar.

The liquids containing the above-mentioned sugars showed
during fermentation the presence of carbonic acid and ethyl-
alcohol.

Finally, it is worthy of mention that this fungus is dis-
tinguished by its power of withstanding high temperatures.
In beer-wort and cane-sugar solutions it develops vigorously at
40° C., and induces an active fermentation at this temperature.

7. OlDIUM LACTIS.

A mould-fungus which has played an important part in
the literature of the physiology of fermentation and in that
of medicine is Oidium lactis, the so-called lactic acid yeast.

Some authors have sought to establish the theory that this
fungus is a stage in the development of species which, under
other circumstances, occur in entirely other forms, and with
quite different properties. It was thus brought into genetic
relation with Bacteria, Chalara (see below), Saccharomyces,
etc. Both Brefeld and Hansen have carried out numerous
investigations with this fungus, and have undertaken culture
experiments, which were continued for a long time without
producing any other than the ordinary Oi'dium-form. Eecently,

FIG. 23.

Oi'dium lactis, after Hansen : 1, Hyphse with forked partitions ; 2, two
ends of hyphse — one with forked partition, the other with commencement
of development of a spherical link ; 3 — 7, germinating conidia ; 6 — 6'",
germination of a conidium, sown in hopped beer-wort in Ranvier's
chamber, and represented at several stages ; at each end germ tubes

[p. 104.]

THE MOULD-FUNGI. 105

it is true, Brefeld has discovered, in several higher fungi, a
formation of conidia resembling chains of Oidium cells ; but
it has not yet been determined whether this also includes
that particular species which we designate Oidium lactis.

Fresenius correctly gave to this species the specific name
lactis (of milk) ; for universal experience goes to show that it
has its ordinary place of abode in milk, where it can in the
majority of cases be found. Up to now, however, no evidence
has been brought forward that this mould-fungus stands in
causal relation to the acid fermentations of milk. Further, it
occurs spontaneously in various other liquids, and among these
in -the saccharine mixtures which find employment in the
fermentation industries, and in these it is able to induce a
feeble alcoholic fermentation.

The often forked, branched, thin-walled, transparent hyphse
(1) form a thick white felt; in the uppermost portions of the
filaments transverse septa are formed close together, after which
the single cells, filled with very refractive plasma, become
detached as conidia (3 to 7, 11 to 14, 17 to 19). When the
fungus grows on solid substrata, the hyphae unite and form
remarkable conical bodies. As a rule, the conidia in longitu-
dinal section are rectangular with rounded corners (3, 6, 1 7 to
19); in a growth of this mould-fungus, spherical, roundish, pear-
shaped, and quite irregular conidia (4, 5, 1 1 to 14) are, however,
also nearly always found. These organs of multiplication, the
only ones known, send out one or more germ-tubes. The
fungus may occur in beer, especially when poor in alcohol.
As the amount of alcohol increases, the conditions for its
growth become more unfavourable ; still, neither wort nor
beer is exposed to the danger of being attacked to any

have developed ; after 9 hours (6'") these have formed transverse septa
and the first indications of branchings ; 11 — 14, abnormal forms ; 15, 16,
hyphse with interstitial cells, filled with plasma ; 17, chain of germinating
conidia ; 18, conidia which have lain for some time in a sugar-solution ;
the contents show globules of oil ; 19, old conidia.

106 MICRO-ORGANISMS AND FERMENTATION.

extent by OicZmm, since it is not able to compete in the
struggle with the concourse of organisms which at once
appear when fermentable liquids are exposed to the germs
of the air.

In numerous investigations with top-fermentation yeast,
I found that this offers a very favourable nutritive material
for this fungus, especially when the yeast is in a quiescent
state at the end of the fermentation. Sometimes a micro-
scopic examination showed an enormous number of conidia.
It is not known what influence such a growth exercises on
the quality of the yeast and the beer ; without doubt it is
advisable to avoid the fungus as much as possible.

8. C. G. Matthews observed that the red colour which
appears on grains of malt, and more particularly when the
quality is not very good, is produced by a Fusarium (probably
grarfiinearum). He cultivated this mould on various substrata.
The fascicular spores are spindle-shaped, curved, and uni- or
multi-cellular ; they are colourless, or only very slightly tinted,
but were embedded in the preparations in a strongly-coloured
mass. The formation of mould commences at the germinal
end of the corn, and spreads from thence more or less over
the surface. When such corns germinate at all, they show an
abnormal development, since they either send out only single
rootlets with a sickly appearance, or the plumule only.
Whilst the spores of Penicillium^ Mucor, Aspergillus, etc.,
are easily distributed over the malt heaps by the air, the
grains attacked by the Fusarium can, according to Matthews,
only communicate the mould to the neighbouring grains,
probably because the spores of this mould have a greater weight,
and more closely adhere to the original mould-growth than
do the spores of the other organisms.

9. Chalara Mycoderma (Fig. 24) is described in Pasteur's
" Etudes sur la biere " as one of the habitants on the surface
of grapes. The mycelium forms a film on liquids, and con-
sists of branched, greyish filaments, often filled with highly
refractive plasma, and which develop at different points conidia

THE MOULD-FUNGI.

107

of unequal form and size. Cienkowski, in his memoir on the
fungi occurring in films, first gave a detailed description of
Chalara. Hansen found that this mould-fungus develops
in ordinary wort and lager beer.

10. A mould-fungus about which a great deal has been
written in the literature of our subject, but whose practical

FIG. 24.

Chalara Mycoderma, after Hansen : 1, a branched hypha, the terminal limb
of which is throwing off conidia ; 2, a hypha, at the upper cell of which
a sterigma, which has thrown off conidia ; 3 — 9. various forms of links
of hyphse, which are separating conidia.

importance certainly stands in inverse ratio to the attention
bestowed on it, is Dematium pullulans (Fig. 25), which was
first described by de Bary, and later more minutely by Loew.
It frequently occurs on fruits, especially grapes, and has a

108

MICRO-ORGANISMS AND FERMENTATION.

branched mycelium, from which buds are thrown out ; these
have a striking resemblance to ordinary yeast-cells (4), and
are able either to propagate through many generations by

FIG. 25.

Dematium pullulans, after Loew : 1, 2, full-grown mycelial threads with
yeast-like cells ; 3, cells of the latter kind developing to mycelial
threads ; 4, cells with yeast-like buds ; 5, appearance of yeast-like cells
on the germ tubes of the brown- walled cells.

yeast-like budding, or to produce germinating threads, which
give rise to a mycelium (3). When this has attained a
certain age, it forms numerous closely-situated transverse

THE MOULD-FUNGI. 109

septa, and gradually becomes brownish or olive-green (5) ; in
this we have the resting stage of the plant. In Hanserfs
air-analyses Dematium was very frequently found, from
spring until late autumn, in wort to which the air had access ;
he observed that when the mould was sown in a saccharine
liquid, it at first only developed mycelial threads ; after some
time, however, the yeast-like cells were separated, without
inducing alcoholic fermentation. Pasteur has very fully
treated of this organism in his " Etudes sur la biere." Since
it occurs so abundantly on the surface of grapes, where the
wine-yeast is developed, and since this often has exactly the
same appearance as the yeast-like cells thrown off by Dema-
tium, it might be imagined that the conidia of the latter
were identical with the wine-yeast cells (Saccharomyces).
In different parts of the above-named work Pasteur expresses
himself differently on this point ; in certain relations he only
puts forward this connection as a supposition, whilst in other
places he regards it as a matter of fact. Here again we
have an example of the attempts previously mentioned to
connect the yeast-fungi (Saccharomycetes) with the mould-
fungi. According to the present methods of research, the
question no longer admits of doubt. The true wine-yeasts
can, under certain conditions, which have now been thoroughly
investigated, produce spores in their interior ; under the same
conditions the conidia of Dematium develop no spores,
and are thus distinguished from the wine-yeast.

1 1 . Finally, we have to mention a mould which may occur,
for example, in fermentable liquids and in fermenting rooms, —
Cladosporium herbarum. This organism sometimes occurs
in very large quantities in fermenting rooms ; some years
ago I found, in a bottom-fermentation room, the ceiling and
a portion of the walls thickly covered with small black
patches ; these consisted of this mould, whose conidia I
consequently always found in the yeast. The plant consists
of a yellowish-brown mycelium, with short, straight, stiff,
and brittle filaments, of which those growing erect can produce

110 MICRO-ORGANISMS AND FERMENTATION.

at their upper extremities conidia of very varying forms —
spherical, oval, cylindrical, straight, or curved. The systematic
position of the mould and its possible genetic connection
with other known fungi is just as little established as its
influence on nutritive liquids. Eriksson states that rye is
sometimes attacked by Cladosporium, and that the mould,
consumed in bread made from rye, or in beer, may give rise to
diseases in the human being.

Concerning these, or at least closely-related forms, Zopf
described exact morphological investigations with numerous
illustrations in his memoir on Fumago, and also in his work on
the fungi. These last-mentioned black, dew-like fungi occur
very frequently on parts of plants. Frank correctly says : —
" We are still quite in the dark with regard to specific
differences, the reason of which is especially to be found in
the frequent polymorphism of these organisms, and in the
fact that the different evolution-forms are scarcely ever
found together."

CHAPTER V.

Alcoholic Ferments.

INTRODUCTION.

IT -does not lie within the scope of a work of this descrip-
tion to give a detailed summary of the knowledge of bygone
times, and it will suffice to pass in review as much only as is
necessary for the proper understanding of the present position
of the subject under discussion. As the investigations of the
last decade originated essentially from questions connected
more or less directly with practice, the results obtained are
also fully entitled to a practical application. It is evident,
however, that this can only be brought about when the
essential results of these scientific investigations are
thoroughly appreciated ; and it is with the object of facili-
tating this that the following resume is given.

The term alcoholic ferment, as commonly used, is very
comprehensive. Mould-fungi, as well as bacteria and budding-
fungi, are able to induce alcoholic fermentation ; but here we
have only to deal with the last-mentioned. Amongst these
budding-fungi are some which also develop mycelium, whilst
with others this form of growth does not as a rule occur ;
among these latter yet another group is included under the
name Saccharomycetes, on account of the property which its
members possess of forming endogenous spores.

In the year 1839 Schwann found that in the case of
certain yeast cells new cells were formed in their interior,
and that these were liberated through the bursting of the
walls of the mother-cells. /. de Seynes (1868) was,

112 MICRO-ORGANISMS AND FERMENTATION.

however, the first who distinctly described the spores in yeast-
cells. Shortly afterwards, in the year 1870, Reess proved
that the formation of spores occurred in several species of
yeast, and stated that the germination of these endogenous
cells took place by budding. As far as the, at that time, very
imperfect methods of experimenting permitted a conclusion
being drawn, it appeared probable that there was a separate
group of such budding-fungi, and to this group Reess gave
the name Saccharomyces.1 The conditions favourable to the
formation of such reproductive organs in the cells were,
however, unknown ; there was no definite method by means
of which their formation could be insured, and experiments
having this for their object were made at random. In the
work already quoted Reess also proposed a system for the
classification of the Saccharomycetes, which he based solely
upon the size and form of the cells. Such a classification
founded upon purely microscopical appearances, has, however,
proved to be useless, and it is impossible to distinguish
between the different species by means of the characters
indicated by Reess. His work has consequently been of no
real practical importance ; and since the essential conditions
for the formation of spores were unknown to him and to his
successor Engel — so that it was purely a matter of chance
whether, in a culture of Saccharomycetes, spore-forming cells
were obtained or not — it is easy to understand the doubt
subsequently expressed as to the existence of spores, and the
disputes which also followed as to whether the yeast used in
practice had or had not lost the property of forming spores.
Finally, Brefeld believed that he had definitely proved that
cultivated yeast was completely deficient in this property.
This confusion was at last dissipated and order established

1 The same author was however less consistent when he admitted
into this group other kinds which did not yield spores, and in this he
was also followed by de Bary in ''Comparative Morphology and
Biology of the Fungi. Mycetozoa and Bacteria," Oxford, 1887. Reess
himself thus at once destroyed the very system, the construction of
which he had just taken in hand.

ALCOHOLIC FERMENTS. 113

when Hansen discovered the conditions regulating the for-
mation of spores, and upon this basis for the first time devised
a method for obtaining them.

Pasteur's " Etudes sur la biere " was published in the
year 1876, and this work advanced in many directions our
knowledge of the phenomena connected with fermentation.
The main portion of this book is devoted to the doctrine,
that every fermentation and every putrefaction is brought
about by micro-organisms, a doctrine which he had defended
with great force in earlier papers. Pasteur's name is with
justice associated with this important doctrine, since it was
mainly through his experiments that its truth has been
confirmed and recognised. The idea, however, can be traced
much further back. Linne and others expressed the belief
that the processes of fermentation and putrefaction, were
caused by living microscopic organisms ; but proof was not
forthcoming until much later. It has already been mentioned
that in the year 1836 Cagniard-Latour proved that the
yeast of beer and wine consists of cells which reproduce
themselves by budding, and that these cells bring about
alcoholic fermentation. Shortly afterwards Schwann arrived
at the same conclusion. In the year 1838 the view was
expressed that different fermentations were caused by different
micro-organisms ; and it was about this time that Turpin
stated that there was " no decomposition of sugar, no fer-
mentation without the physiological activity of vegetation."
I would refer the reader to the above exposition of this
doctrine, which in its historical development is so closely
related to the doctrine of spontaneous generation (see
Sterilisation).

Important discoveries never originate from a single man,
but are really the result of the work of many investigators ;
it is, however, in general much easier to conceive the idea of
some truth than to furnish sufficient proof of its correctness.
Thus, although the doctrine was not new, when in 1857
Pasteur commenced his experiments, some very essential

114 MICRO-ORGANISMS AND FERMENTATION.

connecting links were wanting, as is evident from the fact
that Liebig again gave preference to StahVs experiments in
support of the chemical theory of fermentation. The victory
gained by Pasteur in this dispute constitutes the foundation
of his great fame.

In his "Etudes sur la biere " Pasteur clearly and incontest-
ably proves the significance of the micro-organisms, and he
lays much stress upon the marked influence which bacteria
are capable of exercising upon fermentation and on the
character of the resulting beer. He also treats of the budding-
fungi ; and in the case of some imperfectly described members
of this group, he intimates, as Bail and others had done pre-
viously, that they affect the character of the products of
fermentation in various ways. In this Pasteur is merely
repeating the indistinct views of previous investigators, and
his suggestions take two opposite directions. This is distinctly
seen in his observations on the so-called caseous yeast and the
aerobic yeast. It is possible that in this case he may have
been dealing with distinct kinds of yeast, but it is also
possible that they were merely forms of ordinary brewers'
yeast modified by some treatment to which they had been
subjected. It must not, however, be overlooked that Pasteur
was clear as to his position, and even pointed out the reason
why the question could not be decided, namely, that it was
not then possible to determine ivhether at the starting point
he was dealing with only one or with several species. An
accurate method for the pure cultivation of the different
kinds of yeast had not then been discovered (compare
Chapter I., 7. Preparation of the pure culture). A true
orientation in the world of micro-organisms is consequently
not found in this work, and it is not possible in any
part of Pasteur's statements to find such characteristics for
the budding-fungi on which a scheme of analysis could be
based. Pasteur classed with the Saccharomycetes all the
budding-fungi which showed any marked power of producing
alcoholic fermentation, and it is nowhere clear whether his

ALCOHOLIC FERMENTS. 115

descriptions apply to true Saccharomycetes or to other
budding -fungi. These yeast-fungi, which in our present
system may belong to very different classes, were further
regarded as stages of development of mould-fungi resembling
Demuxtium, but no evidence was given in proof of this view.
Whether or not there are different species of these budding-
fungi (Saccharomycetes, Torula, Dematium), Pasteur leaves
undetermined. His treatment of the botanical problems men-
tioned must on the whole be regarded as having broken down
in the essential points.

The reason above all others why this work was not able to
bring about the reform in brewing indicated in its preface was,
— as will be clear from what has been said above — that from
the position of science at that time, it was not possible to see
clearly into the relations of the different alcoholic ferments
during the process of fermentation. Pasteur was therefore
unable to get beyond the indefinite conjectures and contra-
dictory views of his predecessors. In his review of the micro-
organisms which cause diseases in beer, he speaks only of
bacteria; and the view that these are the only causes of
diseases in beer has since been repeatedly expressed by
Duclaux in 1883, and by other French, English, and
German writers. Pasteur, basing his views on these studies,
recommended brewers to purify their yeast ; and in order to
free it from bacteria, advised its cultivation in a sugar solu-
tion containing tartaric acid, or in wort containing a little
phenol (see below).

In contradistinction to this, Hansen, in the year 1883,
brought forward his doctrine that some of the most dangerous
and most common diseases of loiv-fermentation beer were
caused, not by bacteria, but by certain species of Sac-
charomyces, and that each of the names employed by
Reess, namely, Saccharomyces cerevisice, Sacch. Pastorianus,
Sacch. ellipsoideus, represented not one but several different
kinds or races. He showed that varieties which until
then had been incorrectly grouped under the one name

116 MICRO-ORGANISMS AND FERMENTATION.

Saccharomyces cerevisice gave in the brewery products
having different characters. Starting from this, Hansen
elaborated his method, by means of which a pitching-yeast,
consisting of only one species, is employed. After some
resistance this system has been recognised and introduced
into practice in all countries where the brewing industry
is carried on. Velten of Marseilles, who formerly worked
with Pasteur, has, however, recently attacked this system,
the mistake of which he deems to be that Hansen' s
yeast consists only of one species. He considers it an advan-
tage in Pasteur's purified yeast that the latter consists of
several different kinds, and regards this combination of various
species as necessary in order that the beer may acquire the
desired taste and bouquet. Hansen's latest investigations
(see Chapter I., 7) show how completely this doctrine breaks
down. Hansen proved by experiment that when yeast is
treated with tartaric acid, according to Pasteur's method, the
conditions are so favourable for the development of the
yeasts which produce disease, that finally the culture-yeast
becomes completely suppressed. Pasteur consequently greeted
Hansen's method as an advance, in that he wrote, " Hansen
was tfye first to perceive that beer-yeast should be pure, and
not only as regards microbes and disease-ferments in the
narrower sense, but that it should also be free from the cells
of wild yeasts."

As, however, Pasteur's work always retains its technical
importance, on account of the force with which the influence
of bacteria in the fermentation industries is asserted, so it also
possesses great theoretical interest, especially from the new
theory of fermentation enunciated therein, and which at the
time rightly attracted much attention.

Contrary to Brefeld, who asserted that yeast could not
multiply without free oxygen, and Traube, who indeed granted
that yeast was able to develop without free oxygen, but main-
tained that it then required for its cell-formation the soluble
albuminoids in the liquid, Pasteur stated that the organisms

ALCOHOLIC FERMENTS. 117

of fermentation constitute a group of living beings, whose
function as ferments is directly " a necessary consequence of
life without air, of life without free oxygen "; and further, that
such a fermentation can also take place in a pure sugar solu-
tion. He maintains that the reason why Brefeld could not
get yeast to develop in a moist chamber in an atmosphere of
carbonic acid, was because he was working with old yeast-cells,
whilst it is only possible for yeast to multiply in the absence
of free oxygen when the cells are very young. The minute
quantity of free oxygen which is present in the liquid to
which the yeast is added " rejuvenates the cells and makes
it possible for them to again resume the power to bud, to
preserve life, and to carry on their multiplication without
access of air."

Hence Pasteur makes a distinction between two classes
of organisms : aerobic, those which cannot live without the
presence of free air ; and anaerobic, those which can exist in
the absence of air. According to his view, these latter
constitute " ferments in the true sense of the word."

It would be incorrect to assume that the presence of
alcohol and carbonic acid amongst the products of a fermen-
tation unconditionally presupposes the influence of "organisms
of alcoholic fermentation in the true sense of the term."
The researches of Lechartier and Bellamy, which were
subsequently extended by Pasteur, showed, namely, that
when grapes, oranges, and other fruits on which no yeast^cells
were present, were preserved in vessels filled with carbonic
acid, a development of alcohol and carbonic acid took place.
" The fermentative character is consequently not a condition
of the existence of yeast ; the fermentative power is not
peculiar to cells of a special nature, is no fixed structural
characteristic, but is a property which is dependent upon
external conditions and upon the mode of nutrition of the
organism."

" In short, fermentation is a very general phenomenon.
It is life without air, life without free oxygen; or more

118 MICRO-ORGANISMS AND FERMENTATION.

generally still, it is the necessary result of chemical work
carried out on a fermentable substance, which by its decom-
position is capable of evolving heat ; the heat necessary to
effect this work being borrowed from a part of that which is
liberated by the decomposition of the fermentable substance.
The class of fermentations properly so-called is limited by
the small number of substances which are capable of evolving
heat on decomposition, and which will serve as nourishment
for the lower organisms when the admission of air is excluded "
" (fitudes sur la biere," page 261). This is briefly Pasteur's
famous theory of fermentation.

Fermentations dependent upon oxidation, — such as the
acetic acid fermentation, which, as Pasteur himself had
observed, requires an abundant supply of air, — were con-
sequently not regarded by him as true fermentations. It is
seen, moreover, that he does not strictly adhere to his
definition, in that he emphasises the fact that yeast also pos-
sesses fermentative properties when air is present, although
to a less degree than when oxygen is excluded. The correct-
ness of this under certain conditions has been confirmed in
the case of bottom yeast by Pedersen (1878), and by Hansen
(1879), who came to the conclusion that the amount of
substance in a wort which a definite quantity of yeast can
convert into alcohol and carbonic acid is smaller when the
liquid is aerated during fermentation than when no aeration
takes place. Ed. Buchner (1885) obtained a similar result in
his experiments with bacteria.

Hansen arranged his experiments in such a way that a
rotatory motion was imparted to the liquid which was
being aerated, and the cells thus brought into continual
contact with the vigorous current of air which was blown
through the fluid. Nevertheless, there was a distinct alcoholic
fermentation, and it certainly follows that this was not
induced by life without air.

In NagelVs " Theorie der Gariing " (1879) it is shown
that the admission of oxygen is highly favourable to alcoholic

ALCOHOLIC FERMENTS. 119

fermentation in a sugar solution when no other nourishment
is present, and consequently the yeast does not multiply, or
does so only to a small extent. Ndgeli therefore states
(p. 26) that " Pasteur's theory, that fermentation is induced
through want of oxygen, in that the yeast cells are forced
to take the necessary supply of oxygen from the fermentable
substance, is refuted by all the facts which bear upon this
question."

A. J. Brown, who also holds this view, made a series of
experiments in which fermentations were conducted in pre-
sence of an abundant supply of oxygen, whilst in a duplicate
set - of experiments conducted simultaneously, oxygen was
excluded ; the same number of non-multiplying yeast cells
were present in both cases, and all the other conditions
were kept constant. These experiments showed — contrary
to Pasteur's theory — that the yeast cells exercised a greater
fermentative power in the presence of oxygen than when the
latter was excluded.

Recently Hiieppe and his pupils have also opposed
Pasteur's theory, and have brought forward examples of
fermentation organisms "which can induce the specific
fermentations mostly even more readily when atmospheric
oxygen is present."

Of Nageli's manifold work on the lower organisms, we
will only mention, as connected with the foregoing, the
" molecular-physical " theory of fermentation put forward by
him, and which is essentially a modification of Liebig's
theory. Whilst Pasteur explains fermentation as the result
of activity occurring within the cell, Ndgeli defines fermen-
tation as a transference of the vibrations of the molecules,
groups of atoms and atoms of different compounds (which
themselves suffer no change), contained in the living plasma
to the fermentable substance, whereby the equilibrium of its
molecules becomes disturbed and their decomposition brought
about. In the process of fermentation, the vibrations of the
plasma molecules are thus transferred to the fermentable

120 MICRO-ORGANISMS AND FERMENTATION.

substance. The cause of fermentation is present in the living
plasma, and therefore in the interior of the cells; but it
operates at a moderate distance outside the cell. The
decomposition of sugar into alcohol and carbonic acid takes
place to a small extent within, but mainly outside the yeast
cells. This theory is thus distinctly opposed to that of
Pasteur, and follows on the lines of the theories propounded
by Stahl and Liebig.1

Rayman and Kruis added to our knowledge of the
biology of yeast-fungi by their experiments on beers which,
during a period of several years, had undergone fermentation
with absolutely pure cultures, prepared by Hansen's method.
These investigators found that the fermentation product
obtained by means of pure cultures of Saccharomycetes—
the normal conditions of temperature, etc., obtaining in the
brewery being maintained — is a single alcohol, namely, ethyl-
alcohol. This alcohol remains together with the living yeast
for years in the beer when the latter is preserved at a low
temperature and air is excluded ; when, on the other hand, a
yeast film is allowed to form on the surface through the admis-
sion of air, a vigorous oxidation sets in, and the alcohol becomes
converted into carbonic acid and ivater. In prolonged
fermentations the Saccharomycetes hydrolyse to a variable
extent the albuminoids present in the nutrient fluid, and
they can also oxidise the products to formic and valerianic
acids. The same authors distinguish two reactions in normal
fermentations, namely, a sugar-hydrolysing reaction taking
place in the nutrient medium, and a synthetic (albuminoid)

1 In Bref eld's numerous mycological treatises the budding-fungi
occupy a somewhat prominent position ; thus this naturalist showed
that, many Uttiloffinea, Basidiomycetes, and other fungi can assume a
budding-fungus stage. This had also been previously shown by Bail,
Reess, Zupf, and others ; and since Brefeld did not prove whether these
forms exhibit the property of forming endogenous spores, which is
characteristic of the Saccharomycetes, nor whether they possess any
marked fermentative activity, his indefinite statements that they are
identical with the Saccharomycetes lost all weight.

ALCOHOLIC FERMENTS. 121

reaction taking place in the interior of the organism. They
regard fermentation as an alternate hydration and dehy-
dration.

In all these different theories of fermentation, the main
point of all questions relating to the subject is not touched
upon : — How comes it that, in these microscopic cells, the
plasma, which has the same appearance in the different
species, yet in one cell induces an acetic acid fermentation,
in another butyric acid fermentation ; in a third it induces a
direct fermentation of cane-sugar, whilst in a fourth the
cane-sugar becomes first hydrolysed and then fermented ?
The cause of these different kinds of activity of plasma
is still an unsolved problem.

The theories of fermentation hitherto put forward fail to
give any comprehensive explanation of known facts, and con-
sequently they have here only an historical interest.

From the above resume it will be seen that, at the time
when Hansen commenced his investigations, our knowledge
of the alcoholic ferments was very deficient and untrustworthy.
Consequently the problem had to be attacked experimentally
from the very foundations. Hansen has done this in the
work which he has now carried on for many years.

The previous investigators had certainly gone as far as
was possible along the paths which they had marked out.
When we compare their investigations — especially those of
Pasteur and Reess — with those of Hansen, we find that the
latter attacked the problem from new points of view and
with new methods. He extended his investigations on this
subject far and wide in all directions. His researches have
not only opened up new paths from the scientific standpoint,
but they have also brought about a reform in the fermentation
industry. For these reasons it is but right that they should
form the ground-work of the following section of my book.

122 MICRO-ORGANISMS AND FERMENTATION.

HANSEN'S INVESTIGATIONS.

When Hansen published, in the year 1878, his treatise on
" Micro-Organisms in Beer and Wort," he pointed out the
uncertainty which prevailed in the works of earlier writers,
concerning the true Saccharomycetes ; and he emphasised the
fact that it was not possible to proceed further along the path
which they had pursued, but- that the investigations, and
especially those commenced by Pasteur and Reess, must, if
they were to be carried further, be attacked from a totally
different point of view. It was only in the latter end of the
year 1881 that he succeeded in finding the key to the solution
of the problem. The problem was, in the first place, to
devise a method by means of which one could obtain growths,
each of which was derived from a single cell, in order to
determine by experiment whether these guaranteed pure
cultures exhibited constant characters — that is to say, how far
the Saccharomycetes occur as species, varieties, or races — and,
should this prove to be the case, to determine what these
characters are. When this problem was solved, the next was
to devise a method for the analysis of yeast and to study in
different directions the conditions of life of these organisms.

1. PREPARATION OF THE PURE CULTURE.

In the first chapter of this book it was pointed out
that the idea had been expressed on various sides, that
the only condition for an exact knowledge of the micro-
organisms, hundreds or thousands of which we find in every
drop when examined under the microscope, consists in the
isolation of a single cell, and in working with a pure growth
obtained from this cell. The different methods which had
been employed were also briefly described.

Hansen has repeatedly pointed out in his papers that the
only method ivhich is certain in all cases is to start from
the individual cell and to secure the beginning from this.
He has devised two different methods for this purpose. In

ALCOHOLIC FERMENTS. 123

his first method a liquid medium was employed, and in his
second method a solid medium, for the cultivation ; in both
cases the culture was previously diluted as already described
(Chapter I., 7. Preparation of the pure culture).

With the help of the acquired knowledge of the species
it was possible to submit these methods to a searching
examination, with the result that they proved to be
reliable.

If it is desired to isolate from a mixed growth of different
species those which are in an enfeebled condition it is neces-
sary, as Hansen points out, to employ the dilution method,
using a suitable nutrient fluid, as, for example, wort, the
conditions being then favourable for the growth of the
organisms in question.

If, on the other hand, we wish to separate from a mixed
growth a species which is in a vigorous state of development,
and whose further growth is consequently not dependent upon
specially favourable conditions of nutriment, we can attain
our object more readily and in a shorter time by the employ-
ment of a solid nutrient medium — in this case gelatine and
wort. It has been proved that the addition of gelatine to
wort diminishes its value as a nutritive material for the yeast-
fungi. A series of experiments carried out by Holm show in
fact that, if at the commencement of a fermentation when
the yeast-cells are in their most vigorous state of develop-
ment, some of these cells be introduced into wort-gelatine,
about 4 per cent, of those sown do not develop ; if, on the
other hand, the yeast-cells are taken at the conclusion of a
fermentation, when they are enfeebled, about 25 per cent, of
them give no colonies in wort-gelatine.

The advantage of this method, as employed by Hansen,
for the study of the budding-fungi is that it makes it possible
to directly observe the individual cells under the microscope
and to follow their further development, since the gelatine
plate is enclosed in a moist chamber (compare page 26,
Dilution methods).

124 MICRO-ORGANISMS AND FERMENTATION.

2. THE ANALYSIS.

Throughout the entire series of Hansen's researches a
leading idea obtains, namely, that the shape, the relative size,
and the appearance of the cells, taken by themselves, are not
sufficient to characterise a species, since the same species,
when exposed to different external conditions, can occur in
very different forms and quite different in appearance. On
the other hand, the forms of development of the cells,
regarded from another point of view, constitute very impor-
tant distinctive characters for different species. Thus it is
found that different species under the same treatment behave
differently and assume different forms. This can only be
explained by assuming that there are intrinsic, indwelling
characters in the special cells which exert an influence of
their own.

In the following we give a brief account of the various
means by which Hansen determined the characteristics of
different species. These investigations form at the same time
contributions to the general physiology of the budding-fungi.

(a) The Microscopic Appearance of the Sedimentary
Yeast. — The first examination of a yeast will generally consist
in observing under the microscope the appearance of the sedi-
ment. As examples illustrating what can be ascertained in
this way, we may call attention to the following figures (Figs.
34, 37, 39, 41, 43, 45), representing the young sedimentary
forms of the six species of Saccharomycetes which have been
specially investigated by Hansen. The growths were obtained
by cultivating the cells for some time in wort, then intro-
ducing fresh wort, and by maintaining a temperature of 25°
to 27° C. for 24 hours a vigorous growth was developed. If
we now compare, for instance, the figures representing Sac-
char omyces cerevisice I., with those which illustrate the three
Pastorianus species, we find that, taken as a whole, they show
marked differences. Saccharomyces cerevisice consists mainly
of large round or oval cells, the Pastorianus species form

ALCOHOLIC FERMENTS. 125

mostly elongated sausage-shaped cells. It is, however, a
very different matter when cells of the first species are
mixed with cells of one of the other species ; it is not
then possible, judging from the form alone, to distinguish
the larger and smaller oval and round cells of the Pastori-
anus species from many of the cells of Sacch. cerevisice.
The two species Sacch. ellipsoideus I. and //. consist mainly
of oval and round cells ; sausage-shaped cells, however, also
occur ; and consequently it is in this case likewise impossible
to determine the species by the form of the cells when
these are mixed with Sacch. cerevisice or Sacch. Pastorianus.
. Neither can any conclusions be arrived at by direct
measurements of these sedimentary forms.

A glance at these six groups of figures of pure cultures
shows that we have here three different classes of budding-
fungi, one of which is represented by Sacch. cerevisice,
whilst the second includes the three Pastorianus species,
and the third the two ellipsoid species. This much, but
only this much, is possible from a purely microscopical
examination, and, it must be pointed out, only under the
conditions of cultivation indicated.

(b) Formation of Ascospores. — By Hansels investiga-
tions on the formation of endogenous spores in the Saccharo-
mycetes the first essential link of an analytical method for
the examination of yeast was found. We will give a brief
account of the experimental method adopted and of the
general results obtained.

The formation of spores in yeast-cells has been investi-
gated by various naturalists ; of the many, and in part
contradictory statements, however, the only result which
remained as correct was the fact that Saccharomyces cells
could, under certain unknoiun conditions, form spores in
their interior.

After making a large number of experiments, Hansen was
able to determine the following conditions regulating the
formation of spores in the Saccharomycetes : —

126 MICRO-ORGANISMS AND FERMENTATION.

1 . The cells must be placed on a moist surface and have

a plentiful supply of air.

2. Only young, vigorous cells can exercise this function.

3. The most favourable temperature for most of the

species as yet examined is about 25° C. This
temperature favours spore-formation in all known
species.

4. A few Saccharomycetes likewise form spores when they

are present in fermenting nutrient fluids.
A growth of yeast is developed in the manner described
on page 124. A small quantity is transferred to a previously
sterilised gypsum block ; this block is enclosed in a flat

FIG. 26.

The first stages of development of the spores of Sacch. cerevisiae I., after
Hansen : a, b, c, d, e, rudiments of spores, where the walls are not yet
distinct ; /, g, h, i, j, completely-developed spores with distinct walls.

covered glass and is maintained moist by half filling the
glass with water.1 If it be desired merely to bring about
the formation of spores, the apparatus may be allowed to
remain at the ordinary room-temperature.

Hansen was the first to give an accurate description of the
structure of spores and a detailed account of their evolution,
founded upon observations of individual spores; and he
distinguished three typically different groups of Saccharo-

1 Ascospores can also be obtained when yeast is spread upon sterilised
solidified gelatine, prepared with or without a nutrient solution, kept in
a damp place ; likewise in yeast- water and in sterilised water ; finally,
spore-forming cells also occur in the films of the Saccharomycetes. The
method is evidently not dependent upon these different substrata but upon
the knowledge of the factors which render it possible for the cells to
exercise this function of forming spores.

ALCOHOLIC FERMENTS. 127

mycetes which are distinguished by the mode of germination
or by the form of the spores.

After a certain lapse of time, which varies with the different
species, roundish plasma-particles appear in the cells, and these
are the first indications of spores (Fig. 26). In their further
development, they become surrounded by a wall, which is seen
more or less distinctly in the different species.

In the first type, to which Saccharomyces cerevisice I.
belongs, the spores can expand during the first stages of
germination to such an extent that the pressure which they
exert on each other whilst they are still enclosed in the
mother cell, brings about the formation of the so-called
partition walls (Fig. 27). This causes more or less plasma

FIG. 27.

Spores of Sacch. cerevisise I. in the first stages of germination, after Hansen :
at a, d, e, and g, formation of partition-walls ; at e, f, and g, the walls
of the mother cells have become ruptured ; at g a compound spore
divided into several chambers, the coherent wall is ruptured in three
places.

to become squeezed or wedged between the spores, or the walls
of the spores may be brought into contact. During the further
development, a complete union of the walls may take place, so
that a true partition wall results ; the cell then becomes a
compound spore divided into several chambers.

During germination (Fig. 28) the spores swell and the
wall of the mother-cell, which was originally moderately thick
and elastic, becomes stretched and consequently thinner.
Finally it becomes ruptured, and then remains as a loose or
shrivelled skin, partially covering the spores ; or it becomes
gradually dissolved during germination.

128

MICRO-ORGANISMS AND FERMENTATION.

Budding can occur at any point on the surface of the
swollen spores ; this budding usually takes place after the
wall of the mother-cell has become ruptured or dissolved, but
it also occasionally takes place within the mother-cell. After
the buds have formed, the spores can still remain connected,
or they can soon become detached from each other.

An especially curious and exceptional case is when the
wall separating two spores becomes dissolved, so that a fusion

FIG. 28.

Budding of the spores in Saccharomyces cerevisise I., after Han sen : a, three
spores without the wall of mother-cell ; &, cell with four spores ; at b'
the wall of mother-cell is ruptured ; c, cell with four spores, three of
which are visible ; at c' and c" the ruptured wall of mother-cell is seen ;
d, cell with three spores, at d'" the ruptured wall of mother-cell ;
e — e'"" development of a very strong colony ; / — Ji, other forms of
development ; at h" the wall between the two spores has disappeared.

of the spores results (see Fig. 28, e — e'"" and h — h").
Hansen assumes that the biological significance of this
phenomenon is that the spores, placed under unfavourable
conditions, have a greater chance of forming buds. One
spore plays the part of a parasite to the other. The growing

ALCOHOLIC FERMENTS.

129

together of the spores mentioned above is perhaps the begin-
ning of this process.

The germination of the spores of such species of the
groups Saccharomyces Pastorianus and Sacch. ellipsoideus
as have been examined takes place in essentially the same
way as above described.

A second and quite different type occurs in the case of

a

FIG. 29.
Germination of the spores of Saccharomyces Ludwigii, after Hansen :

represent a gypsum- block culture 12 days old ; d — h, a similar culture,
one-and-a-half months old.

Sacch. Ludwigii (Fig. 29), where the fusion takes place in
the very first stages of germination ; in this case, however, it
is the neiu formations and not the spores which grow
together. These new formations are further distinguished

130

MICRO-ORGANISMS AND FERMENTATION.

from the previous type in that they are not yeast cells, but
mycelium-like growths, — promycelium. The development
of yeast cells takes place from this promycelium, a sharp

FIG. 30.

Saccharomyces Ludwigii, after Hansen. Germinating spores from old gypsum
block cultures. At a and b each spore has developed a germ-filament ; at
c are shown different forms produced by fusion.

partition wall being first formed ; the cell then becomes
detached, and finally its ends become rounded. At the ends
of these cells buds are developed, and these also become
detached at the partition walls.

FIG. 31.
Germination of spores of Saccharomyces anomalus, after Hansen.

In the case of older spores this curious fusion is more
uncommon (Fig. 30). Some germ-filaments develop into a
branched mycelium (group 6).

The third type, which occurs in Saccharomyces anomalus

ALCOHOLIC FERMENTS. 131

(Fig. 31, see also description of the species), is distinguished
from the last-mentioned in that the spores are of a quite
different shape, and resemble the spores of Endomyces
decipiens.1 They somewhat resemble a half sphere with a
rim round the base»

During germination the spore swells and the projecting
rim can either remain or disappear. Buds then make their
appearance at different points on the surface of the spore.

One of the objects of Hansen's investigations was also to
determine in what way the formation of spores was influenced
by different temperatures, with the view to ascertain whether
the different species behave alike, or whether it might not be
possible in this way to discover different characteristics. It
was, therefore, necessary to determine : 1, the limits of
temperature, i.e., the highest and lowest temperatures at
which spores could be formed ; 2, the most favourable tem-
perature, i.e., the temperature at which spores appeared in
the shortest time ; and, finally, 3, the relation of the inter-
mediate temperatures.

In determining the desired intervals of time, the moment
was registered at which the cells showed distinct indications
of the formation of spores (compare Figs. 26 and 32). It is
not possible to make use of ripe spores in these determina-
tions, since no criterion exists for complete ripeness.

The results obtained by Hansen are as follows : —

The formation of spores takes place slowly at low tem-
peratures, more rapidly as the temperature is raised to a
certain point ; when this point is passed their development
is again retarded, until finally a temperature is reached at
which it ceases altogether.

The lowest limit of temperature for the six species first
investigated was found to be O5 to 3° C., and the highest
limit 37-5° C. Hansen also determined the intermediate
temperature and time relations for these six species, and

1 A fungus which is parasitic on the lamellae of certain mushrooms.

132

MICRO-ORGANISMS AND FERMENTATION.

Saccharomycetes with

2, Sacch. Pastorianus I.
5, Sacch. ellipsoideus I. ;

FIG. 32.
ascospores, after Hansen

; 3, Sacch. Past. II.
6, Sacch. ellips. II. ;

1, Sacch. cerevisise I. ;
; 4, Sacch. Past III. ;
a, cells with partition-

wall formation ; b, cells containing a larger number of spores than usual
c, cells showing distinct rudiments of spores.

ALCOHOLIC FERMENTS. 133

found that when these two values are graphically represented
with the degrees of temperature as abscissae and the time
intervals as ordinates, the curves obtained for all six species
had essentially the same form. They sink from the ordinates
of the lowest temperatures towards the axes of the abscissae,
and then rise from these ; at the same time, however, it is
seen from these curves that the cardinal points determined
/more especially from the highest and lowest temperatures,
give characteristic distinctions for the different species ; that
is to say, that the limits of temperature within which the
formation of spores can take place are different for the
various species (compare classification of the genus Sac-
charomyces).

With regard to the time required for the appearance of
the first indications of spore-formation in the six species
investigated under the same conditions of temperature, the
following was observed : At the highest temperature the time
required for the development was in all the species about 30
hours ; at 25° there was also no great difference in the time
required ; at the lower temperatures., however, very evident
differences occurred. Thus, in the case of Sacch. cerevisice /.,
the first indications of spore-formation at 11*5° C. are only
found after ten days ; in the case of Sacch. Pastorianus II.
after 77 hours, and so on.

In all determinations of this kind a very considerable
influence is exerted by the condition of the cells, according
to whether they have been grown at a high or low tempera-
ture, whether they were old or young, feeble or vigorous,
etc., etc. It follows from this that the composition of the
nutrient fluid also exercises an influence. In methodical,
comparative experiments of this nature, a necessary condition
is, therefore, that the previous cultivation of the cells should
always be carried out in the same manner. If these external
conditions be varied, the limits for the reactions of the species
corresponding to such varied conditions must likewise be
determined.

134 MICRO-ORGANISMS AND FERMENTATION.

By means of these experiments Hansen has adduced an
important character for the determination of the Saccha.ro-
mycetes.

A new distinctive characteristic for the species has been
discovered by the same author in the different anatomical
structure of ike spores. Both these characters and others
which are described in the following pages (e.g., film formation,
etc.,) must necessarily be considered in a complete examination
of a Saccharomyces species.

The method given below for the analysis of low breivery-
yeast from, a practical standpoint was based by Hansen on
certain observations of the temperature curves for the
development of spores and on the structure of the spores.
Thus it was found that at certain temperatures the species
employed in the brewery, the so-called cultivated yeasts,
develop their spores later than the so-called wild yeasts,
several species of which also occur as disease-germs in the
brewery. Hansen also found that the structure of the spores
in these two groups is generally different, in that the young
spore of a cultivated yeast has a distinct wall or membrane, and
the contents are not homogeneous, are granular, and exhibit
vacuoles ; in the case of wild yeast, on the other hand, the
wall of the young spore is most frequently indistinct and the
contents are homogeneous and strongly refract light. It
should also be added that the spores of cultivated yeasts are
usually larger than those of wild yeasts.

1. For the continual, daily control of brewery-yeast as
regards contamination with wild species, the following very
convenient method is made use of: — At the conclusion of the
primary fermentation, a small quantity of the fermenting
liquid is removed from the fermenting-vessel in a sterilised
flask ; this is set aside for some hours until the yeast has
settled to the bottom, and the sediment is then spread upon
a gypsum block in the manner described on page 126. This is
then introduced into a thermostat maintained at a tempera-
ture of 25° C. or 15° C.

ALCOHOLIC FERMENTS. 135

It was found namely — and it has been subsequently con-
firmed by the elaborate investigations of Holm and Poulsen —
that the species of cultivated yeasts employed in low-ferment-
ation breweries can be divided into two groups. One group
yields spores later than wild yeast when a temperature of
25° C. is maintained ; the other group, on the contrary, gives
spores in about the same time as wild yeast at the above
temperature, but at a temperature of 15° C. the cells of wild
yeast show spore-formation considerably sooner than the cells
of these cultivated yeasts.

The cultures maintained at 25° C. are examined after an
interval of 40 hours, and those maintained at 15° C. after an
interval of three days.

Experiments of my own show that high breiuery-y easts can
be analysed in a similar manner.

By means of experiments which were undertaken with the
view to determine to what extent Hansen's analytical method
can be relied on for technical purposes, Holm and Poulsen
came to the conclusion that a very small admixture of wild
yeast, about \-2QOth of the entire mass (Carlsberg bottom-
yeast No. L), can be detected with certainty in this manner.
Hansen's previous researches had shown that when, for in-
stance, the two species, Sacch. Pastorianus III. and Sacch.
ellipsoideus //., — which are capable of producing yeast-
turbidity in beer — are present to the extent of only 1 part in
41 of the pitching-yeast, the disease is not developed, provided
that the normal conditions of fermentation and storage have
been maintained; further, that Sacch. Pastorianus 7., which
imparts to beer a disagreeable odour and an unpleasant bitter
taste, can, under the same conditions, scarcely exert its
injurious influence when the admixture of this yeast amounts
to less than 1 part in 22 of the pitching-yeast. Consequently
Hansen's method for the analysis of yeast by means of
ascospore formation gives ample information as to the
presence of these disease ferments.

This method likewise possesses the advantage that the

136 MICRO-ORGANISMS AND FERMENTATION.

analysis can be performed with mixtures such as ordinary
pitching-yeast, and that it can be performed in a short
time.

When the object of the analysis is to more accurately
characterise the different species present in the sample, a
number of cells are isolated by fractionation, and each of the
growths obtained is separately examined.

In an investigation on bottom-yeast during the different
stages of the primary fermentation, published by Hansen in
1883, it was shown that as a rule the wild yeasts are present
in largest amount during the last stages of primary fermen-
tation in the upper layers of the liquid. The samples of the
liquid which are taken from the fermenting vessel for the
analysis of the yeast, must therefore, as stated above, be taken
during the last days of the primary fermentation. If much
time elapses before an analysis is commenced, the yeast must
be introduced into wort, and one or more fermentations carried
out ; and this applies whether the yeast to be examined was
in a dry or a liquid state.1

It is evident, however, that, valuable as the analysis of
yeast is, it must always remain of secondary importance in
the brewery ; the most important link in the system will,

1 The observation mentioned above with reference to low-fermen-
tation yeast has been confirmed by J. Vuylsteke's experiments, in which
fermentations were carried out with mixtures of different Saccharomycetes
in cylindrical glass vessels of about two liters capacity ; by counting
the cells and by means of cultures the relative proportions of the
different species were determined. It is found, however, from the
experiments hitherto conducted by Vuylsteke that the rule mentioned
is not of general application in the case of mixtures of high-fermentation
yeasts with wild yeasts. In some experiments with mixtures of Sacch.
ce?'evisi<e I., Hansen, and Sacch, Pastorianus /., Hansen, the wild yeast
was found to have increased towards the end of the primary fermenta-
tion, whilst in other experiments a diminution of the wild yeast was
observed. On the other hand, all the experiments with mixtures of
Sacch. cerevisiee /., and Sacch. Past. III., showed that the impurity was
greater in the upper layers of the liquid at the end of the primary
fermentation than at the commencement, just as in the case of bottom-
fermentation.

ALCOHOLIC FERMENTS. 137

under all conditions, be the employment of a pure cultivation
of a selected species of yeast.

2. The analysis of the yeast in the propagating appara-
tus, which must be absolutely pure, is carried out as
follows : — At the conclusion of fermentation, samples are
withdrawn, with every precaution, into Pasteur flasks or into
the Hansen flasks employed for sending yeast samples ; from
these, small quantities are introduced into flasks containing
yeast extract, and these are maintained at a temperature of
25° C., the object being to test the yeast for bacteria. The
remainder is set aside for the yeast to settle, the beer is
decanted, and a sample portion of the sediment is introduced
into a sugar solution containing some tartaric acid. After
three or four cultivations in such a solution it is further
cultivated a few times in beer-wort, and then tested for spore-
formation. The smallest traces of wild yeast in the apparatus
are brought into a state of vigorous development by this
treatment (see Chapter I., 7. Physiological methods).

(c) The Formation of Films. — By the observation of the
formation of films, Hansen has found characteristics for the
Saccharomycetes in a manner quite different from that given
above. A new path for the study of these fungi was thus
again opened up, since the statements hitherto made by
different authors in this direction are not in accordance with
their true behaviour.

It is a very generally-known phenomenon, that ferment ed
liquids become coated with films. It is also well-known that
the films formed by the budding fungi — Mycoderma cerevisice,
Mycoderma vini — have especially attracted attention ; and
the frequent mention of such films in the literature of the
subject led to a result well-known also in other branches of
science ; they were spoken of as if well understood for so
long that at last the belief in the actual existence of this
knowledge became firmly rooted. After Hansen had sub-
mitted this question to an experimental investigation, he
showed, however, that this view was erroneous.

138 MICRO-ORGANISMS AND FERMENTATION.

Hansen has treated a large number of films, and amongst
them several forms which are most closely related to different
species of Saccharomyces Mycoderma, which do not produce
endogenous spores. According to de Seynes, Reess, and
Cienkowski, these Mycoderma-species do yield ascospores ; it
is, however, highly probable that these investigators were
dealing with impure films, containing an admixture of true
Saccharomycetes. It is, indeed, a matter of no little diffi-
culty to determine the purity of such a culture if one
does not start from a single cell ; for if Mycoderma
cerevisice is cultivated as sedimentary yeast, the cells
assume an entirely different appearance ; they become filled
to a greater extent with plasma, whilst the cells of the film
are, as is known, poor in plasma and contain strongly-
developed vacuoles. Such forms, whicli are generally re-
garded as Mycoderma cerevisice, readily and quickly form
films ; some simultaneously exhibit distinct signs of fermen-
tation, whilst others do not. On beer and wort these films
are grey and dry in appearance ; afterwards they become
.wrinkled and lighter in colour; air is found freely inter-
mixed between the cells. Some of the varieties of Torula
investigated by Hansen yield similar films ; the film of
Chalara Mycoderma, on the other hand, is glutinous, tough,
and slightly lustrous ; in the case of Monilia — which, as
previously mentioned, can occur with budding cells, and
directly ferments cane-sugar — the film formation is peculiar :
even during vigorous fermentation a film forms on the bub-
bles of foam, spreads gradually over the whole surface, and
sometimes becomes wrinkled. Thus, the cells in the flask first
sink to the bottom as sedimentary yeast, set up a vigorous
fermentation, and again rise with the bubbles of carbonic acid
to the surface, where they enter upon a new phase of develop-
ment. If sterilised lager-beer is infected with this fungus, no
fermentation sets in, and only a thin film resembling dust is
developed ; under other conditions the fungus forms a white,
floury, wool-like layer, as in the case of Oidium.

ALCOHOLIC FERMENTS. 139

The true Saccharomycetes also form films, which, how-
ever, differ somewhat from those mentioned above ; and this
is also the case with some of Pasteur's Torula and with
Saccharomyces apiculatus. From these observations it is
evident that the formation of films is not a peculiarity of cer-
tain species, but must be regarded as a general phenome-
non common to micro-organisms.

In the case of the Saccharomycetes this phenomenon
generally occurs in the following manner: If cultures in
wort are left undisturbed for a shorter or longer time at
the ordinary room temperature, small specks of yeast
gradually appear on the surface of the liquid after the
termination of the primary fermentation ; these can after-
wards coalesce to figures of different forms and sizes, to
isolated patches, the upper surfaces of which are flat and
the under surfaces arched. Finally, they become united to
a coherent and generally light greyish-yellow, glutinous
film, which may extend to the walls of the glass vessel,
forming a complete ring. Such a perfect film-formation
only occurs after the primary fermentation is at an end.
If the flask be shaken, pieces of the film become detached
and sink to the bottom; and in this way a complete layer
can gradually collect at the bottom, whilst the film becomes
continually renewed and assumes a marbled appearance owing
to the younger portions being thin and dark, whilst the older
parts are thick and light.

The conditions under which a film can be formed are a
free, still surface, with direct access of air ; and a vigorous
film-formation presupposes an abundant supply of air. It
follows from this that a far more rapid and vigorous develop-
ment will take place in a Chamberland flask, or in an ordinary
boiling flask with filter-paper tied over the mouth, than in
a Pasteur flask where the admission of air is more limited.
The function of film-formation is thus in this respect
subject to the same conditions as obtain in the case of
endogenous spore-formation.

140 MICRO-ORGANISMS AND FERMENTATION.

Simultaneously with the formation of a film, a decolor-
ation of the wort takes place, the latter becoming of a pale
yellow colour. This reaction takes place most quickly at
the higher temperatures, and occurs most markedly with
those species which give rise to the most vigorous film-
formation.

The preliminary cultivation of the cells is the same as
that previously described (page 124). The liquid is removed
from the growth obtained, and fresh sterilised wort is added ;
the mixture of yeast and wort is agitated, and a drop is
transferred — with the usual precautions — to an ordinary flask
of about 150 c.cm. capacity, half filled with wort, and a
piece of filter-paper is then tied over its mouth. Hansen
exposed flasks treated in this way to different temperatures,
and determined : —

1. The limits of temperature for the formation of films;

2. The approximate length of time required for their
formation at different temperatures ; and

3. The microscopic appearance of the growths at different
temperatures.

The main point in these investigations of the six species
previously mentioned is the microscopic appearance of the
films of these species, formed at the same temperature ; and
here again, when regarded from a different point of view
to that considered in the last section, we have a complete
investigation of the relation between the external interfering
factors and the forms, which proves that we have to do with
so many perfectly distinct types or species.

The examination of the films was made, except when
otherwise stated, when they were so far developed that they
could just be seen with the naked eye.

A glance at the illustrations representing these film-
growths (see page 161 and following pages) will show that
their general character is usually different from that of the
sedimentary forms. For instance, the sedimentary form of
Sacch. cerevisice I. is egg-shaped or spherical, whilst in the

ALCOHOLIC FERMENTS. 141

film, elongated cells quickly appear, and the growth gradually
assumes an appearance perfectly different from that of the
sedimentary yeast.

If we compare the film-formations of the six species, we
find that the films developed at the higher temperatures offer
very few points of difference which are of use in their exami-
nation, Sacch. cerevisice I. and Sacch. ellipsoideus II. being
alone distinguishable from the remainder. It is quite other-
wise, however, when young films developed at a temperature
of 13° to 15° C. are examined. The two species, Sacch.
Pastorianus II. and Sacch. Pastorianus III. — which are
both top-fermentation yeasts, and which in the ordinary cul-
tures cannot be distinguished from each other with certainty
by the form of their cells — exhibit in this case entirely
different forms of growth ; and an equally striking difference
is likewise found between the otherwise similar species Sacch.
ellipsoideus I. and //.

An examination of the limits of temperature for the
formation of films shows that for Sacch. cerevisice I. and
Sacch. ellipsoideus I., these lie within about 38° and 5° to
6° C. ; the limits for the three Pastorianus species are 34°
and 3° C. ; Sacch. ellipsoideus II. has the same lower limit
as the last mentioned species, whilst its maximum tempera-
ture, however, is 38° to 40° C.

The time limits, when compared with those previously
given for ascospore-formation, show that in both cases the
development takes place more slowly at low than at the
higher temperatures ; at temperatures near to the minimum
and maximum limits only a very slight and imperfect film-
formation is ever obtained.

At temperatures above 13° C. the film of Sacch. ellip-
soideus II. develops so rapidly and vigorously that the
flasks with this yeast can be recognised by this alone.
Thus, at 22° to 23° C. the film completely covered the
surface at the end of six to twelve days, whilst in the
case of the other five species a period three times as long

142 MICRO-ORGANISMS AND FERMENTATION.

was required for the formation of films which were generally
more feebly developed. This species and Sacch. Pastori-
anus III. also develop a vigorous film comparatively
rapidly at the ordinary room-temperature, whilst in the
same time the other species are left far behind.

As mentioned above, the film-formations have different
maximum temperatures. This is related to the fact that
the maximum temperature for budding is not the same
for the different species. It was proved that budding and
fermentation can take place at temperatures at which film-
formation no longer occurs. Thus, in the case of Sacch.
cerevisice /., Sacch. ellipsoideus I. and Sacch. ellipsoi-
deus II. Hansen still observed a vigorous fermentation and
budding at 38° to 40° C., and at 34° C. also in the case
of the three species of the group Sacch. Pastorianus. A
relationship is thus shown to exist between the influence
of temperature on budding and fermentation on the one
hand, and film-formation on the other.

(d) The Temperature Limits for the Saccharomycetes. —
Just as the influence of temperature on the development of
spores and films varies with the different species, so it has also
been shown by Hansen's investigations (1883) that both
spores and vegetative cells of different species likewise possess
unequal powers of resistance to hot water. In this respect the
spores have a greater resisting power than the vegetative cells.

In experiments of this nature, as in the cases previously
mentioned, the condition of the cells has a very marked effect
on the results, which are especially influenced according as
old or young cells have been employed. Thus, it was found
that the cells of Sacch. ellipsoideus //., which had been
cultivated in wort for two days at a temperature of 27° C.,
were killed in five minutes when heated to 56° C. in sterilised
distilled water, whilst cells of a similar culture but 2 J months
old were able, under similar conditions, to withstand five
minutes' heating to 60° C. without being killed.

Eipe spores of the same species, which had been developed

ALCOHOLIC FERMENTS. 143

at a temperature of 17° to 18° C., and in the course of eight
days at the same temperature had become partially dried,
withstood a temperature of 62° C. for five minutes, but were
killed at 66° C.

In the case of Sacch. cerevisice I. the vegetative cells are,
under similar conditions, killed by five minutes' heating at
54° C., whilst at 62° C. the spores are killed.

An interesting grouping of Hansen's six species with
reference to a fixed temperature is also found when they are
cultivated in wort under conditions favourable to film-
formation (see page 139). When, for instance, a temperature
of 36° to 38° C. is employed for the development, the three
Pastorianus species will be dead at the end of eleven days,
whilst Sacch. cerevisice I. and the two ellipsoid species will
still be living. From this result it is also evident that the
rule formerly given that top-fermentation yeasts can develop
at higher temperatures than bottom yeasts is incorrect.

Later experiments made by Kayser in some of the
directions mentioned above confirm these results, and they
also show that the yeasts can resist a considerably higher
temperature when in a dry state than in the presence of
moisture. For instance, a pale ale yeast was killed when
exposed for five minutes in a moist condition to a temperature
of 60° to 65° C., whilst when dry it withstood a temperature
of 95° to 105° C. ; in the case of a wine yeast (St. Emilion)
the temperatures were 55° to 60° C. and 105° to 110° C.
The resisting power of the spores was 10° to 20° higher.

Vegetative cells which had developed from the heated
spores exhibited a somewhat greater power of resistance than
normal vegetative cells. This increased resistive power was,
however, not transmitted further, and, on cultivation in beer-
wort, disappeared even in the second generation.

(e) Cultivation on a Solid Nutritive Medium. — Hansen
discovered distinct characteristics for several species of the
Saccharomycetes by suitable cultivation on a solid nutritive
medium. For this purpose he employed small flasks contain-

144 MICRO-ORGANISMS AND FERMENTATION.

ing wort, to which about 5*5 per cent, of gelatine had been
added, the flasks being closed by means of cotton-wool plugs.
When these flasks are inoculated with the six known species
(Sacch. cerevisice /., Sacch. Pastorianus /., //., ///., Sacch.
ellipsoideus /., //.), and set aside at a temperature of 25° C.,
the growths which develop show in the course of eleven to four-
teen days such macroscopic differences that four groups may be
distinguished more or less sharply. Sacch. ellipsoideus I.
stands alone, in that its growth exhibits on the surface a
characteristic net-like structure, which enables this species to
be distinguished by the unaided eye from the other five
species. When gelatine with yeast-water is employed for
such cultures and the experiment conducted at 15° C., Sacch.
Pastorianus //., after sixteen days gives growths, the edges
of which are comparatively smooth, whilst the growths
obtained from Sacch. Pastorianus III. are distinctly hairy
at the edges. A microscopical examination shows that in
this case the two species are also distinguishable morpho-
logically. This is not, however, by any means always the
case with cultures in solid media ; in fact, the differences are
often less marked under such conditions than when nutritive
liquids are employed.

For the Mycoderma species and Sacch. membrancefaciens
Hansen has discovered an important characteristic in their
behaviour in wort-gelatine, in which they form shield-like
colonies readily distinguishable from those of the Saccharo-
mycetes.

In connection with this we may also mention Hansen's
observation that some species, e.g., Sacch. Marxianus and
Sacch. Ludwigii, can develop a mycelium when grown in a
solid medium, whilst others are not able to do so.

In the case of some cultivated yeasts P. Lindner found
distinct differences in their growths on gelatine.

Will and others have also shown that the characteristics
exhibited by cultures on nutritive gelatine are often very
variable.

ALCOHOLIC FERMENTS. 145

(/) The Behaviour of the Saccharomycetes and Similar
Fungi towards the Carbohydrates and other Constituents of
the Nutritive Liquid. Diseases in Beer. — The first striking
proof of the fact that Saccharomyces species can perform
very different work in the nutritive liquid, was obtained after
Hansen's discoveries, by means of pure cultures of the yeasts
prepared in the Carlsberg laboratory, and afterwards in many
other laboratories, and which were subsequently tested in
practice. There are in fact breweries in which a large
number of different species of yeast have been tried on a
large scale and under the same conditions, and where the
attenuation, taste, odour, time of clarifying, and permanence
as regards yeast turbidity, &c., &c., have been found to differ
for each individual species.

Hansen's epoch-making researches on the disease-yeasts
(1883) again showed, from another point of view, the marked
differences amongst the Saccharomyces species in their action
on the nutritive liquid ; he discovered, namely, groups of the
so-called wild yeasts, which bring about detrimental changes
in beer, whilst others were found to be harmless. Amongst
the former, again, there are some which communicate a bitter
taste and disagreeable odour to the beer (Sacch. Pastorianus
/.) without as a rule producing ^urbidity ; whilst others only
fully develop their activity in a late stage of the secondary
fermentation, and cause the beer to become turbid (Sacch.
Pastorianus III. and Sacch. ellipsoideus II.), in that an
abundant yeast deposit forms in a comparatively short time in
the finished beer after it has been drawn off. It is only when
these species — Sacch. Pastorianus /., Sacch. Pastorianus
III., and Sacch. ellipsoideus II. — are introduced into the
wort at the commencement of fermentation that they are
able to induce disease. The addition of disease-yeast to the
beer in the store casks or to the drawn-off beer has no
appreciable effect ; the inoculation of bottled beer with Sacch.
ellipsoideus II. will only take effect when the beer has been
very strongly infected. The main result is that the danger

146 MICRO-ORGANISMS AND FERMENTATION.

of infection lies in the pitching-yeast. These diseases have
led to very great difficulties and have caused considerable
losses in breweries. Hansen's observations on the disease
yeasts have been confirmed by Gronlund, Will, Lasche,
Kokosinsky,Krieger, Windisch, and P.Lindner, and extended
by new examples. The wild yeasts can also bring about
disturbing-effects in top-fermentation breweries. For instance,
the so-called " summer-cloud " of Australian beers is caused,
according to de Bavay, by a wild Saccharomyces species.
This organism causes turbidity and imparts to the beer an
acid, bitter taste.

Eecently Pichi has also detected disease-yeasts in wine.

Just as the mould-fungi exhibit a different behaviour
towards the carbohydrates (see Penicillium, Mucor, Monilia),
so the different Saccharomycetes and similar fungi have been
shown by Hansen's comprehensive investigations to also
exhibit pronounced characteristics in the same direction.
In addition to the true Saccharomycetes we will here also
review Mycoderma cerevisice, Sacch. apiculatus, the Torula
forms, and Monilia.

Hansen examined the behaviour of a large number of
Saccharomycetes towards the four carbohydrates — saccharose
(cane-sugar), maltose, lactose, and dextrose.

His known six species of Saccharomycetes (Sacch.
cerevisice L, Sacch. Pastorianus L, IL, and ///., Sacch.
ellipsoideus I. and //., see page 159) behave as follows: —
They all develop invertase ; they convert cane-sugar into
invert-sugar, which they then ferment ; they ferment maltose
and dextrose, but not lactose. All the bottom-yeasts used in
practice show the same behaviour towards the four sugars
mentioned.

Sacch. Marxianus (page 176), Sacch. Ludivigii (page
179), and Sacch. exiguus (page 176) do not ferment maltose
and lactose ; they invert saccharose and ferment nutritive
solutions of invert-sugar and dextrose.

Sacch. membranaefaciens (page 177) and Mycoderma

ALCOHOLIC FERMENTS. 147

cerevisice (page 200) can neither invert nor ferment the
above four carbohydrates.1

Sacch. apiculatus (page 195) does not invert saccharose,
and of the four sugars mentioned it only ferments dextrose.
It therefore only induces a feeble alcoholic fermentation in
beer wort.

Amongst the Torula forms (page 189) examined by
Hansen there are many which do not secrete invertase, do
not ferment maltose, and which only yield about 1 per cent,
of alcohol (by volume) in beer wort. Other species invert
saccharose. In nutritive dextrose solutions the different
species induce a more or less vigorous fermentation.

Monilia Candida (page 100) possesses no invertive action,
but ferments saccharose (without hydrolysing it), maltose,
and dextrose. It ferments beer-wort, but at the ordinary
room-temperature it only very slowly yields the higher per-
centages of alcohol, as compared with the Sacckaromycetes.

If we now review all these different properties of the
Saccharomycetes we shall see that they fall into two
groups :—

I. Those which develop invertase, and induce alcoholic

fermentation. This group is further sub-divided into
(a) those which not only ferment saccharose and
dextrose, but also vigorously ferment maltose
(Hansen's first described six species, and the
yeasts employed in the brewing industry).
(6) those which ferment saccharose and dextrose, but
not maltose (Sacch. Marxianus, Ludiuigii and
exiguus).

II. Those which do not develop invertase, and do not induce
alcoholic fermentation (Sacch. rnembranaefaciens).

The budding fungi ivhich do not form endospores (non-
Saccharomycetes) show, with reference to the properties of
inversion and fermentation, the most varied characters.

1 According to Lasches experiments, some species of Mycoderma
present in beer are capable of inducing alcoholic fermentation.

148 MICRO-ORGANISMS AND FERMENTATION.

I. The greater majority do not ferment maltose. Many

of these induce a more or less vigorous fermentation in
solutions of dextrose and invert-sugar. Some (Torula
forms) invert saccharose, and many possess no inver-
tive ferment (Mycoderma cerevisice, Torula forms,
Sacch. apiculatus).

II. Only one species (Monilia Candida) ferments maltose,
saccharose (direct), and dextrose, without however
possessing any invertive action.

From the above it is clear that, as pointed out by Han-sen,
the Saccharomycetes cannot be characterised merely as
alcoholic ferments.

When we consider the behaviour of the above-named
fungi in the fermentation industries, it is at once seen that it
is only amongst the Saccharomycetes that species occur which
rapidly and vigorously ferment maltose. The yeasts for
breweries and distilleries must therefore be looked for from
among the true Saccharomycetes. The fungi not included
in the genus Saccharomyces, of which by far the greater
majority do not ferment maltose, are scarcely capable of
plaving any important part in these industries ; on the other
hand they can be employed in the manufacture of wines
from grapes, berries, and fruits, since several of them are able
to induce just as vigorous a fermentation in solutions of
dextrose and invert-sugar, as the Saccharomycetes.

It is perfectly clear from the above that a suitable species
must always be selected.

These different properties of the various species of
budding-fungi are of special importance in analytical
chemistry in cases where solutions containing several different
carbohydrates are under examination. In fact Hansen
expressed the opinion that it will be possible in this way
to arrive at a more exact quantitative determination of the
different carbohydrates in wort. Several chemists have been
recently engaged on this problem, but a true solution has not
yet been found.

ALCOHOLIC FERMENTS. 149

The discovery of isomaltose by C. J. Lintner, jun., has
opened up a new path in the study of the composition of
wort, whilst his investigations are of great importance, in
that they also give us a more intimate knowledge of the
process of fermentation.

Very recently the budding-fungi have been also eagerly
looked for in milk. Grotenfelt discovered a Saccharomyces
(page 180), whilst various budding-fungi (pages 192-194) not
belonging to this genus were found by Duclaux, Adametz,
Kayser, and Beyerinck; they all hydrolyse milk-sugar.
These species have not yet been found in breweries.

Fermi found that certain white and red species of yeast
are capable of exercising a diastatic action. Morris, in
experiments with pressed yeast, arrived at similar results.

The different action of the Saccharomyces species on the
same nutritive liquid (e.g., wort, must) and under the same
conditions, has been further studied by Borgmann, Amthor
and Marx.

According to Borgmann's experiments, the chemical
changes brought about in wort by the two Carlsberg
bottom-yeasts, No. 1 and No. 2, show a pronounced differ-
ence. These two species — which had been in use for some
time in the fermenting-room, and were still practically pure
— were employed for pitching two fermenting vessels con-
taining wort from the same brew ; the fermentation took
place under conditions which enabled a true comparison to
be made, and the resulting beer was stored as usual. The
differences in the chemical products were especially pro-
nounced in the proportion of free acid (No. 1 contained
in 100 c.c., 0-086, and No. 2, 0-144 acid, calculated as
lactic acid), and glycerine (No. 1 contained 0-109 and
No. 2, 0-137).

As a result of these experiments, Borgmcmn points out
that the ratio between the alcohol and glycerine in these
two beers differs from that previously found in beers, the
ratio obtained from previous analyses being : —

150 MICRO-ORGANISMS AND FERMENTATION.

Alcohol. Glycerine.
Maximum ... ... 100 5'497

Minimum 100 4-140

whilst the Carlsberg beers gave the following numbers : —
No. 1. No. 2.

Alcohol. Glycerine. Alcohol. Glycerine.

100 2-63 100 3-24

It is thus seen, that, as Borgmann also points out,
good beer may be produced in which the ratio of glycerine
to alcohol is lower than the previously-admitted minimum.
A series of eight different species of Saccharomyces, and
amongst them six " cultivated " yeasts, all in absolutely pure
cultures, were investigated by Amthor with reference to their
chemical action on beer-wort. His results again confirmed
Hansen's principle, that in practice a selection must always
be made. The fermentations were conducted in Pasteur
flasks of one liter capacity under the same conditions and
in two series, the first of which corresponded to the primary
fermentation in the brewery, and the second at the same
time also to the secondary fermentation. The alcohol,
extract, specific gravity, attenuation, glycerine, nitrogen,
reducing substance, and the degree of colour, were deter-
mined in the fermented worts. The tables show, as pointed
out by the author, palpable differences in the work accom-
plished by the different species. The percentage of alcohol
(by volume) varied within the limits 4-34 and 6'02 (3-55
to 5*94 at the end of the primary fermentation), the extract
between 8-27 and 11-23 (8'49 to 12-61 at end of primary
fermentation), the attenuation between 36*7 and 53-3 (28-8
to 52-1 at eod of primary fermentation) ; the percentage of
glycerine showed very striking differences and fluctuated
between 0*08 and 0'15 ; and likewise the amounts of nitrogen
and reducing substance, and to some extent also the degree of
colour, showed considerable differences.

A very considerable number of Saccharomycetes occurring

ALCOHOLIC FERMENTS. 151

in must — absolutely pure cultures of which were prepared by
Hanserfs method — were investigated by Marx, both from a
botanical point of view and with reference to their chemical
action on the nutritive liquid. The time required for spore-
formation was very different for the different species, and
likewise the number of spore-forming cells and the number
of spores in individual cells exhibited striking and constant
differences. In connection with this, it is especially of
interest that the pure cultivated species show distinct
differences in fermentative power and in the production of
volatile substances, which impart a special bouquet to wine,
and finally in their power of resistance to different acids
and to elevated temperatures. As marked differences in
taste are produced by not a few species, Marx is justified
in emphasising the practical importance of such investiga-
tions, since it thus becomes possible, by the addition of
yeasts of known properties to wine-must, to produce wines
having definite characters as regards taste, &c., independent
of the locality.

More recently Amthor has also investigated a number of
absolutely pure cultures of wine yeasts, and has detected
typical differences both with regard to spore-formation and
to the time of duration of the fermentation, finally also in
the chemical composition of the wines produced. Similar
results have also been obtained by Jacquemin, Rommier,
Martinand, and Rietsch, in France ; Muller-Thurgau, in
Switzerland ; Nathan and Wortmann, in Germany ; Mack
and Portele, in Austria ; Forti and Pichi, in Italy ; the com-
parative experiments conducted by these authors having
been partly carried out on a large practical scale.

(g) Variations in the species of the Saccharomycetes. —
Hansens numerous investigations have proved that the Sac-
charomycetes are affected in various ways by external in-
fluences. From the results recorded in the previous sections,
we are perfectly justified in saying that there are a number of
species, not only of the so-called wild yeasts (species which

152 MICRO-ORGANISMS AND FERMENTATION.

were formerly described under the general names Sacch.
Pastorianus, Sacch, ellipsoideus, &c.), but also of well-
characterised top- and bottom-yeasts, which are employed in
practice. It is a point of great practical interest that
species cultivated in beer-wort, the cultivation of which has
been uninterruptedly continued for several years, have shown
no, or at most but slight, changes. At the same time that
Hansen arrived at these results, he also discovered that it
was possible, by suitable treatment, to produce variations in
different directions ; also the individual peculiarities of the
cells in an absolutely pure culture can here assert them-
selves. Some of these changes are only temporary, and
disappear under suitable treatment when the species re-
assumes its original character. Others become more deeply
rooted, and it is then only under especially favourable con-
ditions that the culture can be deprived of its newly-
acquired properties. In certain cases it was not possible,
even after years of methodical treatment, to re-convert a
culture to its original state.

1. As is known, the data regarding the time required
for the appearance of the first indications of spore-forma-
tion in the previously-described six species, are subject to
the condition that the growth has been previously cultivated
in wort for 24 hours at a temperature of 25° C. At the
same time that Hansen published (1883) the temperature
curves for these six species, he also found that cultures
which had been grown in wort at the above temperature,
but for two days instead of one, developed spores more
slowly and more sparingly than usual. If, however, such
cultures are subsequently treated in the manner first
described, the culture again assumes its normal condition.
We have here, therefore, an example of a very feebly-rooted
variation.

2. In a gelatine culture of " Carlsberg bottom-yeast
No. 1 " both oval and elongated sausage-shaped cells are
often found, so that according to Reess the presence of two

ALCOHOLIC FERMENTS. 153

species must be assumed. If colonies of each kind are
separately introduced into flasks containing wort, growths
are again obtained which consist partly of egg-shaped
and partly of " Pastorianus " cells. Reinserts experiments
showed that when these latter cultures were repeatedly re-
cultivated in fresh flasks the cells still partly retained their
sausage-shape for a lengthened period. When such a culture
was introduced into a yeast-propagating apparatus, the growth
obtained from it still showed an admixture of these cells ;
these disappeared, however, after the yeast from the propa-
gating apparatus had been introduced into an ordinary
fermenting vessel. In this case, therefore, the variation is
more strongly rooted, and only disappears after the yeast has
been propagated through a series of fermentations.

Another example in the same direction is that of a species
of Sacch. cerevisice (a bottom-yeast) which, after a lengthened
and difficult development, was subsequently cultivated in
wort at a temperature of about 27° C. when the cells obtained
exhibited their ordinary appearance ; when cultivated at
7*5° C., however, grouped colonies with mycelium-like
branches were obtained. This is an interesting example of
the influence of temperature on the form of yeast cells.

3. As an example of a much more deeply-rooted change
in the nature of the cells, Hansen's observations on Sacch.
Ludivigii may be mentioned. When single individuals taken
from an absolutely pure culture are again separately cultivated
as pure cultures, it is possible to obtain growths which exhibit
great differences in their power of forming spores. By a
methodical choice of single cells Hansen succeeded in obtaining
growths which, under the known conditions, completely failed
to yield spores; on the other hand, he found that when, starting
from the same original growth, a yeast speck which had
sprung from a spore-yielding cell was chosen and further
developed, a growth was obtained which was forthwith capable
of yielding an abundance of spores. By such methodical
selection, three varieties were separated from this species, one

154 MICRO-ORGANISMS AND FERMENTATION.

of which was characterised by its high capacity of forming
spores ; in the second this property had nearly disappeared,
whilst the third did not form spores at all. After numerous
cultivations in wort, the third form returned, but only slowly,
to its original condition, in which it was able to form spores ;
when it was cultivated in a solution of dextrose in yeast-
water, however, this property was immediately re-acquired.

Another example of physiological transformation is the
following : The three species described under the group
Saccharomyces Pastorianus form under certain conditions a
dough-like sediment similar to those of the other Saccharo-
mycetes, under other conditions, however, a film-like, wrinkled,
or a caseous sediment consisting of small lumps (Pasteur's
levure caseeuse), that is to say, sediments of very different
appearance, and yet produced by the same species. In the
last-mentioned cage, the fermenting wort also assumes a very
characteristic appearance, and, contrary to what ordinarily
occurs, remains bright throughout the fermentation, so that
the yeast flakes can be observed to rise from the bottom to
the surface and to again sink. If this curious sedimentary
yeast is repeatedly cultivated by new fermentations in wort, it
can be again transformed into the dough-like form.

A similar physiological transformation occurs in the^m-
formations of the Saccharomycetes (p. 137).

4. At the beginning of the year 1889, Hansen published1
the results of a series of experiments which were undertaken
with the view to discover the conditions causing variation, and
by experiment to bring about the formation of new races, and
if possible new species. These studies are being continued
in his laboratory. The following account is taken partly from
the source mentioned and partly from more recent publica-
tions.

Hansen found in the case of several Saccharomyces species,
that when their cells were cultivated in aerated wort at a

1 Centralbl. f. Bakt. u. Parasitenk. Bd. V., p. 665, 1889.

ALCOHOLIC FERMENTS. 155

temperature approaching their maximum temperature, they
became affected in such a manner that they lost their power
of forming spores, and the same applies also to the innumer-
able generations gradually formed in new cultures at the
optimum temperature. Yet the cells had a vigorous appearance
and were further cultivated under very varied conditions.

Similar changes were also brought about by cultivation
on a solid nutritive medium. These newly-formed varieties,
as Hansen provisionally calls them, have not only lost their
power of spore-formation, but at the same time also their
property of forming films.

- These investigations also have a practical bearing on the
breiuing industry, although in a direction different from that
of Hansen's earlier researches. The Carlsberg bottom-yeast,
No. 2, well-known to the brewing world, is one of the species
which loses the property of spore-formation when it is subjected
to the above-mentioned treatment. In the case of this yeast,
it has been proved by numerous experiments that, simul-
taneously with the change mentioned, the plasma of the cells
also undergoes transformation in other directions. The
newly-formed growth gives a more feeble fermentation, the
higher percentages of alcohol being produced more slowly than
usual ; in short, after the yeast has been treated as described,
it works in a different manner than before such treatment.

No objection can be raised to the view that we are here
possibly dealing with the formation of new species. We
know in fact that the species are not fixed and unchangeable,
as was generally assumed in Linnets time, but that the
characters of a species are only constant under certain con-
ditions. The complete elucidation of these important and
intricate problems can, however, only be effected by a series
of experiments carried on through a long period of time and
varied in different directions.

In order to guard against any misunderstanding, it may
not be superfluous to remind the reader that these remarkable
changes are only brought about by a long-continued and

156 MICRO-ORGANISMS AND FERMENTATION.

violent interference with the vital process of the cells, and
that they do not occur so long as the development takes
place in the normal manner.

An example of the persistence with which Saccharomyces
cells retain, under normal conditions, the property of spore-
formation, is met with in breweries and distilleries. We have
here species of yeasts which have lived through hundreds of
years, and have developed an infinite number of generations
under conditions which, as a rule, have not permitted the
exercise of the above-named function, and yet the power to
do so has always been persistently retained.

(A) Gelatinous Formation secreted by the Budding-
fungi. — Under certain but as yet undetermined conditions,
the colonies produced by the budding of yeast cells can unite
to irregular masses which sink to the bottom more quickly
than the single yeast cells (breaking and clarifying in the
brewery). This behaviour is undoubtedly related to a phase
in the development of yeast cells which Hansen discovered
in 1884. He discovered that not only the Saccharomycetes
but also other budding-fungi are able to secrete a gelatinous
netiuork, which can be seen as threads or plates, and in
which the cells lie imbedded (Fig. 33, A, B). If, for example,
some moderately thick brewery yeast is placed in a glass and
allowed to remain covered up in such a manner that it slowly
dries, and then a trace of this yeast mixed with water, the
network can be clearly seen (Fig. 33, A). The formation
also occurs in the gypsum block and gelatine cultures. I
have myself very frequently observed this remarkable for-
mation, after Hansen had called my attention to its nature,
in the yeast samples which are sent to my laboratory in filter-
paper enclosed in envelopes.1 Hansen also found it in the

1 This method of preserving a sample of yeast for some time is very
convenient. A small piece of filter-paper is rapidly passed through a
flame several times, a few drops of yeast are poured on to it, and it is
then folded up, and afterwards wrapped in several layers of paper which
have been similarly treated.

ALCOHOLIC FERMENTS.

157

film-formations of nearly all species. An ordinary microscopic
examination of the pitching-yeast in a brewery does not
show this formation ; with the help of staining, however, its
presence can be readily detected (Fig. B). When the yeast
was repeatedly washed, it was no longer possible to detect the

,a

FIG. 33.

Yeast cells with gelatinous network, after Hansen : A, network obtained by
partial drying ; 1, portion formed of threads, from which the cells have
become detached ; 2 and 3 show that the network can also form complete
walls ; such a formation is seen between a and b ; a is a vegetative
cell, b is a cell with two spores ; 4, shows three cells, a, imbedded
in the network. £, network with yeast cells; the latter were stained
by methyl-violet ; the network is not stained. Some of the yeast
cells are still in the meshes, but most have become detached.

network by staining ; but if the water was removed, and the
yeast set aside for a time, the gelatinous masses could after
suitable treatment be readily seen. By varying the conditions
of nourishment of the cells, the development could be pro-
moted or hindered, and the chemical composition modified.

158 MICRO-ORGANISMS AND FERMENTATION.

The whole behaviour suggests the zoogloea-formation of
bacteria.

The Microscopic Appearance of a Yeast-cell. — As an
introduction to the systematic description of the separate
species of Saccfiaromyces, we give the following general
description of the Saccharomyces cell.

The microscopic appearance of a yeast cell as it most
frequently occurs in a fermenting liquid is a spherical or oval
figure, which, by the swelling out of its wall, gives rise to one
or more buds, which detach themselves sooner or later from
the mother cell. This cell is consequently surrounded by a
membrane which can vary somewhat in the different stages
of the development of the cell, but rarely in a noticeable
degree. It is otherwise, however, with the contents of the
cell. The contents present the simplest appearance when the
cell is observed in its most vigorous state of growth. The cell-
contents then consist of clear homogeneous plasma. As the
processes of multiplication and fermentation continue, different
bodies appear in this plasma; partly clear portions filled with
sap (vacuoles), partly larger and smaller particles, some of
which can be shown to be fat globules, whilst others appear to
be of a similar nature to the plasma. These granules have been
minutely described by Raum. This granular appearance of the
plasma increases with the further development of the cell, and
at a very advanced stage of the fermentation, when the cell has
almost come to a state of rest, the plasma may become reduced
to a thin layer on the inner side of the wall, whilst a large
vacuole occupies the remaining space, and contains numerous
small and large grains, many of a fatty nature. If such cells
are again brought into a fermentable liquid, they soon exhibit
a highly characteristic appearance during the period which
precedes the macroscopic phenomena of fermentation. The
grains disappear, and numerous fine plasma-threads appear in
the clear cell-sap, and gradually circumscribe rounded
vacuoles ; finally these disappear, and the cell again becomes
filled with clear homogeneous plasma-

ALCOHOLIC FERMENTS. 159

As in most vegetable cells, a cell-nucleus (first discovered
by Schmitz) is also found in the yeast cell, and its presence
can be proved by staining with osmic acid or with picric acid
and hcematoxylin. According to Hansen this cell-nucleus is
either spherical or disc-shaped. In old film-formations of
Saccharomycetes, he found cells which distinctly showed the
nucleus without any treatment. — Janssens observed the
partition of the cell-nucleus both in the budding and in the
spore-formation of the Saccharomycetes.

CLASSIFICATION OF THE GENUS SACCHAROMYCES.

"BUDDING FUNGI, mostly without a mycelium, the indivi-
dual species of which occur with cells of different form and
size. Under certain treatment, and sometimes also ^vithout
any previous treatment, cell-nuclei are seen. Under certain
conditions the cells develop ENDOGENOUS SPORES ; the germi-
nating spores of most species grow to budding cells; in
exceptional cases a promycelium is first formed. Number
of spores 1 to 10, most frequently 1 to 4. Under favourable
conditions the cells secrete a gelatinous network, in ^vhich
they lie imbedded.

The greater number of the species induce alcoholic fer-
mentation.

SACCHAROMYCES CEREVISLE I.
(Figs. 34—36.)

This and the five following species (Sacch. Pastorianus
I., II., and III., Sacch. ellipsoideus I. and 77.), all develop
invertase ; with this they effect the hydrolysis of saccharose
to invert-sugar, and they ferment the latter. They produce
a vigorous fermentation in dextrose solutions, and likewise in
maltose solutions, especially when a nutrient liquid such as
yeast-water is added. All are vigorous alcoholic ferments

1 This top-fermentation yeast must not be confused with Uansen's
Carlsberg bottom-yeast No. I.

160

MICRO-ORGANISMS AND FERMENTATION.

which in the course of fourteen days at the ordinary room-
temperature, readily produce 4 to 6 per cent (by volume)
of alcohol in beer wort. They are unable to ferment lactose.

Saccharomyces cerevisice /., is an old English top-fermen-
tation yeast, which is employed in breweries in London and
Edinburgh.

The young growth of sedimentary yeast (Fig. 34) developed
in wort, consists essentially of large round and oval cells ;
really elongated cells do not occur under these conditions.

FIG. 34.

Saccharomyces cerevisise I. Hansen. Cell-forms of young sedimentary
yeast, after Hansen.

Ascospore-formation (Figs. 26—28, 32, 1) :—1
At 37 '5° C. no ascospores are developed.

„ 36—37°
35
33-5
30
25
23

16-5
11—12
9

the first indications are seen after 29 hours.

25
23
20
23
27
50
65
10 days.

no ascospores are developed.

1 The preparation of the growth of a Saccharomyces species for these
investigations must be made in the following manner : — After the cells
have been cultivated for some time in ordinary wort (about 14 per cent.

ALCOHOLIC FERMENTS. 161

Spores strongly refractive to light. Wall of spores very
distinct. Size of spores 2-5 — 6 /*.

Film-formation : —

At 38° C. no film-formation occurs.
„ 33 — 34° „ feebly-developed film-specks are

seen after 9 — 18 days.

,,26—28 „ „ „ „ „ 7—11 „

,,20-22 „ „ „ „ „ 7-10 „

" 13~15 " \ (Fir ^ I " » 15-3° »

„ 6- 7 „ J ( ^ { „ „ 2- 3 months.

„ 5 „ no film-formation occurs.

.Microscopic appearance of the cells in the films : —
At 20 — 34° C. : Colonies frequent ; sausage-shaped and
curiously formed cells occur.

FIG. 35.

Saccharomyces cerevisiae I. Hansen. Film-forms at 15 — 6° C.,
after Hansen.

At 15—6° C. (Fig. 35) : The greater number of the cells
resemble the original cells ; isolated cells of different form.

In old cultures of films all forms occur, including greatly
elongated mycelium-like cells (Fig. 36, p. 162).

SACCHAROMYCES PASTORIANUS I. HANSEN.
(Figs. 37, 38.)

Bottom-fermentation yeast.

Sedimentary forms grown in wort: — Mainly elongated,

Ball.) at the ordinary room-temperature, the young vigorous cells obtained
are introduced into fresh wort, in which they are allowed to develop
for about twenty-four hours at 25° to 27° C. This growth is used for
the gypsum-block culture.

FIG. 36. Sacch. cerevisise I. Hansen. Cell-forms in old cultures of films, after Hansen.

ALCOHOLIC FERMENTS.

163

sausage-shaped cells, but also large and small round and oval
cells (Fig. 37).

It frequently occurs in the air of the fermenting-rooms.
It imparts to the beer a disagreeable bitter taste and
unpleasant odour ; it can also produce turbidity, and can
interfere with the clarification of the beer in the fermenting
vessel.

FIG. 37.

Saccharomyces Pastorianus I. Hansen. Cell-forms of young sedimentary
yeast, after Hansen.

Ascospore formation (Fig. 32, 2) : —
At 31-5° C. no ascospores are developed.

„ 29.5 — 30-5° „ the first indications are seen after 30 hours

27
24
26
35

29

27-5

23.5

18

15

10

8-5

7

3-4

0-5 „ no ascospores are developed.
Size of spores 1*5 — 5 p.

50

89

5?

5 days

7 „
14

164 MICRO-ORGANISMS AND FERMENTATION.

Film-formation : —

At 34° C. no film-formation occurs.
„ 26 — 28° „ feebly-developed film-specks are

seen after 7 — 10 days.

„ 20 22 55 ,5 55 55 ,, 8 lO 5,

„ 13—15 „ j ™ 3g) f „ 15-30 „

,,6—75,) I „ „ 1— 2 months.

» 3- 5 „ „ „ „ „ 5— 6 „

like Fig. 38, but without the large colonies.
„ 2 — 3 „ no film-formation occurs.

FIG. 38.

Saccharomyces Pastorianus I. Hansen. Film-forms at 13 — 15° C., from
Holm's drawing in Hansen's Memoir.

ALCOHOLIC FERMENTS.

165

Microscopic appearance of the cells in the films : —
At 20 — 28° C. nearly the same forms as in the sediment-
ary yeast.

At 13 — 15° C. strongly-developed mycelium-like colonies
of very elongated sausage-shaped cells (Fig. 38) are moderately
frequent.

In old cultures of films the cells are smaller than in the
sediment ; very irregular and sometimes almost thread-like
cells are found.

SACCHAKOMYCES PASTOKIANUS II. HANSEN.
(Figs. 39, 40.)

Fie. 39.

Saccharomyces Pastorianus II. Hansen. Cell-forms of young sedimentary
yeast, after Hansen.

Feeble top-fermentation yeast.

Sedimentary forms grown in wort : — Mainly elongated
sausage- shaped cells, but also large and small oval and round
cells (Fig. 39).

It frequently occurred in Hansen's analyses of the air in
the brewery ; appears to belong to the species which do not
cause diseases in beer.

Ascospore-formation (Fig. 32, 3) :—

166

MICRO-ORGANISMS AND FERMENTATION.

At 29° C. no ascospores are developed.
„ 27 — 28° „ the first indications are seen after 34 hours.

?) 25 ,,

55 27 ..

36

"**

1 I'D „
'

" - ^ 55

0*5 „ no ascospores are developed.
Size of "the spores 2 — 5 M.

77

'
17

FIG. 40.

Saccharomyces Pastorianus II. Hansen. Film-forms at 15 — 3° C. , after
Holm's drawing in Hansen 's Memoir.

Film-formation : —

At 34° C. no film-formation occurs.
„ 26—28° „ feebly-developed film-specks are

seen after 7—10 days.
„ 8 — 15 „
10—25

., 20 — 22
„ 13—15
„ 6—7
55 3- — 5
2 — 3

„ -\ „ „ r „ „ — „

„ Y (Fig. 40) - „ „ 1— 2 months.

„ j „ „ I. „ „ 5 6 „

„ no film-formation occurs.

ALCOHOLIC FERMENTS.

167

Microscopic appearance of the cells in the films : —

At 20 — 28° C : Nearly the same forms as in the sediment-
ary yeast ; also irregular sausage-shaped cells.

At 15—3° C. : Mostly oval and round cells.

In old cultures of films the cells are smaller than in the
sediment; very irregular and sometimes almost thread-like
cells are found.

Streak cultures of this species in gelatine yeast-^vater
give, after sixteen days at 15° C., growths with compara-
tively smooth edges, and in this respect it also differs from
the following species.

SACCHAROMYCES PASTORIANUS III. HANSEN.
(Figs. 41, 42.)

FIG. 41.

Saccharomyces Pastorianus III. Hansen. Cell-forms of young sedimentary
yeast, after Hansen.

Top-fermentation yeast.

Sedimentary forms grown in wort: — mostly elongated,
sausage-shaped, but also large and small oval and round
cells (Fig. 41).

It was separated from a bottom-fermentation beer which
showed yeast-turbidity, and has been proved by Hansen to be

168 MICRO-ORGANISMS AND FERMENTATION.

one of the species which produce this disease. Recent experi-
ments of Ha/nsen show that this disease-yeast possesses another
peculiar property ; namely, when the fermenting wort has an
opalescent appearance, the addition of Sacch. Pcistorianus III.
will in certain cases effect a clarification.

Ascospore formation (Fig. 32, 4) : —
At 29° C. no ascospores are developed.
„ 27 — 28° „ the first indications are seen after 35 hours.
,, 2b*5 „ „ ,, ,, „ 60 „

9;? 90

55 ^^ 55 55 5? 55 55 ^° 55

)5 ^2 „ „ ,, ,, „ -9 „

n44
55 55 5> 55 5? 55

55 16 ,, „ ., „ „ 53 ,,

„ 10*5,, „ „ „ „ 7 days.

?5 "'^ 55 55 55 5? 55 " y>

„ 4 „ no ascospores are developed.

Size of the spores 2 — 5 p.

Film-formation : —

At 34° C. no film-formation occurs.
„ 26 — 28° „ feebly -developed film-specks are

seen after 7 — 10 days.

9H 99 Q 1 *>

55 ^U -^ 55 55 55 55 55 " ^ " 55

„ 13-15 „ } „ „ r „ „ 10—20 „

„ 6—7 „ (Fig. 42) „ „ 1— 2 months.

5? 3 o „ / „ ,, I. „ „ o o ,.

„ 2 — 3 „ no film-formation occurs.

Microscopic appearance of the cells in the films : —

At 20 — 28° C. : Nearly the same forms as in the sediment-
ary yeast.

At 15 — 3° C. : Strongly-developed colonies of elongated
sausage-shaped or thread-like cells, which closely resemble a
mycelium in appearance (Fig. 42).

In old cultures of films, the cells have the same forms as
at 15 — 3° C.5 and are often still thinner and more thread-
like.

ALCOHOLIC FERMENTS.

169

170

MICRO-ORGANISMS AND FERMENTATION.

Streak cultures of this species in gelatine yeast-iuater
give, after sixteen days at 15° C., growths with distinctly
hairy edges.

SACCHAROMYCES ELLIPSOIDEUS I. HANSEN.
(Figs. 43, 44.)

Bottom-fermentation yeast.

Sedimentary forms grown in wort : — mostly oval and round
cells ; sausage-shaped cells rare (Fig. 43).

FIG. 43.

Saccharomyces ellipsoideus I. Hansen. Cell-forms of young sedimentary
yeast, after Harisen.

Occurs on the surface of ripe grapes.
Ascospore-formation (Fig. 32, 5) : —
At 32'5° C. no ascospores are developed.

3O5 — 31*5° „ the first indications are seen after 36 hours.

23

29.5
25

18
15
10-5
7-5

4 „ no ascospores are developed.
Size of the spores 2—4 /*.

21 „
33 „
45 „
4 J days.

11 „

ALCOHOLIC FERMENTS.

171

FIG. 44.

Saccharomyces ellipsoideus I. Hansen. Film-forms at 13—15° C., from
Holm's drawing in Hansen's Memoir.

172 MICRO-ORGANISMS AND FERMENTATION.

Film-formation : —

At 38° C. no film-formation occurs.

„ 33 — 34° „ feebly-developed film-specks are

seen after 8 — 12 days.

„ 26 — 28 „ „ „ „ „ 9 — 16 „

„ 20-22 „ „ „ „ „ 10—17 „

„ 13—15 „ (Fig. 44) „ „ „ 15—30 „

„ 6 — 7 „ „ „ „ „ 2 — 3 months.

„ 5 „ no film-formation occurs.

Microscopic appearance of the cells in the films :—
At 20—34° C. and 6—7° C., the cells are smaller and
more sausage-shaped than in the sedimentary yeast.

FIG. 45.

Saccliaromyces ellipsoideus II. Hansen. Cell-forms of young sedimentary
yeast, after Hansen.

At 13 — 15° C., freely-branched and strongly-developed
colonies of short or long sausage-shaped cells, often with
verticillated branches (Fig. 44).

In old cultures of films, the cell forms are the same as
at 13—15° C.

Streak cultures of this species in wort-gelatine (wort with
the addition of about 5*5 per cent, of gelatine) give — in
contradistinction to the other five species — in the course of
eleven to fourteen days at 25° C., a characteristic net-like
structure, by means of which it can be distinguished by the
naked eye from other species.

ALCOHOLIC FERMENTS.

173

SACCHAROMYCES ELLIPSOIDEDS II. HANSEN.
(Figs. 45, 46.)

Usually bottom-fermentation yeast.

Sedimentary forms grown in wort: — mostly oval and round
cells ; sausage-shaped cells rare (Fig. 45).

It was separated from beers which showed yeast-turbidity ;
is a species which causes y east-turbidity , and has been shown
by Hanserfs experiments to be more dangerous than Sacch.
Pastorianus III.

Ascospore-formation (Fig. 32, 6).
At 35° C. no ascospores are developed.

the first indications are seen after 31 hours.

55 °*

) O<±

55 L-

ue iiiou

lilUHJC

55

33

55

55

55

55

31-5

55

55

55

55

29

55

55

55

55

25

55

55

55

55

18

55

55

55

55

11

55

55

55

55

8

55

55

55

55

27 „

55

23 „

55

22 „

55

27 „

55

42 „

55

5J days.

55

9

4 no ascospores are developed.
Size of spores 2 — 5 n.

Film-formation :

At 40° C. no film-formation occurs.
„ 36 — 38° „ feebly-developed film-specks are

seen after 8 — 12 days.

„ 33-34

55 55 55 55

55

3— 4 „

„ 26-28

55

55 55

55

55

4— 5 „

„ 20—22

55

55 55

55

55

4— 6 „

„ 13-15

55

(Fig. 46.) -

55

55

8-10 „

„ 6— 7

55

55 55

55

55

1 — 2 months.

„ 3- 5

55 '55 55 ^ 55

55

5— 6 „

„ 2- 3

„ no film-formation occurs.

174 MICRO-ORGANISMS AND FERMENTATION.

Microscopic appearance of the cells in the films : —

At all temperatures, the same forms as in the sediment ;
at and below 15° C. the cells are only slightly more elongated
(Fig. 46).

In old cultures of films there are colonies of short and
long sausage-shaped cells, often with verticillated branches.

Related to this species are two ellipsoid species, described
by Will, and which are also disease-yeasts. One of these, a
bottom-fermentation yeast, gives colonies in wort-gelatine,
which when young form — whether on the surface or embedded
in the gelatine — a network with large meshes ; afterwards
they become denser in the middle, with irregularly-fringed

FIG. 46.

Saccharomyces ellipsoideus II. Hansen. Film-forms at 28—3°,
after Hansen.

edges ; sometimes, however, compact colonies with regular
outline are formed under the same conditions. The maximum
temperature for spore-formation is 39° C. ; at the optimum
temperature (34° C.), the first indications of spores are seen
after eleven hours. The lower limit for spore-formation is
4 to 5° C. The vegetative cells are killed when heated in
sterilised wort for half an hour at 70° C. The temperature
limits for film-formation are 41° and 4° C. In old films
especially are found numerously-branched clusters, consisting
of very much elongated cells. This species imparts a rough
bitter after-taste to beer and also causes turbidity.

The second ellipsoid species which was obtained from a
beer showing yeast-turbidity, gives colonies in wort-gelatine,

ALCOHOLIC FERMENTS. 175

some of which are sharply defined, whilst in others the out-
line is indistinct. The temperature limits for spore-formation
are 32° and 0*5° C., the optimum temperature being 24° C.
The temperature limit for the vitality of the vegetative cells
in wort is 70° C. In old films very numerously-branched
clusters occur. Besides causing yeast-turbidity, this species
also imparts a sweetish, disagreeable, aromatic taste to beer,
and a bitter, astringent after-taste. The yeast sediment
always has a dark colour.

SACCHAROMYCES ILICIS. GRONLUND

which was found on the fruit of Ilex Aquifolium, is a bottom-
fermentation yeast, consisting mainly of spherical cells. The
temperature limits for spore-formation are 8° and 38° C. The
spores have no vacuoles. In the films, slightly-elongated cells
are found. Streak cultures on gelatine have a floury, but
otherwise a variable, appearance. This species, grown in wort,
imparts a disagreeable, bitter taste. According to Schjerning
it contains invertase, and induces alcoholic fermentation in
solutions of saccharose, dextrose, and maltose. In ordinary
beer wort it can produce about 2*8 per cent, alcohol (by
volume).

SACCHAROMYCES AQUIFOLII. GRONLUND
was also found on the fruit of Ilex Aquifolium. It is a top-
fermentation yeast, and consists of large round cells. The
temperature limits for spore-formation are 8° and 31° C. ;
the spores contain vacuoles. In the films, spherical and egg-
shaped cells alone occur. Streak cultures in gelatine vary in
appearance, some being glossy and some floury. This species
imparts to wort a disagreeable, sweet taste, with a bitter after-
taste. It inverts saccharose and induces alcoholic fermentation
in solutions of saccharose, dextrose, and maltose. In ordinary
beer-wort it can produce about 3*7 per cent, alcohol (by
volume).

SACCHAROMYCES PYRIFORMIS. MARSHALL WARD
(see ginger-beer plant, page 71).

176 MICRO-ORGANISMS AND FERMENTATION.

SACCHAROMYCES MARXIANUS. HANSEN.

This species, which was discovered by Marx on grapes, and
described by Hansen, develops in beer-wort in the form of
small oval cells, essentially similar to those of Sacch. exiguus
and ellipsoideus. Elongated, sausage-shaped cells, often in
colonies, soon appear, however, and if the culture be set aside
for some time small mould-like particles are formed, and partly
swim in the liquid, and partly settle to the bottom. These
particles consist of mycelium-like colonies of essentially the
same nature as the film formations of the six species previously
described ; they are also built up of cells, which are readily
separated at the point of union. The ascospores are kidney-
shaped, spherical, or oval. After cultivation for two to three
months in wort contained in the two-necked flasks, there
were only traces of film-formation with only a few sausage-
shaped and oval cells.

This yeast is one of those species which develop a my-
celium under certain conditions of culture on a solid nutritive
medium.

In beer-wort it yields only 1 to 1*3 per cent, (by volume)
of alcohol, even after long standing. It does not ferment
maltose ; it inverts saccharose ; and in nutritive solutions of
the latter, and of dextrose, it yields considerable quantities of
alcohol.

SACCHAROMYCES EXIGUUS (REESS). HANSEN

develops in wort a growth, the cell-forms of which most closely
correspond to the species described by Reess under the above
name. It is, however, impossible to determine whether Reess
was really dealing with this species, since any Saccharomyces
species may, under certain conditions, form a preponderating
number of similar small cells.

This species only gives scanty spore-formation and weak
film-formation, but it yields a well-developed yeast-ring. The
cells of the film resemble those of the sedimentary yeast, but
short sausage-shaped and small cells are more frequent.

ALCOHOLIC FERMENTS. 177

Haiisen found this species in pressed yeast. Its behaviour
towards the sugars is similar to that of the last species, though
it develops a greater fermentative activity in solutions of
saccharose and dextrose. In wort, it also yields only small
quantities of alcohol. It does not ferment maltose solutions.
It inverts saccharose.

Experiments of a practical nature, which were conducted
by Hansen, have shown that this species does not produce any
disease in beer, even when present in considerable quantities
either at the beginning or end of the primary fermentation,
or when it is added after storage of the beer.1

.Some other species examined by Hansen can likewise
ferment saccharose and dextrose, but not maltose and lactose.

Saccharomyces Joergensenii, described by Lasche, also
belongs to the group of the Saccharomycetes, which may be
termed Sacch. exiguus. The growth consists of small round
and oval cells. The optimum temperature for spore-formation
is 25° C., the temperature limits being 8° and 30° C. At tem-
peratures above 30° C. the growth rapidly dies. A true film-
formation was not observed ; in old cultures only a very feeble
yeast ring was formed, and this consisted of round and oval
cells. In gelatine it yields colonies which resemble those of
low-fermentation brewery yeast. Wort-gelatine becomes slowly
liquefied. The streak-culture is dirty grey in appearance, with
smooth edges. This species ferments saccharose and dextrose,
but not maltose. When it is mixed with cultivated yeasts and
grown in wort, it consequently becomes suppressed and cannot
therefore produce any disease in beer.

SACCHAROMYCES MEMBRAN^EFACIENS. HANSEN.

This peculiar species, which occupies a special place amongst
the Saccharomycetes, when grown in wort, yields a strongly-
developed light grey, wrinkled film, which very quickly covers

1 This is of special interest, as Sacch. exiguus was formerly regarded
as a disease-producing species.

N

178 MICRO-ORGANISMS AND FERMENTATION.

the whole surface of the liquid, and which consists mainly
of sausage-shaped and elongated oval cells; these have
strongly-developed vacuoles, and have a more or less emptied
appearance. Between the colonies is an abundant admixture
of air.

The spores are very abundantly developed, not only under
the ordinary conditions of cultivation, but also in the films.
They are irregular in form, and at the ordinary room-tem-
perature they germinate in a Kanvier chamber after ten to
nineteen hours.

On wort-gelatine, the cells form dull grey specks, often
with a faint, reddish tinge, which are rounded, flat and spread
out, and wrinkled. The colonies embedded in the gelatine
present, however, a quite different appearance. The gelatine
becomes liquefied by this fungus, although only slowly.

This species is incapable of fermenting either saccharose,
dextrose, maltose, or lactose, and neither does it invert saccha-
rose. It was found in the slimy secretion on the roots of
the elm, and shows considerable resemblance to the species
Mycoderma cerevisice and Mycoderma vini, but it is a true
Saccharomyces.

Koekler found this species in very impure well-water.
Pichi has described two species which very closely resemble
Sacch. membrancefaciens.

SACCHAROMYCES HANSENIL ZOPF

was discovered by Zopf amongst the fungi of cotton-seed
flour. It forms very small spherical spores, which are mostly
developed singly, and at most in pairs, in the mother-cell. It
does not induce alcoholic fermentation in fermentable nutrient
sugar solutions, but on the other hand crystals of calcium
oxalate are observed in the sediment. Zopf found such
crystals in nutrient solutions of galactose, grape-sugar,
cane-sugar, milk-sugar, maltose, dulcite, glycerine, and
mannite.

ALCOHOLIC FERMENTS. 179

SACCHAROMYCES LUDWIGII. HANSEN.
(Figs. 29, 30.)

This characteristic species, which was discovered by Ludwig
in the viscous secretion of the living oak, is the only one of
the known Saccharomycetes which can be recognised solely by
means of a microscopic examination. The following description
is from Hansen's investigations. The cells are very variable
in size, are elliptical, bottle-shaped, sausage- or frequently
lemon-shaped. Partition walls can occur in all the complex
cell-combinations. The vegetative growths in wort-gelatine
are — like those of nearly all the Saccharomycetes — round,
light grey, or faintly yellow. In wort it only yields 1'2 per
cent, (volume) of alcohol after a long continued fermenta-
tion ; and this is in accordance with the fact that maltose is
not fermented by this species. In dextrose solutions, on the
other hand, it yields alcohol up to 10 per cent, by volume.
It inverts saccharose, but does not ferment solutions of lactose
and dextrin, and it does not saccharify solutions of starch.
It readily develops spores in aqueous solutions of saccharose,
in wort-gelatine, in yeast-water, and in wort ; in the latter
case even when no film has formed.

Spore-formation (Figs. 29, 30) occurs most rapidly at a
temperature of 25° C. It is characteristic of this species
that, especially in the case of the young spores, a fusion of
the germinated spores often occurs, and these new forma-
tions develop germ-filaments (promycelium), from which
new yeast-cells become gradually marked off by sharp trans-
verse septa. At the ends of these cells, buds are developed,
and these again become marked off by transverse septa.

In old cultures there is often a strong tendency to form
mycelium, but portions are only exceptionally found the
cells of which are firmly united together, and which show
only slight constrictions ; these portions have distinct,
straight, transverse walls. Each cell of such colonies can

180 MICRO-ORGANISMS AND FERMENTATION.

form buds and spores. Amongst them are also found
irregular cells, and very large many-branched cells.

It is also characteristic of this species that when kept in a
solution of saccharose the cells die within two years., whilst
most of the other Saccharomycetes examined can be pre-
served in this liquid for a much greater length of time.

SACCHAROMYCES ACIDT LACTICI. GROTENFELT.

Grotenfelt has described under this name a species of
Saccharomyces which, when added to sterilised milk, pro-
duces an intense curdling with formation of acid ; on gelatine
and agar-agar it forms white porcelain -like colonies, and on
potatoes it yields broad, moist, whitish-grey patches, which
soon become brown. In puncture cultivations in gelatine
short flask-shaped growths develop from the point of inocu-
lation into the gelatine. The cells are elliptical, 2*0 to 4*35 p
in length, and 1*50 — 2 -90 p- in breadth.

When a solution of milk-sugar was inoculated, with the
addition of calcium carbonate, and the product distilled,
alcohol could be detected. In a neutral 3 per cent, solution
of milk-sugar, Saccharomyces acidi lactici yielded O'lOS per
cent, of acid in eight days.

SACCHAROMYCES MINOR. ENGEL.

The vegetative cells are completely spherical, in size up
to 6 /* in diameter, and are united in chains or in groups
composed of but few cells (6 to 9). Spore-forming cells
7 to 8 ^, and containing 2 to 4 spores of 3 ^ in diameter.

This organism is, according to the above author, the most
active ferment in the fermentation of bread.1

1 Decisive experiments on the essential active factors in the fermen-
tation of bread have not yet been made. In the manufacture of white
bread, ordinarily " pressed-yeast " is used; this consists in the main of
alcoholic ferments, and according to the generally-accepted view the
yeast is the only active ferment. In the manufacture of black bread,
and in some countries also of white bread, so-called leaven is employed ;

ALCOHOLIC FERMENTS. 181

SACCHAROMYCES ANOMALUS. HANSEN.

(Figs. 31 and 47).

This very curious species was found by Hansen in an
impure brewery yeast from Bavaria. It gives a rapid and
vigorous fermentation in wort, and even at the beginning of
the fermentation develops a dull grey film. During fermen-
tation the liquid acquires an ethereal, fruity odour.

this is made by kneading together flour, bran, and water, and allowing
the mass to undergo spontaneous fermentation. It contains bacteria in
large numbers, and also yeast-like cells, and amongst the latter alcoholic
ferments. Very opposite views have, however, been expressed with
regard to the importance of these different organisms in the fermentation
of black bread.

According to Chicandart (1883) and Marcano the active ferment is a
bacterium. Boutroux attributed the fermentation to the activity of both
bacteria and budding fungi ; later he regarded alcoholic yeast as the
chief cause. Laurent regarded the so-called Bacillus panificans as the
main cause of the fermentation of bread. Dilnnenberyei-' s investigations
led to the conclusion that the budding fungi must be looked upon as the
only essential organisms of fermentation in bread. The rising of the
dough is accordingly caused in the first place by the carbonic acid
liberated by the alcoholic fermentation ; further by the expansion of
the air and the vapourisation of the alcohol, water, and volatile fatty
acids formed by the bacteria. Peters found four different budding fungi
in leaven, and one of these has been identified with Saccha?'omyces minor
Engel. The second is of about the same size as Saccharomyces minor ;
the cells are egg-shaped, and in nutrient liquids develop to moderately
large, many-branched colonies ; it yields spores abundantly. In addition
to the above, a species of Mycoderma and a species related to Saccharo-
myces cerevisice were also found. Peters describes several species of
bacteria occurring in leaven, but none of them has all the properties of
Laurent's Bacillus panificans; on the contrary, these properties were
found divided amongst various bacteria. Laurent, therefore, was pro-
bably dealing with impure cultivations. These bacteria gave no alcoholic
fermentation, and no appreciable evolution of gas in sterilised dough.

The above experiments constitute a good preliminary to the decisive
experiments on the cause and action of the rising of dough.

The diseases of black bread which have been investigated by
Ujfelmann, Kretschmer, and Niemitowicz, e.y., vigorous growth of
mould, sliminess caused by an exuberant growth of bacteria, may no
doubt be partly attributed to impure leaven, in which the most various
organisms will thrive.

182 MICRO-ORGANISMS AND FERMENTATION.

The cells grown in wort are small, oval, and sometimes
sausage-shaped, and in their microscopic appearance they
resemble the Torula species. When the development has
gone on for some time many of the cells both in the sediment
and in the film are found to contain spores.

The spores are developed on various substrata, both liquid
and solid, and also under conditions where abundant nutri-
ment is present. In an ordinary gypsum-block culture a
moderately abundant development of spores is obtained after
forty hours at 25° C.

The form of the spores is highly characteristic (Fig. 47) ;
it resembles a hemisphere with a projecting rim round the
base. On germination the spores swell and develop buds
(see Fig. 31).

FIG. 47.

Spores of Saccharomyces anomalus. after Hansen. Some spores are free,
others enclosed in the mother-cells. At the bottom, on the right-hand
side, are three spores, surrounded by the burst wall of the mother-cell.

After Hansen had drawn attention to the above curious
Sacckaromyces species, it, and probably other allied species,
were also observed by Holm, Lindner, and Will, who likewise
found it in impure brewery yeast. Yeasts yielding hat-shaped
spores appear in fact to be by no means uncommon.

As was previously mentioned, the spores of this fungus
resemble those of Endomyces decipiens, and a relationship
perhaps exists between this Saccharomyces and the fungus
named. As yet, however, no proof has been forthcoming
in support of this.

SACCHAROMYCES CONGLOMERATUS. KEESS.

This species is described by Reess as follows : — " Eound
budding cells, of 5 to 6 p diameter, united in clusters, which

ALCOHOLIC FEKMEtfTS. 183

are formed from two old cells which, before budding in the
direction of their common longitudinal axis, usually simul-
taneously throw out several buds as branches. The asci very
frequently remain united in pairs, or each united to a vegeta-
tive cell. Spores 2 to 4, which on germination again
give rise to clusters. Occurs on decaying grapes and in
wine-yeast at the commencement of fermentation. Fermen-
tative action doubtful."

In Hansen' s cultures of film-formations of the Saccharo-
mycetes, colonies of the above-mentioned appearance were
found in old films of the six species first investigated by
hi-m. And since Hansen never found a definite species
among his cultivations which could be identified as Reess's
Saccharomyces conglomeratus, he is inclined to assume
that the cell-colonies mentioned of the different Saccharo-
mycetes are identical with this species.

The different races or species of yeast may be divided
into two groups, according to the kind of fermentation to
which they give rise, namely : — bottom-yeasts and top-yeasts.
In spite of many assertions to the contrary, it has not hitherto
been possible to bring about an actual conversion of top-yeast
into bottom-yeast, or vice versa. The investigations of Hansen
and Kuhle show that it is certainly possible for a bottom-
fermentation yeast to produce transitory top-fermentation
phenomena ; these, however, quickly disappear with the pro-
gressive development of the yeast. When formerly it was
stated that by the continued cultivation of, e.g., bottom-yeast
at an elevated temperature, this could be converted into top-
yeast, these old experiments can only be explained on the
assumption that the bottom-yeast employed was impure and
contained an admixture of top-yeast, which at the elevated
temperature gradually developed at the expense of the
bottom-yeast, until it finally constituted the chief portion
of the yeast.

184

MICRO-ORGANISMS AND FERMENTATION.

As examples of two different bottom-fermentation species
of yeast, the Carlsberg bottom-yeasts " No. 1 " (Fig. 48) and
" No. 2 " (Fig. 49) employed in the Old Carlsberg brewery at

FIG. 48.
Carlsberg bottom-yeast No. 1, after Hansen.

Copenhagen, may be more minutely described. Distinct
differences are noticeable even in the ordinary microscopic
examination.

The cells of the species No. 1 (Fig. 48) are mostly some-
what elongated, but there are also smaller characteristic

FIG. 49.
Carlsberg bottom-yeast No. 2, some cells with spores, after Hansen.

pointed cells. When the yeast taken from the fermen ting-
vessel is washed with water and placed for a short time under
ice, the contents of all the cells will quickly assume a granular
appearance, and if the yeast is kept in this manner for
several days the number of dead cells will very rapidly increase.
The cells of the species No. 2 are, under normal conditions,
roundish oval, some being almost spherical. Here and there
giant-cells occur (left-hand side of figure). In a yeast-mass

ALCOHOLIC FERMENTS. 185

washed with water the cell-contents long remain clear, and
only slightly granular, and if the yeast be kept for a long
time in this way only very few dead cells will be found.

The gelatine cultures of both species form colonies, having
the ordinary appearance of the Saccharomycetes.

On gypsum blocks the species No. 2 develops spores much
more quickly and abundantly than No. 1 species.

The fermentation phenomena also differ. No. 2 gives
thick, high foam and a dense, firm layer on the surface ; No. 1
gives a low foam and the liquor is often exposed in places.
No. 2 clarifies comparatively quickly ; No. 1 clarifies slowly.
No. 2 forms a firm layer at the bottom of the fermenting-
vessel, whilst No. 1 gives a fluid sediment. In the primary
and secondary fermentations No. 2 gives a more feeble
fermentation than No. 1.

The finished beer obtained with the two yeasts in the
same brewery shows marked differences. With regard to
taste, the beer obtained with No. 2 yeast is preferred by most;
but this is a matter of opinion ; at all events the taste is
different in the two cases. Finally the two species give very
different results as to the stability of the beers with regard
to yeast turbidity. The beer prepared with the No. 1 yeast
is decidedly more stable in this respect than that prepared
with No. 2 yeast. Consequently No. 1 is especially suited
for lager and export beers, and No. 2 for running beers.
These characteristics have always been found to remain
unchanged for years.

From the above description of the microscopic relation-
ships of these two types of bottom-yeast it must on no
account be assumed that we are able, by means of a micro-
scopic examination of an unknown species of yeast, to
determine whether it will give a high or a low attenuation, or
whether it will clarify slowly or quickly, etc., etc. Hansen's
investigations have, on the contrary, proved that it is
impossible to establish any general rule by this means, since
species which give a high attenuation may have the same

186 MICRO-ORGANISMS AND FERMENTATION.

microscopic appearance as species which give a low attenua-
tion. It will only be possible to form an opinion in this
direction when our knowledge of the structure of the plasma
is much more advanced. Statements to the contrary which
have hitherto appeared in the literature of the subject are
simply erroneous assertions.

A preliminary grouping for practical purposes of the
different species or races of bottom- and top-fermentation
beer-yeasts which have been prepared in pure culture by
Hanserfs method in my laboratory, is as follows : —

A. — BOTTOM-FERMENTATION SPECIES.

1. Species which clarify very quickly and give a feeble

fermentation in the fermenting-vessel ; the beer holds
a strong head. The beer, if kept long, is liable to
yeast -turbidity. Such yeasts are only suitable for
draught-beer.

2. Species which clarify fairly quickly and do not give a

vigorous fermentation ; the beer holds a strong head ;
high foam ; the yeast settles to a firm layer in the
fermenting vessel. The beer is not particularly stable
as regards yeast-turbidity. These yeasts are suitable
for draught-beer and partly for lager beer.

3. Species which clarify slowly and attenuate more

strongly ; the beer has a good taste and odour ;
the yeast deposit is less firm in the fermenting
vessel. The beer is very stable against yeast-
turbidity. These yeasts are suitable for lager beer,
and especially for export beers which are not pas-
teurised or treated with antiseptics.

B. — TOP-FERMENTATION SPECIES.

1. Species which attenuate slightly and clarify quickly.

The beer has a sweet taste.

2. Species which attenuate strongly and clarify quickly.

Taste of beer more pronounced.

ALCOHOLIC FERMENTS. 187

3. Species which attenuate strongly, clarify slowly, and
give a normal after-fermentation. The beer is
stable against yeast-turbidity.

As a very significant result of practical experience, and
one which shows how pronounced are the characters of many
species of cultivated yeast, the fact may here be mentioned
that generally speaking the above grouping holds good, even
under the different practical conditions obtaining in widely
separated countries. For instance, the Carlsberg yeast No. 1
gives everywhere a beer which is very stable as regards yeast -
turbidity ; other species, which clarify more rapidly, have
been found to retain this property everywhere under normal
brewery-conditions.

An example of the permanence of the specific properties
under very different external conditions has also been given
by Irmisch in a comparative examination of two bottom-
yeasts. One of the species gave a low attenuation and
multiplied to a very small extent in the wort, whilst
the other, on the contrary, gave a high attenuation and
possessed the power of multiplication in a high degree ; the
course of the fermentation in the two cases also showed
marked differences. These differences still obtain on varying
the concentration of the wort or the quantity of the yeast, at
very different temperatures, also when cultures are employed
which have been grown in wort containing diastase, under
various conditions of aeration with ordinary wort, and with
a specially prepared wort very poor in maltose, in the
presence of grains during fermentation, and in solutions of
cane-sugar. Likewise in fermentations which were carried
on for six months, an examination of the product showed
that the typical differences of the two species had not
disappeared.

Besides the beer top-yeasts, there are also certain high-
fermentation species which are employed in distilleries and
in yeast factories. In recent years a number of distillery
yeasts have been prepared in the author's laboratory in

188 MICRO-ORGANISMS AND FERMENTATION.

pure culture. They exhibited marked differences in their
sedimentary forms and in ascospore-formation. The species
which were introduced into practice also differed in this
respect. Delbruck, P. Lindner, and Stenglein have also had
the same experience.

Belohoubek, Schumacher and Wiesner, have carried out
microscopical and chemical investigations of yeasts of the kind
last mentioned, and Belohoubek's " Studien iiber Presshefe "
(Prague, 1876) especially contains accurate descriptions of
the appearance under the microscope of ordinary pressed
yeast in the different stages of its development, and observa-
tions on the microscopic indications of the quality of the
manufactured yeast, so far as can be judged from the contents
of the cells. The decomposing yeast cells show a change in
the colour and consistence of their plasma ; this gradually
becomes darker and liquid, the vacuoles become larger, and
the sharp outlines between the vacuoles and the plasma
gradually disappear, the plasma shrinks from the cell-wall
and finally collects in irregular masses in the cell-fluid ;
these also disappear at last, and finally the cell- wall is
dissolved. According to the above authors, there also occur
in pressed-yeast, cells which suddenly develop a number of
small vacuoles ; these " abnormal vacuolar " cells quickly
perish.

OTHER BUDDING-FUNGI.

(Torula, Saccharomyces apiculatus, Mycoderma

cerevisice and vini.)

In the following pages we give a review of some other
fungi, which are of more or less importance in the fermen-
tation industries, and which resemble the Saccharomycetes
in that they multiply by budding ; these species develop a
mycelium only exceptionally. On the other hand they are
all distinguished from the Saccharomycetes by the absence of
the property of forming endogenous spores which characterises
the latter.

ALCOHOLIC FERMENTS. 189

The forms examined by Hansen, and which produce a
mycelium, must strictly be classed with the mould-fungi.
Since, however, their position amongst the moulds has not
yet been systematically determined, these species may, on
practical grounds, be described in this place.

TORTJLA.

The yeast-like forms which Pasteur figured and described
under the name Torula, are widely distributed and there-
fore not unfrequently occur in physiological analyses connected
with fermentation. They occur in both spherical and more
or less elongated forms, and are distinguished from the genus
Saccharomyces, as was first pointed out by Hansen, in that
they are unable to form endogenous spores. In most cases
they multiply only by budding, in some few cases also by the
formation of mycelium.

Hansen has observed many different species, and has
described the following more in detail : —

The first occurs in wort, the cells being either single or in
colonies of a few cells. Some cells have a large vacuole in
the middle, and this sometimes contains a small strongly-
refractive particle. The size of the cells varies considerably
(1*5 to 4-5 /*). The species does not secrete invertase, and
causes a scarcely perceptible alcoholic fermentation in beer-
wort.

The second species has, under the same conditions, larger
cells (3 to 8 /x) than the first ; they resemble the foregoing,
except that the contents of the cells grown in wort are often
very granular.

The third species which, under the microscope, resembles
the last, produces under the same conditions as much as 0-88
per cent, by volume of alcohol ; it gives a distinct head with
evolution of carbonic acid, but it cannot invert cane-sugar.

The fourth species (2 to 6/*) inverts cane-sugar and
produces a little more than 1 per cent, by volume of alcohol

190

MICRO-ORGANISMS AND FERMENTATION.

in wort with considerable frothing ; it does not, however,
ferment maltose.

The fifth species, which in the form and size of its cells
resembles the first, develops a uniform, dull grey film on wort

FIG. 50.

Torula, after Hansen : sedimentary forms after one day's cultivation in
beer-wort at 25° C.

and yeast-water at the ordinary room temperature, likewise
on lager beer and even on liquids containing as much as 10
per cent, of alcohol. It inverts cane-sugar and forms a slight
film on the solution. It does not, however, excite any
appreciable alcoholic fermentation.

FIG. 51.

Torula, after Hansen : sedimentary forms after one day's growth in beer-
wort at 25° C.

A sixth species (Fig. 50), which forms spherical and oval
cells, gives a distinct fermentation in beer-wort, yielding as
rriuch as 1-3 per cent, by volume of alcohol. It does not
ferment maltose solutions. It inverts cane-sugar and in 10
per cent, and 15 per cent, solutions of this sugar in yeast-
water it yields respectively 5*1 and 6'2 per cent, (volume) of
alcohol after fourteen days' cultivation at 25° C. ; the last
growth yielded 7 per cent, (volume) of alcohol after two

ALCOHOLIC FERMENTS.

191

months. Dextrose solutions of the above concentration and
under similar conditions gave 6*6 and 8*5 per cent, of alcohol
by volume.

The seventh species (Figs. 51 and 52) was found in the soil
under grape-vines. The sedimentary cells are most frequently
oval and in part larger than those of the last species. The
cells of the films are partly very irregular in form. This
Torula produces only 1 per cent, (volume) of alcohol in
wort, does not ferment maltose, and neither ferments nor
inverts cane-sugar. In 10 per cent, and 15 per cent, solu-
tions of dextrose in yeast-water it gives 4*6 and 4'5 per

FIG. 52.

Torula, after Hansen : same species as Fig. 51. Film-formation on a wort
culture ten months old.

cent, by volume of alcohol after 15 days at 25° C., and
4-8 and 4*7 per cent, after 28 days. In two other flasks
4*8 and 5-3 per cent, of alcohol had been produced after
long standing. Hansen assumes that this species takes
part in vinous fermentation, and considers it probable
that species such as the sixth and seventh, which produce
a vigorous fermentation in dextrose solutions, take part in
the fermentation of grape-juice and other fruits. On the
other hand they have probably little importance in breweries
and distilleries since they are unable to ferment maltose.
Another species of Torula (Torula Novae Carlsbergice),

192 MICRO-ORGANISMS AND FERMENTATION.

the cells of which exhibit very different forms, has been
described by Gronlund. It imparts a disagreeable bitter
taste to wort. According to Schjerning's investigations it
inverts cane-sugar, and induces alcoholic fermentation in
solutions of cane-sugar, dextrose, and maltose. In ordinary
brewery-wort it can produce about 4*7 per cent, (volume) of
alcohol.

Torula species which contain no invertase, yield only
about 1 per cent, (volume) of alcohol and do not ferment
maltose, are found widely distributed in nature. Those
which have been examined ferment solutions of dextrose.

Eelated to the above are the red-coloured budding-fungi
(the pink yeast of medicinal bacteriology) universally dis-
tributed in atmospheric dust ; several species of these are
known ; Kramer, for instance, found in must a top-fermen-
tation torula-yeast which produces a red colouring-matter
soluble in water. It ferments dextrose and in a 10 per
cent, solution it yields 4-5 per cent, by volume of alcohol ;
it inverts cane-sugar, directly ferments maltose, but has no
action on lactose.

These different species cannot be distinguished by the
microscope alone either from each other or from the round
cells of the Saccharomycetes. Pasteur distinguished the
Torula-forms from the other yeasts, because the species
which he examined excited only a very feeble alcoholic
fermentation. It will be seen, however, from the above
that there are also species with pronounced fermentative
activity.

Hansen assumes with some degree of probability that
they are derived from the higher fungi, and in his cultiva-
tion experiments he has, as mentioned above, in a few cases
observed the development of a mycelium,.

Duclaux found a yeast-fungus in milk \vhich . induces
alcoholic fermentation in a solution of lactose. A con-
version of lactose into galactose was not observed. This
fungus appears to be most closely related to the Torula

ALCOHOLIC FERMENTS. 193

species. The cells are 1'5 to 2'5 p in diameter, and almost
spherical. According to Duclaux's experiments, this yeast
is more aerobic than the ordinary alcoholic yeasts. Even
with strong aeration of the liquid, the whole of the milk-
sugar is used up in the alcoholic fermentation. In a 5
per cent, solution of milk-sugar 2*5 per cent, of alcohol
was formed in eleven days at 25° C. The most favourable
temperature for the fermentation of a neutral solution is 25°
to 32° C., whilst at 37° to 40° C. the fermentation ceases.
Small quantities of acid have a retarding influence on
the fermentative activity of this yeast.

Adametz likewise describes a budding-fungus which
ferments milk-sugar. Since this fungus does not yield
endogenous spores by Hansen's method, it is classed in the
group of non-Saccharomycetes. The cells are of about the
same size as those of Saccharomyces cerevisice, and are
spherical and elliptical. The colonies grown on peptone-
gelatine are round with slightly jagged borders and are of
a dark brown colour. A puncture-cultivation in wort-
gelatine yields a dull, flat mass on the surface and a
vigorous growth in the punctured channel, and from this
numerous rays penetrate into the gelatine. In sterilised
milk, this fungus induces fermentation phenomena within
24 hours at 50° C., in 48 hours at 38° C., and in about
four days at 25° C. In this fermentation, the milk-sugar
is alone decomposed.

Both of the species mentioned above have been more
closely investigated by Kayser, together with a ne^v species
which likewise ferments lactose and also belongs to the
non-Saccharomycetes. All three yield colonies on gelatine,
which are more spread out than those of beer- and wine-
yeasts ; in the middle of the colonies there is a thicker
portion, whilst the border resembles mycelium. In milk
and in neutral liquids, when sufficiently aerated, they
induce an appreciable fermentation at 25° to 30° C. The
milk does not coagulate or become viscous during the

194 MICRO-ORGANISMS AND FERMENTATION.

alcoholic fermentation. All three species ferment lactose,
galactose, cane-sugar, glucose, invert-sugar, and finally
maltose, but the last only with great difficulty. In
the fermentation of milk-sugar with these yeasts, the
resulting liquids are as rich in alcohol as the strongest
beers. Kayser remarks that it may perhaps be possible
to make practical use of this observation and by means
of these fungi to convert the large quantities of whey,
obtained in the manufacture of cheese, into an alcoholic
liquor.

Beyerinck has described two yeasts which also ferment
milk-sugar, and which must be provisionally regarded as
non- Saccharomycetes ; these are Saccharomyces KepTiir,
which occurs in kephir-grains and consists of longish,
variously-formed cells, and forms slightly jagged colonies on
gelatine; and Saccharomyces Tyrocola, which consists of
small roundish cells, and forms snow-white colonies on
gelatine. Beyerinck found that these two species secrete a
particular invertive ferment (lactase) which inverts not only
cane-sugar but also milk-sugar ; it does not, however, invert
maltose. Lactase can be prepared as follows : — A five per cent,
solution of milk-sugar, containing also nutrient salts and
asparagine, is fermented with kephir-yeast ; the product is
filtered and the ferment is precipitated from the filtrate by
the addition of alcohol. According to Schuurmans Stekhoven,
however, the enzyme of Beyerinctis kephir-yeast does not
invert milk-sugar.

It was previously proved by Bourquelot that Aspergillus
niger contains a soluble chemical ferment which has some
similarity to the invertase of beer-yeast, but is distinguished
from the latter by its property of converting maltose into
dextrose. He points out, however, that two ferments may
possibly be present, just as diastase is now regarded as a
mixture of several ferments. A doubt of this nature will
always arise when a soluble chemical ferment is found to
vary in its action.

ALCOHOLIC FERMENTS. 195

SACCHAROMYCES APICULATUS. KEESS.
(Fig. 53.)

As already pointed out, the name of this ferment is
incorrect according to our present views, for only those
budding-fungi which yield endogenous spores are considered
to belong to the Saccharomycetes, and the fungus in question
does not possess this property. We will, however, provisionally
retain the old generic name, as has been done by Hansen,
until the systematic classification has been further developed.

As is known, this ferment was the subject of one of the
finest and most thorough biological investigations of our
time, for Hansen was enabled, after several years' work, to
determine both its habitat in nature and its regular migra-
tions at the different seasons of the year. The reason that
this species was selected for the investigation was that, whilst
other species occur in very varied and uncertain forms, which
makes the study of their occurrence in different localities
very difficult, this ferment can be recognised with certainty
by its form, since it always occurs in cultures with lemon-
shaped cells ; this is the typical form of the species.

Sacch. apiculatus occurs abundantly in wine-yeast,
especially during the first stages of the fermentation, also in
Belgian spontaneously-fermented beer ; in nature it is found
abundantly on ripe, sweet, succulent fruits.

If a little of such a growth in a drop of nutritive liquid
is examined under the microscope, the development of the
fungus can be followed. This is, as was first pointed out by
Hansen, very characteristic (compare Fig. 53). It is seen
that the buds formed from the typical lemon-shaped cells
may be either lemon-shaped (a, 6, c, e, /) or oval (a — c) ;
it is also noticed that the oval cells must first form one or
more buds before they are able to assume the lemon-shape
(e — -/), and finally, that the lemon-shape of a cell attained
by budding (&, k', k") may be lost again on the formation
of a new bud (&'"). Under other conditions the cells can

196 MICRO-ORGANISMS AND FERMENTATION.

F* r '

4 »*•

I '* i 1 'i

j?

FJO. 53.

Saccliaromyces apiculatus, after Hansen. Budding cells : a — a", a cell
which in the course of 3£ hours developed a bud at its lower extremity ;
b — b", a similar series, showing the development of a bud at the upper
extremity of the mother cell, whilst a bud had been previously formed
at the opposite end ; c is a chain of cells, c' is the same three-quarters of
an hour later ; the lowest bud had, like those above it, assumed the typical
form of the species, but in the figure it is seen from the end, so that
its longitudinal axis is at right angles to the plane of the paper ; d — d",
development during 1| hours ; «—«'", during 2£ hours ; /—/"", during
3 hours ; in e—f, it is seen that the oval cells first develop a bud and
only subsequently assume the typical lemon-shape ; g — m, abnormal cells
and series of development.

ALCOHOLIC FERMENTS. 197

assume quite different forms, sausage-shaped, half-moon-
shaped, bacteria-like, &c. (g — m). Is there now any rule
in this apparent confusion of forms ? It was shown above
that the fungus can form two kinds of buds, and that the
oval buds must develop one or more new buds before they can
assume the typical form. The question then is : Under what
conditions are those two kinds of buds developed ? It was
shown by means of culture experiments that the lemon-
shaped buds are developed especially during the first stages
of the culture, but are afterwards crowded out by the oval
forms.

We will now give a further description of the fungus
from a physiological and biological standpoint.

Saccharomyces apiculatus is a bottom-fermentation
yeast, which is capable of exciting alcoholic fermentation
in beer-wort ; the fermentation in this liquid is, however, a
feeble one, only 1 per cent, (volume) of alcohol being
produced, whilst Saccharomyces cerevisice (bottom-yeast)
under the same conditions gives 6 per cent, by volume of
alcohol. This arises from the fact that Sacch. apiculatus
cannot ferment maltose. Hansen also found that it does not
secrete invertase. On the other hand it excites a vigorous
fermentation in 15 per cent, and 10 per cent, solutions of
dextrose in yeast water, and in one experiment as much as
3 per cent, (volume) of alcohol was formed. After three
months the liquid still contained sugar, whilst the amount
of alcohol had not increased during the last one and a half
months. The fungus was thus unable to complete the
fermentation. In another of Hansen's experiments as much
as 4'3 per cent, (volume) of alcohol was produced.

It was found from experiments, in which a mixture of
this .fungus with Saccharomyces cerevisice was grown in
beer-wort, that, being the weaker species, it was crowded
out by the latter, although it retarded the Sacch. cerevisice
to no small degree.

In flasks with the same beer-wort, and at the same tern-

198 MICRO-ORGANISMS AND FERMENTATION.

perature, and each containing one species, Saccharomyces
apiculatus will multiply to a greater extent than Sac-
charomyces cerevisice in a given interval of time.

At the critical time of the year, the ferment, if
present in the wort in considerable quantities, may exist
for a length of time, side by side with Saccharomyces
cerevisice, and will no doubt retard its action a little; but
when the beer is transferred to the lager cellar, the fungus
remains inactive in the alcoholic liquid and frequently
perishes.

The most interesting phases in the life of this ferment
are the conditions of its occurrence in nature, which have
also been explained by Hansen. Microscopic investigations
and culture-experiments showed that in the summer the
ferment was abundantly developed on sweet, succulent
fruits (cherries, gooseberries, strawberries, grapes, plums,
&c.) during their ripening. On the contrary it was only
very exceptionally found on the same fruits so long as
these were unripe. Since it is found in a vigorous condi-
tion of budding on the above-mentioned ripe fruits, but is
never or only exceptionally found on other fruits, leaves,
twigs, &c,, it is perfectly clear that Saccharomyces apicu-
latus has its true habitat on the ripe fruits named. This
was also further proved by the fact that it always, without
exception, occurs in the soil under the cherry trees, plum
trees, vines, and other plants on the fruits of which it is
found; whilst, on the other hand, it was only extremely
seldom that it was found in the numerous samples of
soil from other and most diverse localities. The ferment
is brought on to and into the earth at such places by
the fallen fruit and by the rain, and the question then
arises does it also winter there ? The answer was obtained
in two ways : partly by taking numerous samples of soil
from these places during winter and spring — these, when
introduced into flasks containing wort, gave in by far the
greater number of cases a vigorous growth of our ferment

ALCOHOLIC FERMENTS. 199

—partly by introducing, with every precaution, cultures of
Saccharomyces apiculatus into the soil and leaving them
during the winter. In the spring and early summer the
soil was again examined, and culture experiments proved
that the ferment was still alive in all the samples. Thus,
it was proved that the ferment can winter in the soil, and
it was also previously shown that it practically only occurs
at the stated places in the soil. In some more recent ex-
periments of Hans en's , vigorous growths of the ferment in
well-closed Chamber-land filter-tubes were placed below the
surface of the earth. After three years, the contents of the
tubes were introduced into sterilised wort, and a vigorous
development of the ferment was obtained. The period of its
life can thus extend beyond a year.

Finally, it remained to be proved that the soil is its true
habitat during the winter ; in order to prove this, Hansen
examined the dust from the most diverse places from January
to June, also the dried fallen fruits of many plants, and finally
also various excrements. The seventy-one analyses gave a
negative result, and thus furnished the proof that the true

O JT

vsinter habitat of the ferment is the soil under the previously
mentioned plants. It retains its ordinary appearance during
the long winter-time, and in the summer it is again carried
into the air by the united action of insects and the wind, and,
through these two means of transport, it becomes further
distributed from fruit to fruit.

It is evident that at the time when the ferment appears
in abundance on the ripe fruits mentioned, it may also be
carried by the wind to other places, and thus also on to
unripe fruits. Even in his first memoir, Hansen stated that
the reason of the rare occurrence on unripe fruits might be
that the ferment quickly perishes, partly from want of
nourishment and partly from the drying up of its cells.
Subsequently he proved by experiment the correctness of this
view. He stirred up with water partly old and partly young
cells, and placed them in thin layers, either on object-glasses

200 MICRO-ORGANISMS AND FERMENTATION.

or on thin pulled-out tufts of cotton-wool, which were then
allowed to dry, protected from the sun. In less than twenty-
four hours all the cells had perished. It is self-evident that
the isolated cells lying on the unripe fruits are still more un-
favourably placed than in the experiment. If, however, thick
layers of the cells are wrapped in cotton-wool or filter-paper,
they will continue to live for a long time, as they do in the
earth — in filter-paper, for instance, over eight months.

No complete investigations have been published on the
life-history of other alcoholic ferments. Saccharomycetes
occur very generally on fruits containing a sweet juice.
For several years Hansen has carried on experiments similar
to those described, with species of Saccharomycetes which
often occur in fruit gardens, namely Sacch. Pastorianus /.,
Sacch. ellipsoideus /., also with Carlsberg bottom-yeast No. 1,
and with some top -fermentation beer-yeasts. He always
found that the yeasts sown in the soil in September were
still alive after a year. Some species had formed spores at
the surface of the soil. Further experiments will probably
show that the true Saccharomycetes also have their habitat on
fruits during the summer and in the soil during the winter.

In antithesis to these direct observations of Hansen, is
Pasteur's statement that the wine-yeasts are unable to live
in the soil during the period from one season to the next.
Where the yeasts come from which are found on the grapes
at the time of ripening, Pasteur was unable to say.

MYCODERMA CEREVISLE AND VINI.

It is characteristic of these species that they very readily
form films on various alcoholic liquids. Under the above
names are included a number of different species, some of
which can excite a feeble alcoholic fermentation ; they
behave differently towards lager beer, some causing disease
whilst others do not.

The Mycoderma cerevisice (Fig. 54) examined by Hansen,
and which is very generally met with in the Copenhagen

ALCOHOLIC FERMENTS.

201

breweries, forms variously-shaped cells. The cells are usually
transparent and less refractive than the true Saccharomycetes ;
in each cell there are generally one, two, or three highly
refractive particles, which often have a quivering, rolling
motion. This micro-organism forms a dull, greyish, wrinkled
film on wort and beer, and does not excite alcoholic fermen-
tation ; neither does it invert solutions of cane-sugar.

The colonies on the surface of the gelatine are bright,
grey, dull, and spread out like a film or hollowed like a shell.
By means of this macroscopic appearance Mycoderma is
readily distinguished from the ordinary Saccharomycetes

FIG. 54.

Mycoderma cerevisise from Copenhagen breweries. Drawn from nature

by Holm.

which, on the same medium, form bright greyish-yellow
colonies with a dry or lustrous surface, and of a more or less
arched form. Sacch. membrancefaciens (page 177), which
differs so markedly in its biological behaviour, and which very
rapidly gives a strong film on the liquid, alone resembles
Mycoderma also in its behaviour on plate cultures.

The form of film described above was obtained by Hansen
when lager beer was exposed in open vessels at temperatures
between 2° and 15° C. ; at 33° C. a development still
occurred, but at temperatures above 15° C. this species gave
place more and more to competing forms. Therefore, since
low temperatures are favourable to its development, it will

202 MICRO-ORGANISMS AND FERMENTATION.

readily thrive in the storage cellar, especially as lager beer
forms a much more favourable medium for its growth than

O

wort. This is seen to be the case when traces of a pure film
are introduced into lager beer and wort contained in open
vessels and left to develop; the culture in the lager beer
nearly always remained pure, whilst in the wort various other
species made their appearance.

In Hansels comprehensive series of experiments on
Carlsberg beer, it was always found that both lager and export
beers were attacked by this fungus ; but there was never the
smallest sign that the beer had acquired any disease from this
source. The fungus was widely distributed just at those
periods when the beer was found to be particularly stable and
of good flavour. This has also been confirmed by numerous
experiments on lager and export beers conducted by Gronlund
and A. Petersen, and those carried out in the laboratory of
the author. It is self-evident that we are here only speaking
of beer which has been properly treated. In imperfectly
closed bottles and casks Mycoderma cerevisice will of course
rapidly develop a film, which alone is sufficient to destroy the
product.

Belohoubek was the first who found that under certain
conditions Mycoderma can cause considerable injury in the
brewery. Subsequently Kukla described a curious cloudiness
in lager beer, which had the appearance of a cloud of fine
dust in the liquid, and which manifested itself either during
storage or after tapping ; he attributes this disease to Myco-
derma, and he further assumes that it is the weak wort and
also a particular composition which specially favour the
development of Mycoderma. It is to be hoped that further
investigations will throw more light on this subject.

Hansen had previously expressed the opinion that the
name Mycoderma cerevisice denotes not one, but several
different species, and Lasche's experiments have confirmed
this. The latter investigator describes four different species
which he isolated from cloudy beers. They are distinguished

ALCOHOLIC FERMENTS. 203

from the species described by Hansen by the fact that they
produce alcohol in beer-wort ; one yields 0*26 per cent, by
volume, two yield 0-79 per cent., and the fourth produces
2*51 per cent. Lasche concludes from his experiments that
these four species cause diseases in beer, namely, turbidity
and changes in taste and odour ; in this respect they also
differ from Hanserfs Mycoderma. Lasche is inclined to
assume that the chemical composition of the wort has no
influence on the disease caused by Mycoderma, since, in his
experiments, the disease was produced in worts of high
extract and in worts of low extract, in worts which were rich
in sugar and in worts containing little sugar.

It is frequently stated that the chemical activity of certain
species of Mycoderma on the surface of vinous liquids is a
process of oxidation by which alcohol is converted in some
cases into carbonic acid and water, in others into acetic acid ;
fatty acids are also said to be formed, and these are oxidised
and ethereal salts produced (Schulz).

Winogradsky found that the Mycoderma occurring on
tvine, prepared in pure culture by Hanserts method, changes
its form with the composition of the nutritive solution ; he
experimented partly with solutions, the mineral constituents
of which remained constant whilst the organic substances
varied, and partly with solutions in which the reverse was
the case.

Although De Seynes, Reess, Engel, and Cienkowski
claimed to have found ascospores in Mycoderma, it has not
since been possible to bring about this formation. It would
appear from the figures given that the fat globules which
occur in many unicellular fungi during the resting stage had
been mistaken for spores ; in some cases the mistake appears
to have arisen through the presence of an admixture of true
Saccharomycetes. The old name Mycoderma is therefore
more appropriate to this fungus than the new term
Saccharomyces.

CHAPTEE VI.

The Application of the Results of Scientific
Research in Practice.

IT is universally acknowledged that the process of fer-
mentation plays a very important part in all branches of
the fermentation industries. The better insight which has
been gradually gained in this direction has been brought
about through the development of the science of the
organisms of fermentation. This gradual development may
be divided into three great periods.

The investigations of the first period all relate to the
important question, whether living organisms can come into
existence by spontaneous generation. The second period is
noted for Pasteur's classical researches. In the third period,
dating from 1879, and which was founded by Hansen, a
reform was for the first time carried out.

1. The first period (1745 to 1857) gave rise to the theory
of sterilisation and its foundation in practice (see
page 9).

Spallanzani's discoveries in connection with spontaneous
generation formed not only the starting-point of modern
bacteriology (compare p. 10), but they also acquired great
importance in practical life. In 1782 Scheele proved that
vinegar can be preserved unchanged after it has been heated,
and Appert (1810) likewise showed that beer, wine, and other
liquids can be preserved by similar treatment. It was further
shown that air can be purified by passing it through a
strongly-heated tube (Schwann) or through a cotton-wool

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 205

filter (Schroder and Dusch). From this, the result was also
arrived at that water can be purified by a similar process,
provided the filter is sufficiently dense.

2. The Pasteur period dates from 1857. The great
merit of this investigator was that he proved that bacteria
exert a disturbing influence in different fermentations, and
that they can produce diseases in liquids which are under-
going alcoholic fermentation.

It is therefore necessary to proceed in such a manner that
infection of this nature is avoided, and this is best attained
by preventing the access of impure air to the liquids. The
consequence of this doctrine — as regards the brewery — is the
abandonment of open coolers and refrigerators, the aeration
of the wort by air which has been previously sterilised, and
the purification of the air in the fermen ting-rooms.

The statements in Chapter VII. of Pasteur's " Etudes sur
la biere " (1876), regarding the importance of the oxidation of
the wort during cooling, are also of practical value. By means
of direct determinations of the amount of oxygen in the wort,
Pasteur showed that a certain quantity of oxygen, partly in
the free state, and partly combined in the wort, exerts an
influence on the course of the fermentation and on the
brightening, but that when the proportion of oxygen in the
wort exceeds certain limits it can act injuriously on the
character (force et arome) of the beer (page 377).

Although several investigators have undertaken elaborate
researches in this direction, it has not hitherto been possible
to establish any fixed rules for practical guidance. These
must be determined by trial experiments for each individual
case.

Scheele's and Appert's method for the treatment of vinegar,
wine, and beer, at elevated temperatures, was taken up by
Pasteur, and through his great authority obtained a wide
application (the so-called Pasteurising). Kecently milk has
also been treated in this manner, especially since Koch proved
that the tuberculosis bacillus is so widely distributed.

206 MICRO-ORGANISMS AND FERMENTATION.

The experiments on aeration described in " Etudes sur
la biere " gave rise to extensive series of investigations which
yielded valuable information on the fermentative and repro-
ductive power of yeast in the presence of varying amounts
of air. This relation plays an important part in the
distillery and in pressed-yeast works. No results, however,
have as yet been obtained, which can be directly applied in
practice.

The reason why the method proposed by Pasteur for the
purification of yeast has acquired no real importance for
practical purposes, has been already stated (page 24).

3. With Hansen's investigations on the alcoholic ferments
there began, as Aubry says, a new era in the history of the
fermentation industries. In the year 1883, Hansen demon-
strated that the universally dreaded yeast-turbidity, and like-
wise the disagreeable changes in taste and odour, in fact some
of the commonest and worst diseases of beer, are not caused
by bacteria, by the water, by the malt, by the particular
method of brewing, &c., as was then commonly believed,
but that these diseases must be attributed to the yeast itself ;
for in such cases the pit ching-y east contains, in addition to
the cultivated species, other Saccharomycetes, which act as
disease germs (Sacch. Pastorianus L and III., Sacch.
ellipsoideus //.). A basis was thus formed for the neiv
system.

He subsequently showed that the name Saccharomyces
cerevisice embraces many different — both bottom- and top-
fermentation — races or species, which communicate very
different characters to beer.

As the rational result of these scientific investigations he
completed the third link of his new system — his method for
the pure cidtivation of yeast. If it were possible to free the
impure yeast mass from wild yeasts as well as from bacteria
and mould-fungi, we should still not attain all we desire ; for
if the purified yeast contains several species of Saccharo-
myces cerevisice, we are still dealing with a mixture which

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 207

is just as uncertain as before the purification, and, in addi-
tion, the composition of such a yeast-mass is always liable
to change during fermentation. In fact, Hansen has shown
in recent investigations that cases occur in which two yeasts,
each of which by itself gives a faultless product, will when
mixed give rise to disease in the beer. He made these
experiments with the two species of Carlsberg bottom-yeast
No. 1 and No. 2 (see page 184) ; in one set of experiments
the pitching-yeast consisted chiefly of No. 1 with a small
admixture of No. 2, and in the other set, the reverse was
the case. It was found that in all cases the small quantity
of. the admixed yeast, whether No. 1 or No. 2, made the
beer less stable as regards yeast turbidity, than when the
chief constituent of the pitching-yeast was employed alone.
Thus the two cultivated yeasts under these conditions behaved
in such a manner as to produce effects similar to those brought
about by the wild yeasts (Sacch. Pastorianus III. and Sacch.
ellipsoideus II.). We can therefore only obtain true uni-
formity in working when a suitable species has been
obtained from the yeast-mass by systematic selection, and
cultivated by itself (compare pages 26-32).

The systematic studies which Hansen has carried on for
many years on the constancy of the characters of different
species of yeast, have proved that, under the conditions of
the brewery, they only undergo slight changes, which are
of no importance in practice, and this result has been con-
firmed by various investigators.

On the other hand, he found that, when the conditions
of the life of the yeast are disturbed by a systematic and
more vigorous treatment, it was possible to produce
varieties (see page 151), which remained more or less
constant in their properties, and even to produce new
species. As a result of these investigations Hansen obtained
useful varieties of some brewery yeasts.

The biological and physiological characteristics of the
species discovered by Hansen led him also to a method

208 MICRO-ORGANISMS AND FERMENTATION.

for the practical analysis of breiuery yeast (page 134), by
means of which it is possible to ensure in time against
foreign yeasts prevailing. It was previously proved by his
experiments on a large scale that the forms which produce
yeast turbidity may be present to the extent of one part
in forty-one of the pitching-yeast, and the species (Sacch.
Pastorianus I.) which produces a disagreeable odour and
an objectionable bitter taste to the extent of one part in
twenty-two, without exercising any injurious influence, pro-
vided the brewing operations are conducted under normal
conditions. It has been found, however (by the experi-
ments of Holm and Poulsen\ that it is possible, by Hanserts
analytical method, to detect with certainty the presence of
1 -200th part of wild yeast.

From numerous analyses carried out by this method,
it has been shown that the rules which were formerly
generally accepted for judging a sound fermentation do
not suffice for determining the presence of disease-germs,
since both the head of the liquid, and the attenuation,
breaking, and brightening may be satisfactory in spite of
the yeast being strongly contaminated with disease-germs.

The question — how long will a pure culture remain in its
original good state ? — can evidently not be answered in a
general way. Hansen found that different races differ in
their power of resisting infection; likewise the length of
time during which a yeast will remain pure and good will
vary for the same species in dissimilar fermenting rooms.
We also know that the season plays an important part, and
that the time of year when wild yeasts, bacteria, and
moulds are most abundant in the air, is especially dangerous.
Infection is also known to occur at other times of the year,
especially from utensils, &c. ; disease-germs often gain admis-
sion to the brewery through the open coolers ; cask sediments
form another source of contamination. Most frequently,
however, brewers introduce disease-germs into the fermenting
vessels with the pitching-yeast which they obtain from other

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 209

breweries. The analysis will, however, always indicate infec-
tion long before it has become dangerous, so that a new, pure
cultivation of the same yeast can be introduced in good time.
A still greater certainty is attained by the continuous working
of the yeast-propagating apparatus described below. The
main result achieved is that we now no longer proceed
flap-hazard, and are not compelled to leave ike fermentations
to take their chance, as was formerly the case.

Since the various species of yeast differ in their power
of resisting competing disease-germs, it is in many cases of
great importance to be able, at short intervals, to introduce
into, the brewery a considerable quantity of a suitable and
absolutely pure species of yeast, which has been previously
selected by systematic experiments. This object is attained
by means of the yeast-propagating apparatus devised by
Hansen and Kiihle, and which, when once charged with an
absolutely pure cultivation, will work continuously for years.
The apparatus (Fig. 55) is described in detail with directions
for use in Hanserts " Untersuchungen aus der Praxis der
Gfarungsindustrie " x ; it consists of three principal parts with
the connecting tubes, namely: — 1, the arrangement for
aerating the wort, consisting of the air-pump (A) and air-
vessel (B) ; "2, the fermenting-cylinder ((7), and 3, the wort-
cylinder (D).

The air, which is partially purified by previous filtration,
is pumped into the air-vessel, from which it can be passed to
the wort-cylinder or to the fermenting-cylinder. In both
cases it has to pass through sterilised cotton-wool filters
(g, m). The wort-cylinder is connected by piping directly
with the copper, from which the hopped wort is run boiling
hot into it ; it is then aerated in the closed cylinder and cooled
bv water from p. The wort is then forced into tine fermenting-
cylinder, which, like the wort cylinder, is constructed on the

1 Published by R. Oldenbourg, Munich: 1st part, 2nd edition, 1890;
2nd part, 1892. In this work will also be found a description of Hansen 's
complete system.

210

MICRO-ORGANISMS AND FERMENTATION.

same principle as the ordinary two-necked flask. It is fitted
with a doubly-bent tube (c, d), which dips into a vessel con-
taining water, a vertical glass tube (/, -£,/) for measuring the
height of the liquid in the cylinder, an appliance (6, 6) for

FIG. 55.

Yeast-propagating apparatus devised by Hansen and Kuhle : A, air-pump ;
B, air-vessel ; O, ferment ing -cylinder ; a, window ; b b b, stirrer ; o c,
doubly-bent tube ; d, vessel containing water ; I, cock for drawing off
the beer and yeast ; / /, glass tube connected at e and h with the
cylinder, and graduated for the measurement of fixed quantities of
liquid ; g, filter ; i, india-rubber tube placed at the middle of the glass
tube ; j, tube with rubber connection for introducing the pure culture ;
k k, connection with the wort- cylinder D ; in, filter ; n n, doubly-bent
tube ; o, vessel containing water ; p p, cold water tube ; u, tube with
cock («) for introducing wort ; t, outlet-tube for the water used in
cooling ; q, cock (the wort is allowed to rise to this height) ; r, cock
for drawing off wort.

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 211

stirring up the settled yeast, and a cock (I) for drawing off
the beer and the yeast. At about the middle of the cylinder
there is a small side-tube (j) fitted with india-rubber con-
nection, pinch-cock, and glass-stopper. When a portion of
the wort has been forced into the fermen ting-vessel, the pure
yeast — which is forwarded to the brewery in a flask specially
constructed for this purpose— is introduced through the rubber
tube at^'; this is again closed, and the remainder of the wort
may then be added either at once or after the lapse of a few
days, according to the quantity of yeast which has been
introduced.

Where it is necessary to regulate the temperature during
fermentation, the fermenting-vessel is surrounded by a copper
water-jacket.

By means of this simple apparatus it is possible to obtain,
at short intervals, absolutely pure pitching-yeast sufficient for
about eight hectoliters of wort. As already stated, the
apparatus, when once started, works continuously. For
further details I refer the reader to the exact description in
Hansen's work mentioned above.

A modification of the propagating apparatus has been
devised by Bergh and Jorgensen (Fig. 56). The filtered air
passes through the three-way cocks at A, B, and (7, into the
two cylinders A and B. The upper cylinder holds about 50
liters, and the lower cylinder 160 liters. A is provided with
a stirrer (E), a tube (a) for introducing the yeast and with-
drawing samples. The bent tube F is for the exit of the
carbonic acid. The tube G P connects the two cylinders, and
the connection can be made or unmade by means of the cock
G. H is the outlet for the water used in cleaning A.

The cylinder B is surrounded by a cast-iron jacket made
in two parts ; the upper portion serves as a water-jacket for
cooling the wort and for regulating the fermentation ; the
lower portion is used as a steam-jacket, and is provided with
a cock at 0 for the entrance of the steam, and another at 8
for the outlet. M is a ring-shaped tube provided with small

212

MICRO-ORGANISMS AND FERMENTATION.

holes ; this is connected with the cold-water main during the
cooling of the wort ; the water flows out at N. The stirrer
J is set in motion by means of toothed wheels. The height
of the liquid in the cylinder is indicated by means of a float,

FIG. 56.
Yeast-propagating apparatus devised by Bergh and Jorgensen.

connected with which is a pointer and arc L. Connected
with the top of the cylinder is the bent tube K. At the
bottom is the cock Q, which is in connection with the pipe b.
Both the bent tubes dip into the vessel R, which is filled with
water.

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 213

The wort is introduced into the lower cylinder, where it
is treated in the ordinary manner. The pure culture is
introduced into the upper cylinder, and is then washed down
into the lower cylinder by means of a little wort, which is
forced from B into A, and then back again into B. When a
vigorous multiplication of the yeast has set in, the liquid is
stirred up and a portion forced into A ; this is to be used to
start the next fermentation. The cylinder B thus serves
alternately as fermenting-cylinder and wort-cylinder.1

Other modifications have been devised by Brown and
Morris, Elion, Kokosinski, and Van Laer', more widely
different are the forms devised by P. Lindner and Marx.2

In order to be able to send to a distance the selected
pure cultures in a liquid condition, a special form of flask
was devised by Hansen. The yeast can be sent to great
distances in these flasks, and there is no difficulty in safely
transferring it from the flask to the cylinder of the propa-
gating apparatus.

In sending small quantities of pure cultures, and in
such a manner that they may be safely and readily employed
for further cultivation, the small Hansen flasks (page 20)
are employed. They are connected, in the flame, with the
Pasteur flask in which the pure culture has developed. A

1 Both the above described forms of apparatus are manufactured by
Messrs. Burmeister and Wain, of Copenhagen; Hansen and Kuhle's
apparatus is also made by W. E. Jensen, of Copenhagen.

2 An apparatus which has now become of considerable importance as
a link in Hanserfs system of pure yeast cultivation is the closed cooler
mentioned above, by means of which it is possible to introduce the wort
into the fermenting-vessel absolutely pure and properly aerated. Ap-
pliances having this for their object were devised by Velten shortly after
the publication of Pasteur's " Etudes," and were constructed in accordance
with Pasteur's theoretical views, but hitherto they could not acquire any
practical importance ; for what was the use of having a pure wort when
the disease-germs were again introduced with the yeast ? The conditions
which render such appliances useful are only now attaine d through the
introduction of pure yeast, and the open coolers will therefore gradually
disappear in the future.

214 MICRO-ORGANISMS AND FERMENTATION.

trace of the yeast is transferred to the cotton-wool, and
the flask is again closed in the flame with the asbestos
stopper, which is then coated over with sealing-wax. When
the culture is to be used, the flask is again connected with
a Pasteur flask containing wort, and the yeast is rinsed into
the latter.

This method has proved especially valuable for sending
absolutely pure yeast to tropical countries. In many cases
it would otherwise have been impossible to send pure
cultivations to Australia, South America, and the most
distant countries of Asia.1

It is a fact of the greatest importance, that even after
the lapse of years, the perfectly identical yeast once selected
can always be had again, a sample of the pure culture
being preserved in the laboratory in a 10 per cent, solution
of cane-sugar (page 20). In such a solution, the cultivated
yeasts can be kept alive and unchanged in their properties
for years.

With regard to the preservation of micro-organisms on
solid substrata, Percy Frankland found that bacterial fer-
ments sometimes completely lose their fermentative power
after repeated cultivation in solid media. In some cases
the fermentative power disappeared after a single plate-
cultivation.

The absolutely pure and systematically selected races of
yeast, prepared in large quantities for industrial purposes,
are now — Hansen having made his first experiment in 1883

1 The use of sterilised filter-paper for sending yeast samples is for
quite a different object. This method is used either for sending an
impure brewery-yeast to a laboratory in order that a pure culture may
be prepared from it, or a sample of a pure culture may be conveniently
sent in this manner, enclosed in an envelope ; it is clear, however, that a
sample sent in this way can no longer be depended on as absolutely pure,
and the sample can therefore only serve as material for a new pure
cultivation.

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 215

in the well-known Old Carlsberg brewery at Copenhagen —
employed in numerous breweries in all beer-producing
countries, not only in Europe, but also in America, Asia,
and Australia.

Since Hansen's system was first carried out in a bottom-
fermentation brewery, it naturally first found application
in breweries of the same kind, and it is here that it has
reached the highest degree of perfection. Any one who
wishes to become acquainted with ike application of this
system to one or the other branch of the fermentation
industries, should therefore take as the starting-point of
his studies the results achieved in bottom-fermentation
brewing.

The advance was subsequently introduced into top-
fer 'mentation brewing, and it was found that here also
the system offers the same advantages. At first the same
objection was again raised which had been brought forward
against pure cultivations of bottom-yeast, namely, that it
could not produce a secondary fermentation, as the latter
was supposed to be caused by the so-called wild species of
yeast; the objection, however, again fell through when the
question was submitted to the practical test of experiment.
There are, in fact, top-fermentation yeasts which give
a vigorous secondary fermentation, and others which
produce only a feeble after-fermentation. A species should
therefore be systematically selected which will answer the
required conditions.

The new system afterwards continually spread, and has
now forced its way into distilleries, pressed-yeast factories,
and into the different vinous fermentations.

Hansen's epoch-making investigations of the last decade
have given rise to a very extensive literature, the contents
of which may be summed up under three headings : —
Treatises of a purely ephemeral nature, the only object of
which is clearly to oppose the work of the Danish investi-
gator and to bring obstacles against the introduction of

216 MICRO-ORGANISMS AND FERMENTATION.

this reform into science and practice ; other treatises, which
discuss the subject, but which show a misunderstanding of
the separate links or of the kernel of the system ; and,
finally, a number of valuable investigations, the object of
which is to throw light on the system from various sides
and thus to facilitate a true conception and the practical
application of the system.

It would lead us too far to discuss these different sides
of the literature. In concluding this description, I will
confine myself to a few quotations from the highest
authorities who have thrown light on the subject from
various points of view, and have merited the greatest
praise in helping the extension of the system in different
countries.

Professor Lintner, sen., gives the following review of
the situation in 18851: —

"Now that different breweries have employed pure
cultivations of the Carlsberg yeasts, and that the Scientific
Station at Munich has also introduced pure cultivations of
Munich yeasts into various breweries, the results obtained
may be summarised as follows : —

"1. By contamination with so-called wild yeasts, a
brewery-yeast, normal in other respects, may grad-
ually become incapable of producing a beer of
good flavour and with good keeping properties.

" 2. A contamination of this kind can occur through
wild yeasts present in atmospheric dust during
summer and autumn, or the wild yeast may be
introduced with the pitching-yeast or with cask
sediment.

" 3. By means of Hansen's methods of analysis and pure
cultivation, it is possible to isolate from a contami-
nated yeast, the desired brewery-yeast in a good and
pure condition.

1 Zeitsclirift f . d. ges. Brauw., 1885, p. 399.

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 217

" 4. The pure cultivated yeast possesses in a marked
degree the properties of the original yeast previous
to contamination, both as regards the degree of
attenuation, and the taste and keeping properties
of the beer.

" o. Different races of normal bottom-fermentation yeast
(Sacch. cerevisice) exist with specific properties,
which are constant for each race and form dis-
tinctive characteristics."

Professor Aubry, director of the Scientific Brewing Station
at Munich, wrote (1885)1 : — "In addition to the breweries
mentioned (Spatenbrau and Leistbrau in Munich), a large
number of breweries at home and abroad have carried out
experimental fermentations with pure Carlsberg yeast. The
results which were expected were naturally not attained in all
cases, the degree of attenuation was found to be too low in
the greater number of cases,2 the taste was not the one locally
desired, &c., &c., but all the reports which reached us were
favourable as regards the keeping properties, brilliance, and
the freedom of the beer from any taste of yeast. The good
properties of the yeast have brought about its permanent in-
troduction into many breweries, as, for instance, the Liesinger
brewery, at Liesing, near Vienna. In the present brewing
season the Spaten brewery in Munich has made extensive
use of yeast obtained from Carlsberg, and a great part of the
pitching-yeast used in the brewery of the Franziskanerkeller
in Munich, during the winter, was also derived from pure
cultures of Carlsberg yeast. The course of fermentation and
the results with regard to the taste, condition, and keeping
properties of the beer, answered all requirements. The
property of giving a somewhat low attenuation appears to
be characteristic of the yeast, for it remains constant.
The taste of the beer at first differs somewhat from the

1 Zeitschrift f. d. ges. Brauw., 1885.

2 Carlsberg bottom-yeast No, 2, a quick clarifying species.

218 MICRO-ORGANISMS AND FERMENTATION.

ordinary Munich taste, but approaches more nearly to this
with later generations ; it remains, however, soft and
agreeable.

Dr. Will writes (1885)1 : — " If now, as I trust I have made
clear, it is possible to detect with certainty the species of
yeast which have an injurious influence in the brewery, we
must make practical use of this knowledge, and only employ
pitching-yeasts which do not show the above-mentioned
characteristics for the injurious species which so frequently
and actively exert a disturbing influence in the brewery.
This, however, will only be possible when yeast cells endowed
with the properties of normal bottom-yeast are isolated from
the ordinary brewing yeast, and further cultivated with the
exclusion of every contamination ; in other words, ivhen only
pure cultivated yeast is employed in the brewery. Hansen
is entitled to the greatest praise in this particular direction,
since he has pointed out a way and devised a method which
enabled him to attain the desired end. The far-reaching
results which were obtained in Old Carlsberg with pure
cultivated yeast have already caused many other breweries
to employ only pure cultivated yeast, and the results in
general have given satisfaction when varieties of normal
bottom-yeast were chosen which corresponded with the
requirements as regards attenuation and taste.

" It is to be hoped, therefore, that the value of pure
cultivated yeast may become recognised in ever increasing
circles, and many old prejudices regarding the yeast over-
come ; also that the smaller breweries, which have besides
many difficulties to contend with, will not resist the conviction
that a number of calamities may be avoided by the intro-
duction of pure cultivated yeast into an otherwise well-
conducted brewery. The amount expended will yield a
liberal interest."

Dr. Reinke, who is at the head of the experimental

1 Allgem. Brauer- tmd Hopfenzeitung, 1885.

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 219

brewing station of the Royal Agricultural College in Berlin,
made the following significant statement of the situation in
1888 1:—

" Without the exact study of Hanserts pioneering inves-
tigations, and without their utilisation, no one at the present
time is able to permanently resist the competition in the
brewing industry. Hanserfs researches have brought about
a revolution in the brewery, especially with regard to the
treatment of the yeast."

Professor Bclohoubek, of the Bohemian Polytechnic at
Prague, says in his well-known biography of Hansen, 1 889 2 :

" No one will be surprised that the establishment of the
principle of pure yeast-cultivation, and the truly crushing
criticism concerning the general custom of leaving fermenta-
tions in the brewery to chance which until then prevailed,
and, above all, that the actual introduction into the brewery of
pure cultivated yeast prepared by Hansen's method, produced
at first astonishment amongst practical men — with some
honourable exceptions — then ridicule, and finally provoked
hostile opposition ; for it is known to the initiated what
obstinate conservatism there is in brewing circles, where
all innovations and reformatory efforts are not only met
with passiveness and mistrust, but are sometimes most
tenaciously resisted. Fortunately many important factors
were united in the struggle against the opposition, which
finally suffered a decided defeat in spite of the support
of some theoretical specialists, more particularly in North
Grermany and Austria-Hungary. It was chiefly the correct-
ness of Hansen's views which contributed to this victory,
and which completely convinced the most eminent authori-
ties of Europe on the science of fermentation; secondly,
the fact that able experts also outside Denmark began
to experiment with pure yeast-cultivation; thirdly, the

1 Chemiker-Zeitung, 29 Dec., 1888, p. 1749.

2 Zeitschrift f. d. ges. Brauw., Munich, 1889, p. 505.

220 MICRO-ORGANISMS AND FERMENTATION.

highly favourable results which were obtained in the brewery
with pure yeast; and, finally, also the fact that in 1887
Professor Hansen, in conjunction with Captain Kuhle, suc-
ceeded in devising a pure yeast apparatus which enabled
them to produce large quantities of the pure yeast which had
been prepared on a comparatively small scale in the laboratory.
At the present time hundreds of breweries obtain a pure
cultivated standard yeast from institutions in which pure
cultivations of beer-yeast are prepared, and thousands of
breweries do the same indirectly in that they obtain their
pitching-yeast from the above breweries. The universal
employment of pure yeast in the brewing industry is
therefore now only a question of time.

" If we now weigh with the most complete objectiveness
the significance of these facts as applied to the conditions
obtaining in European bottom-fermentation breweries, we are
compelled to acknowledge that the reform introduced by
Professor Hansen is still more far-reaching than is generally
assumed. A result of this reform is already being discussed
in brewing circles, namely, the abandonment of open coolers
in all breweries where pure yeast is employed, as these freely
permit of the contamination of the wort with micro-organisms
and especially with bacteria and the so-called " wild " yeasts.
It is therefore proposed to filter the hopped wort, or to
separate the suspended matter (cooler-deposits) by another
method, to saturate the wort with filtered air, and to cool it
by artificial means. But these are by no means all the
precautions which must be adopted in order to guard against
further infection of the wort in the fermenting-rooms and in
the lager-cellar. Only when these questions have been
solved — perhaps by means of closed fermenting-vessels of a
suitable material, by the sterilisation of the fermenting and
storage vessels, by a more rational arrangement of the fer-
menting-rooms and lager-cellars, and by the ventilation of
these by means of filtered air, &c. — only then will it be
possible amongst beer producers and consumers to enjoy to

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 221

the full the great advantages of having a beer of better quality
and keeping properties than the present beer, advantages
which are a result of the employment of pure yeast.

" The above statements concerning the importance of
pure cultivated yeast, refer throughout to beer bottom-
yeast only. There could be no doubt even from the
first that it would also be possible to employ Hanserts
method of pure yeast-culture to top-fermentation yeast,
and with the same result ; this has since been proved ex-
perimentally by Alfred Jorgensen, and pure top-yeast has
proved just as successful in the brewery as pure bottom-
yeast. The writer of these lines is convinced that the
introduction of pure cultivated species of yeast into dis-
tilleries, and especially into pressed-yeast factories, will give
very advantageous results. In distilleries — other conditions
being maintained the same — better fermentations and a
greater yield of alcohol in comparison with the average now
attained are to be expected, whilst in pressed-yeast factories
a better yield of yeast should result from a successful selec-
tion of a pure cultivated species, and possibly the employment
of clear mashes will then be found preferable to mashes con-
taining the grains as now employed."

In Dr. H. Bungener's treatise " La levure de la biere "
1890,1 the following statement occurs, contrasting the old
with the new period : — " In France, Hanserts system has been
eagerly taken up by L. Marx, A. Fluhler, and Kokosinski. In
some breweries, it has been recently introduced, and it will
soon be adopted by others. We are convinced that its intro-
duction into all the larger breweries of France, and in fact
everywhere else, will only be a question of time. It has in
fact been established, that it ensures regular working and
a good result in one of the most important stages of the
manufacture, where hitherto chance and, in consequence, also
uncertainty prevailed."

1 Moniteur scientifique du Dr. Quesneville, Juillet-Aoftt, Paris, 1890.

222 MICRO-ORGANISMS AND FERMENTATION.

Prof. C. Lintner, jun., of the Technical College, Munich,
writes (1891 )l : — "In the abstracts relating to advances in
the brewing industry, the epoch-making investigations of the
Danish savant Emil Chr. Hansen, and their application in
the breweries, have been frequently reported. A connected
account of Hansen's reform and methods, however, has not
yet appeared in this journal, though such an account would
be by no means undesirable, considering the great importance
which the subject has acquired during the seven years since
its introduction into the brewing industry. Hitherto, the
brewery has mainly benefited from Hanserts system, which,
however, has also already found its way into the distillery and
pressed-yeast factory, and these branches of the fermentation
industry will also be greatly benefited by its introduction."

In England some of the most celebrated authorities have
frankly acknowledged the value of Hansen's investigations.
Amongst these is Professor Percy Frankland, who has
expressed himself as follows2 :—

"Emil Christian Hansen, of Copenhagen, has enor-
mously extended our knowledge of the alcohol-producing
organisms or yeasts; he has shown that there are a num-
ber of distinct forms, differing indeed but little amongst
themselves in shape, but possessing very distinct proper-
ties, more especially in respect of the nature of certain
minute quantities of secondary products to which they give
rise, and which are highly important as giving particular
characters to the beers produced. Hansen has shown how
these various kinds of yeast may be grown or cultivated in a
state of purity even on the industrial scale, and has in this
manner revolutionised the practice of brewing on the
continent. For during the past few years these pure
yeasts, each endowed with particular properties, have been
grown with scrupulous care in laboratories equipped ex-

1 Dingl. Polytechn. Journal, Jahrg. 72, Bd. 279, Heft 9.

2 Royal Institution of Great Britain. Meeting, February 19, 1892.

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 223

pressly for this purpose, and these pure growths are thence
despatched to breweries in all parts of the world, particular
yeasts being provided for the production of particular varieties
of beer. In this manner scientific accuracy and the certainty
of success are introduced into an industry in which before
much was a matter of chance, and in which nearly everything
was subordinated to tradition and blind empiricism."

The system has now been introduced into top-fermentation
breweries in all countries. After its adoption some time ago
by various American and Australian breweries, which are
worked on the English system, W. JR. Wilson succeeded
(1892) in carrying out this important reform in a London
brewery, both primary and secondary fermentation being
effected by a single selected species of yeast. According to
the reports in English journals numerous breweries in Great
Britain have successfully adopted Hansen's system of pure
cultivated yeast.

The following is taken from a report by J. C. MacCartie
of Melbourne1 : —

" The Burton yeast2 yields a mild ' round ' flavoured beer
of great brilliancy and stability, and one that is excellently
suited for bottling. I now come to a matter that should be
of interest to your readers. Mr. de Bavay and I read with
some astonishment the statements made by certain scientific
gentlemen in England, concerning the difficulty or impossi-
bility of obtaining after- or secondary fermentation when one
type of yeast alone — say Sacch. cerevisice — is used; for there
has not been the slightest difficulty in obtaining secondary
fermentation in ' stock ' or bottled ales, where the Australian
or ' Burton ' yeasts have been used here.

" I have with Mr. de Bavay over and over again examined
both ; stock ' and bottled ales fermented with pure Burton
yeast, and that secondary fermentation was vigorous in them,

1 The Brewers' Journal, 1889, No. 291, p. 489.

2 A " Burton " species obtained in pure cultivation in the laboratory
of the author of this book from English yeast.

224 MICRO-ORGANISMS AND FERMENTATION.

any one who saw the foam and ' head ' on the beers could not
doubt.

" Mr. de Bavay tells me that he frequently obtains a well-
marked secondary cask-fermentation in a fortnight from
racking the beer, and this in cases where the yeast used
was fresh from the laboratory, and therefore practically free
from the slightest intermixture of other types of yeast."
(De Bavay^s brewery in Melbourne is worked on the top-
fermentation system.)

MacCartie, therefore, basing his opinion on these facts,
has no doubt but that within a few years Hansen's system
will also be adopted in all the important top-fermentation
breweries of the world. His detailed account is of great
interest, since it again affords proof, obtained in actual
practice, that there are differences in the species of Saccharo-
myces cerevisice, and thus it also proves the necessity for
making a selection from these species with reference to
practical requirements.

W. R. Wilson writes l :—

" Some gyles have been prepared with pure yeast, and
have been compared against ale brewed at the same time, but
pitched with ordinary yeast. It is admitted on all hands
that, so far as can be at present ascertained, the pure-yeast
ale is immeasurably superior to the other. Hansen's pure
yeast system applied to English high fermentations, appears
to be equally suitable for ales, porters, and stouts."

With regard to the employment of the system in top-
fermentation breweries in North France and Belgium, the
following statements may be quoted.

Dr. E. Kokosinski, director of the laboratory at Lille,
writes 2 : —

" In August, 1888, after three years' preparatory study,

1 The Brewers' Journal, 1892, p. 527.

2 Application industrielle de la me"thode Hansen a la fermentation
haute dans le Nord de la France. Compt. rend, de la Station scientifique
de Brasserie, Gand, 1890.

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 225

I introduced pure cultivated yeast for the first time into a
top-fermentation brewery in Lille. Shortly afterwards, at
the beginning of 1889, I also introduced it into some other
breweries in Lille, Roubaix, Douai, and St. Omer, and at the
present time there are fifteen breweries in North France in
which pure yeast is employed, and all, without a single
exception, obtain excellent results ivith it"

He summarises the results of his practiced experience
iwth beers obtained ivith the help of pure cultivated top-
yeast, as follows : —

" 1. They have the particular flavour which the brewer
wishes to obtain ;

" 2. This taste is uniform and always remains constant ;
it is characterised by great pureness :

u 3. The clarification takes place more readily and more
quickly ;

" 4. The beers are more resistant to the action of bacteria
and have a greater stability.

" It follows from the above that if Hanserts method has
rendered great service in bottom-fermentation, it is now
about to do the same also for top-fermentation, and in the
latter case it will be of far greater value, for in top-fermenta-
tion we have not the advantage of the low temperature which
obtains in bottom-fermentation, and which tends to check the
action and the development of disease germs."

Professor van Laer, of the scientific station at Ghent,
expresses himself in a similar manner1 : —

" If, to the practical results obtained by myself, we add
those obtained by Messrs. Alfred Jorgensen and Kokosinski,
we are justified in stating that the pure yeast question has
been solved both for top- fermentation and bottom-fermenta-
tion, and that its general application is merely a question of
time."

1 Application industrielle de la methode Hansen a la fermentation
haute en Belgique, 1890. Compt. rend, de la Station scientifique de
Brasserie, Gand, 1890.

226 MICRO-ORGANISMS AND FERMENTATION.

Similar opinions on the importance of Reinserts work
have been expressed by other famous zymotechnologists such
as Delbruck (Berlin), Fluhler (Lyon), Griessmayer (Munich),
Langer (Modling), Maercker (Halle), Marx (Marseilles),
Schwackhofer (Vienna), Thausing (Vienna), and others.
Several of his former opponents have become his warmest
supporters.

Finally it inay be mentioned that the numerous and very
different types of top-yeast which have been prepared in pure
culture in the analytical section of my laboratory since 1884,
have given just as satisfactory practical results as the pure
bottom-yeasts, when the yeast has been treated with the
necessary care.

The French opponents to the employment of Hansen's
system have in fact taken up their position on the old stand-
point of 1876, when neither the wild yeasts nor the very
different types of cultivated yeasts were known.

In the above we have only spoken of the brewing industry.
Hansen's discoveries are, however, already being applied to
other branches of industry in which alcoholic fermentation
plays a part. Thus experiments have been made at many
places in pressed-yeast factories and in distilleries, and in a
great number the system has been already introduced with
success. In wine- fermentation a beginning was made, as
stated above, in 1888, by a pupil of Hansen's, L. Marx, of
Marseilles ; subsequently other investigators have worked in
the same direction in France, and also Muller-Thurgau in
Switzerland, Forti and Pichi in Italy, Mach and Portele
in Austria, and Wortmann in Germany. Nathan (Wurtern-
berg) and Kayser (France) have likewise made extensive
experiments in connection with the fermentation of fruit-
wines.

A result of Hanserfs epoch-making discoveries was the
establishment of special laboratories, the object of which is
to prepare absolutely pure material for employment in
practice, to carry out control analyses for the brewery, and

RESULTS OF SCIENTIFIC RESEARCH IN PRACTICE. 227

to instruct the younger generation in the true understanding
and the proper application of his discoveries. Such institu-
tions have now been established in -almost all countries, and
are partly private and partly supported by state ; they have
already produced a number of able teachers, analysts, and
technologists, who are working with energy and judgment
with a view to more widely circulate in science and in practice
the views of the Danish investigator.

Hanserfs investigations have indirectly exercised an in-
fluence in connection with the dairy ; as was mentioned
in a previous chapter, pure cultures of lactic acid bacteria
have been successfully employed for the souring of cream.
Finally, also, in the tobacco fermentation, Schloesing and
Suchsland have made experiments with the view to produce
a definite aroma in tobacco leaves by the addition, during
the fermentation, of pure cultures of certain species of
bacteria.

The idea which underlies all these reformatory investiga-
tions, is the principle which has been recognised and carried
out for centuries in horticulture and agriculture, namely, that
in order to obtain the desired species of plant, the pure seed
should be sown free from the seeds of all other plants.

BIBLIOGEAPHY.

ADAMETZ, L. — Saccharomyces lactis, eine neue Milchzucker vergarende

Hefeart.— Centralblatt f. Bakt. Bd. V. Nr. 4. 1889.
Die Bakterien normaler und abnormaler Milch. — Oesterr. Monats-

schrift fur Tierheilkunde und Tierzucht. XV. Nr. 2. 1890.
Untersuchungen iiber Bacillus lactis viscosus, einen weitverbreiteten

milchwirtschaftlichen Schadling. — Berliner landwirtschaftl.

Jahrbiicher. 1891.
AHJLBTTRG. — Ueber Aspergillus oryzae.— Mitteil. der deutschen Gesell-

schaft fur Natur-und Volkerkunde Ost-Asiens, 16. Heft, 1876;

und Dinglers polyt. Journal, 230. Bd., S. 330. 1878.
AMTHOR, C. — Studien iiber reine Hefen. — Zeitschrift fur physiologische

Chernie. Bd. XII. 1888.
Ueber den Saccharomyces apiculatus. — Zeitschr. fur physiologische

Chemie. Bd. XII. 1888.

Ueber Weinhefen. — Zeitschr. fur angew. Chemie. 1889.

Beobachtungen iiber den Saccharomyces apiculatus. — Chemiker-

Zeitung. Nr. 38. 1891.
APPERT. — Le livre de tons les manages ou 1'art de conserver, pendant

plusieurs anne"es, toutes les substances animales et ve"ge"tales. —

4e 6dit. (1° <§dit. 1810). Paris, 1831.
ATKINSON. — The chemistry of sake-brewing in Japan, the processes of

preparation of Koji and of fermentation. — Tokio, 1881.
AUBRY, L. — Ein Beitrag zur Klarung und Bichtigstellung der

Ansichten iiber reine Hefe. — Zeitschr. f. d. ges. Brauwesen.

Nr. 7. Munich, 1885.

Noch ein Wort iiber reine Hefe.— Ibid. Nr. 12, 1885.

Ueber desinfizierende Stoffe. — Ber. d. Wiss. Stat. Munich, 1887.

1889.
Beobachtungen iiber Hefe. — Mitt, der Wissenschaftl. Station fur

Brauerei, Munich. — Zeitschr. f. d. ges. Brauwesen. Nr. 4. 1892.
BAIL, TH.— Ueber Hefe.— Flora Nr. 27, 28. 1857.
BAINIER, G. — Observations sur les Mucorinees. — Ann. d. sc. nat. Bot.

Se>. VI., T. XV. 1883.

BIBLIOGRAPHY. 229

BARY, DE. — Comparative Morphology and Biology of the Fungi,

Mycetozoa and Bacteria. — English translation by H. E. F.

Garnsey, M.A. Revised by Isaac Bayley Balfour, M.A., M.D.,

F.K.S.— Oxford, at the Clarendon Press, 1887.
— Lectures on Bacteria. — Sec. Edition. By H. E. F. Garnsey and

I. B. Balfour.— Oxford, 1887.
BAU, A. — Ueber die scheinbare Zunahme des Dextringehaltes in

Bierwiirzen wahrend der Garung. — Wochenschr. f. Brauerei.

Nr. 28, 1890.
— Ueber die scheinbare Zunahme des Dextringehaltes in Bierwiirzen

wahrend der Garung, sowie iiber die Bestimmung der Dextrose

und des Dextrins in ihnen. — Wochenschr. f. Brauerei. Nr. 42,

1890.
— Die Bestimmung von Maltose, Dextrose und Dextrin in Bierwiirze

und Bier mittels Reinkulturen von Garungsorganismen. —

Centralbl. f. Bakt. und Parasitenk. V. Jahrg. IX. Bd. 1891.
— Beitrage zur Physiologie der Monilia Candida. — Woch. f. Brau.

Nr. 43, 1892.
— Ueber die Bestimmung der vergarbaren Substanz in Bierwiirzen. —

Chem. Ztg. Nr. 80-82, 1892.
— Ueber die Verwendung der Hefe zur quantitativen Bestimmung

garfahiger Substanzen.— Chem. Ztg. Nr. 23, 1893.
— Ueber die Bestimmung der Isomaltose. — Chem. Ztg. Nr. 29, 1893.
BECHAMP. — De I'influence de 1'oxygene sur la fermentation alcoolique par

la levure de biere.— Compt. rend. LXXXVIII. p. 430. 1879.
BELOHOUBEK, A. — Ueber die Erzeugung von Reisbier. — Bay. Bierbr.

1870.
— Einige Worte iiber den Bau und die Einrichtung der Brauereien. —

Prague, 1875.

— Studien iiber Presshefe. — Prague, 1876.
— Ueber die Erzeugung von Presshefe in Kartoffel-Brennereien. —

Oesterreichische Brennereizeitung. 1878.
— Ueber die Obergarung von Bierwiirzen. — Prague, 1881.
—Ueber den Emfluss der Hefe auf die Qualitat des Bieres und iiber

die Bedeutung der reinen Samenhefe fur die Brauindustrie in

Bohmen und Mahren. — Vortrag. Bohm. Bierbrauer. Prague, 1885.
Dr. Emil Chr. Hansen, eine biographische Skizze. — Zeitschr. f. d.

ges. Brauwesen, Munich, Nr. 24, 1889.

(Also a number of Memoirs on Fermentation in the Bohemian language.)
BERTHELOT. — Sur la fermentation alcoolique. — Ann. de chim. et de

phys. L. p. 322. 1857.
BEYERINCK, M. W — Die Laktase, ein neues Enzym. — Oentr. f. Bakt.

u.Parasit. VI. Bd. Nr. 2. 1889.
Verfahren zum Nachweise der Saureabsonderung bei Mikrobien. —

Centralbl. f. Bakt. und Parasitenkunde. IX. Bd. Nr. 24. 1891.

230 BIBLIOGRAPHY.

BIERNACKI, E. — Ueber die Eigenschaft der Antiseptica, die Alkohol-

garung zu beschleunigen, und iiber gewisse Abhangigkeit ihrer

Kraft von der chemischen Baustruktur, der Fermentmenge und

der Vereinigung mit einander. — Pfliigers Archiv. XLIX.

BILLROTH. — Untersuchungen iiber die Vegetationsformen von Cocco-

bacteria septica. 1874.
BLANKENHORN UND MORITZ. — Untersuchungen iiber den Einfluss der

Temperatur auf die Garung. — Ann. d. Oenologie 4. Bd. 1874.
BORDAS. — Sur une maladie nouvelle du vin en Algerie. — Compt. rend.

CVI. 1. 1888.

BORGMANN, E. — Zur chemischen Charakteristik durch Reinkulturen
erzeugter Biere. — Fresenius, Zeitschrift fur analytische Chemie,
B. XXV. Heft 4.
BOURQUELOT, E. — Sur les proprietes de 1'invertine. — Journ. de pharm.

et chim. VII. p. 131. 1883.

— Sur le non deMoublement prealable du saccharose et du maltose
dans leur fermentation lactique. — Journ. de pharm. et de chim.
VIII. p. 420. 1833.
— Recherches sur les proprietes physiologiques du maltose.— Compt.

rend. d. s. de 1'Ac. d. sc. T. 97. Paris, 1883.
— Rech. sur les proprietes physiologiques du maltose. — Journ. de

1'anat. et de la phys. 1886.
— Les microbes de la fermentation lactique du lait. Le kephir. — Journ.

de pharm. et de chim. XIII. 1886.

— Sur la fermentation alcoolique du galactose. — Journ. de pharm. et
de chim. XVIII. p. 367. 1888.

Sur la fermentation alcoolique du galactose. — Compt. rend. CVI.

S. 283. 1888.

— Les fermentations. — Paris, 1889.
BOUTROUX. — Sur la fermentation lactique. — Compt. rend. T. LXXXVI.

1874.

— Sur une fermentation nouvelle du glucose. — 1880.
— Sur Thabitat et la conservation des levures spontanees. — Bull, de la

Soc. Linneenne de Normandie. 3. s£r. VII. 1883.
— Fermentation panaire. — Compt. rend. des. s. de 1'Acad. d. sc. T.

CXIII. 1891.
BRAEUTIGAM, W. — Untersuchungen iiber die Mikroorganismen in

Schlempe und Biertrebern. — Inaug.-Diss. Leipzig, 1 886.
BREFELD. — Botan. Untersuchungen iiber Schimmelpilze. — 1-4. Heft.

Leipzig, 1872-1881.

— Mucor racemosus und Hefe. — Flora 56. Jahrg. 1873.
—Ueber Garung I., II., III.— Landw. Jahrbiicher III., IV., V., Bd.

1874, 1875, 1876.

— Methoden zur Untersuchimg der Pilze. — Ber. d. mediz.-phys.
Gesellsch. zu Wiirzburg. 1874.

BIBLIOGRAPHY. 231

BREFELD. — Beobachtungen iiber die Biologie der Hefe. — Sitzb. d.

Gesellschaf t naturf orschender Freunde zu Berlin. Bot. Ztg. 1875.
— Botanische Untersuchungen iiber Hefenpilze (Fortsetzung der

Schimmelpilze).— 5. Heft. Leipzig, 1883.

— Untersuchungen auf dem Gesamtgebiete der Mycologie. (Fortset-
zung der Schimmel- und Hefenpilze). — 6 — 10. Heft. Leipzig,

1884—1891.
BRE.TCHA. — Mikrosk. Analyse des Kiihlgelag'ers. — Zeitschrift des

Brauindustrie-Vereins f. d. K. Bohmen. 1879.
BROWN, A. J. — The chemical action of pure cultivations of Bacterium

aceti. — Trans. Chem. Soc., 1886, p. 172. Chemical News LI1L,

p. 55. 1886.
— On an acetic ferment which forms cellulose. — Trans. Chem. Soc.,

1886, p. 432.
— = — Some experiments on the numerical increase of yeast cells. —

Transact, of the Laboratory Club. Vol. III., Nr. 4. London, 1890.
— On the influence of oxygen on fermentation. — Proceed, of the

Chem. Soc. Nr. 107, p. 33. 1892.
BROWN, HORACE T., and MORRIS, G. H. — On the non-crystallisable

products of the action of diastase upon starch. — Trans, of the

Chemical Society, 1885, p. 527.
BUCHNER, ED. — Ueber den Einfluss des Sauerstoffes auf Garungen. —

Zeitschr. f. phys. Chemie IX. S. 380. 1885.
BUNGENER, H. — La levure de biere. — Moniteur scientifique du Dr.

Quesneville. Paris, 1890.
— und WEIBEL, L. — Einiges iiber die Zusammensetzung des Wiirze-

Extraktes. — Allg. Brauer- und Hopfenzeitung Nr. 5. 1891.
BUSGEN. — Ueber Aspergillus Oryzae. — Botan. Centralblatt. Jahrg. VI.,

Nr. 41, S. 62. 1885.
CAGNIARD-LATOUR. — Journal de 1'Institut. 1 835 — 1 837.

— Me"moire sur la fermentation vineuse. — Compt. rend. IV. S. 905.

1837.
CALMETTE. — Contribution a I'etude des ferments de 1'amidon. — La levure

chinoise. Ann. de llnst. Pasteur. T. VI., p. 604. 1892.
CHAPTAL. — L'art de faire le vin. 1807.
CHICANDART. — La fermentation panaire. — Monit. Scientif. Quesneville.

Oct., 1883.
CIENKOWSKY. — Die Pilze der Kahmhaut. — Bull. d. Petersb. Akademie.

1873.
Zur Morphologic der Bakterien.— M<§m. de 1'Acad. de St. Peters-

bourg. S. VII., T. XXV. 1877.

Die Gallertbildungen des Zuckerriibensaftes. — Charkow, 1878.

COHN, F.— Beitrage zur Biologie der Pflanzen.— 1. Bd. 2. H. 1872 ;

1. Bd. 3. H. 1875 ; 2. Bd. 2. H. 1876 ; 2. Bd. 3. H. 1877. (Investi-
gations on bacteria).

232 BIBLIOGRAPHY.

COHN, F. — Ueber Kephir. — Sitzber. der Schles. Ges. fur vaterland.

Kultur. Breslau, 1883.
— Ueber Schimraelpilze als Garungserreger. — Jahrb. d. Schles.

Gesellsch. f. vaterland. Kultur zu Breslau. LXI.? S. 226. 1884.
CONN, H. W. — Ueber einen bittere Milch erzeugenden Mikrokokkua.

—Centralbl. f. Bakt. u. Parasitenk. JX. B. 1891.

DAVID. — Ueber Rotweingarungspilze. — Ann. d. Oenologie. 4. Bd. 1874.
DELBRTJCK. — Die Sauerung des Hefengutes. — Zeitschr. f. Spiritus-

industrie. 1881.
— Der Charakter des Bieres, auch des Weissbieres. — Woch. f. Brauerei.

Nr. 6. Berlin, 1885.

— Die Carlsberger reingeziichtete Hefe. — Ibid. Nr. 10. 1885.
— Zur Wirkung der Kohlensaure-Entwickelung auf die Garung. — Ibid.

Nr. 42. 1886.
— Die Reinzuchthef e und die Presshef ef abrikation. — Zeitschr. f .

Spiritusindustrie. Berlin, 1889.
DONATH. — Ueber den invertierenden Bestandteil der Hefe. — Ber. d.

deutsch. chem. Ges. VIII., p. 795. 1875.
DOWDESWELL. — On the occurrence of variations in the development of

a Saccharomyces. Journ. Roy. Microsc. Soc. — Ser. II., vol. V.

London, 1885.

DUCLAUX. — Memoire sur le lait. — Ann. de I'lnstitut agronomique. 1882.
— Chiraie biologique (Microbiologie). Paris, 1883.
— Fermentation alcoolique du sucre de lait. — Ann. d. 1'Instit. Pasteur.

Nr. 12. 1887.
DtiNNENBERGER, C. — Bakteriologisch-chemische Untersuchung liber

die beim Aufgehen des Brotteiges wirkenden Ursachen. — Botan.

Centralblatt. Bd. 33. 1888.
EHRENBERG. — Epistola de Mycetogenesi. — Nov. act. Acad. nat, cur.

1821.

EIDAM. — Der gegenwartige Standpunkt der Mykologie. 1871, 1872.
ELION, H. — Beitrage zur Kenntnis der Zusammensetzung von Wiirze

und Bier.— Zeitschr. f. angew. Chemie. Heft 11. 1890.
— Die Bestimmung von Maltose, Dextrose und Dextrin in Bierwiirze

und Bier mittelst Reinkulturen von Mikroorganismen.— Centralbl.

f. Bakt. u. Parasitenk. IX. Bd. 1891.
ENGEL. — Les ferments alcooliques. — These pour le Doct, es. sc. nat.

1872.
FERMI, C. — Beitrag zum Studium der von den Mikro organismen

abgesonderten diastatischen und Inversions fermente. — Centralbl.

f. Bakt, u. Paras. XII., p. 713. 1892.
FERNBAGH, A. — Sur le dosage de la sucrase. — Formation de la sucrase

chez Aspergillus niger. — Ann. de ITnst. Pasteur, p. 1. 1890.
— Sur 1'invertine ou la sucrase de la levure. — Ann. de 1'Inst. Pasteur.

T. IV., p. 641. 1890.

I
BIBLIOGRAPHY. 233

FITZ. — Ueber die alkoholische Garung durch Mucor Mucedo und M.
racemosus. — Ber. d. deutschen chem. Ges. VI., p. 48, 1873, und
VIII., p. 1540. 1875.
— Ueber Schizomyceten-Garungen. — Ber. d. deutschen chem. Gesellsch.

Bd. 9 bis 17. 1876—1884.

FLUGGE.— Die Mikroorganismen. — Leipzig, 1886.
FEFHXER, A. — Conference faite a la Societe des sciences iudustr. de

Lyon. — Section de Chimie. 1886.
FOKKER, A. P. — Ueber das Milch saureferment. — Fortschr. der Medicin.

Nr. 11. 1889.
— Ueber das Milchsaureferment. — Centralbl. f. Bact. u. Parasitenk.

VI. Bd., p. 472. 1889.
— Onderzoekingen omtrent melkzuurgisting. — Ned. Tijdschr. v.

Geneesk., p. 88 and 509. 1890.
— - — Onderzoekingen over melkzuurgisting. — Weekblad van het Ned.

Tijdschr. v. Geneeskund. Nr. 4 and Nr. 19. 1890.
FORTI, 0. — Contribuzione alia conoscenza dei lieviti di vino. — Le stazioni

sperimentali agrarie Italiane. Vol. XXI., fasc. III. 1891.
FOTH, G. — Einfluss der Kohlensaure auf Garung und Hefebildung. —

Wochenschr. f. Braueri, p. 73 and p. 305. Berlin, 1887!
— Ueber den Einfiuss der Kohlensaure auf das Wachsthum und die
Garthatigkeit der Hefe und ihre Bedeutung fiir die Konservirung
des Bieres.— Woch. f. Br., p. 263. 1889.

FRAENKEL, C. — Grundriss der Bakterienkunde. — 3 Aufl. Berlin, 1890.
FRANKLANB, P. F. — New method for determining the number of micro-
organisms in air. — Proc. Roy. Soc. XLL, p. 443.
—Filtration of the air.— Philos. Trans, of the Roy. Soc. Vol. 178,

p. 113. 1887.
— and Fox, J. J. — On a pure fermentation of mannite and glycerine.

—Proc. Roy. Soc., London. Vol. XL VI. 1889.
— Micro-organisms in their relation to chemical change. — Roy. Inst. of

Great Britain. Meeting Feb. 19, 1892.
— and MAC GREGOR. — Fermentation of Arabinose with the Bacillus-

ethaceticus.— Trans. Chem. Soc., 1892, p. 737.
— and LUMSDEN. — Decomposition of Mannitol and Dextrose by the

Bacillus ethaceticus.— Trans. Chem. Soc., 1892, p. 432.
FRESENIUS. — Beitraege zur Mycologie. 1850-63.
FREUND, P. — Die Reinzuchthefe und der Geschmack des Bieres. —

Oesterr. Brauer- und Hopfen- Zeitung. 1890.
FRIEDLANDER. — Mikroskopische Technik. 1884.

GAYON. — Faits pour servir a 1'histoire physiologique des moisissures. —
Mem. de ia Soc. des sciences phys. et naturelles de Bordeaux.
1878.

— Sur la fermentation produite par le Mucor circinelloides. — Ann. de
Chim. et de phys. XIV., p. 258. 1878.

234 BIBLIOGRAPHY.

GAYON. — Sur Inversion et sur la fermentation du sucre de canne par

les moisissures. — Bull. Soc. cliim. XXXI., p. 139. 1879.
— Sur un precede' nouveau d'extraction du sucre des m6lasses. — Ann.

agronomique. 1880.
GAYON et DUBOURG. — Recherches sur la reduction des nitrates par les

infiniment petits.— Nancy, 1886.
— De la fermentation de la dextrine et de 1'amidon par les Mucor. —

Ann. de 1'Inst. Pasteur. Nr. 11. 1887.
— et DQBOURG. — Sur la fermentation alcoolique du sucre interverti. —

Compt. rend, de 1' Academic. T. CX., p. 865. Paris, 1890.
— et DUPETIT. — Sur un moyen nouveau d'empecher les fermenta-
tions secondaires. — Compt. rend., 8 Novbr., 1886.
GIUNTI, M. — On the effect of light on the acetic acid fermentation. —

Le Stat. speriment. Agrar. Ital. XVIII.

GOLDEN, K. E.— Fermentation of bread.— Botan. Gaz., p. 204. 1890.
GRAWITZ. — Beitrage zur systematischen Botanik der pflanzlichen

Parasiten mit experimentellen Untersuchungen iiber die durch sie

bedingten Krankheiten. — Virchows Archiv. B. 70. 1877.
GRIESSMAYER. — Hansen als Reformator der Garungsindustrie. — Allg.

Brauer- und Hopfenzeit. Nuremberg, 1890.
GROTENFELT, G. — Studien iiber die Zersetzung der Milch. — II. Ueber

die Virulenz einiger Milchsaurebakterien. III. Ueber die Spaltung

von Milchzucker durch Sprosspilze und iiber schwarzen Kase. —

Fortschr. der Medicin. Nr. 4. 1889.
GRUBER, M. — Eine Methode der Kultur anaerobischer Bakterien, nebst

Bemerkungen zur Morphologie der Butter sauregaruug. — Centr.

f. Bakt. p.li67 f. 1887.
— Ueber die Methoden der Priifung der Desinfektionsmittel. — Centr.

f. Bact. XI. B., p. 115. 1892.
GRONLUND. — Ueber Organismen in der Kiihlschiffwu'rze. — Allg. Zeits.

f. Bierbr. u. Malzfabr. Vienna, 1885.
Ueber bitteren, unangenehmen Beigeschmack des Bieres. Z. f . d. ges.

Brauwesen. 1887.
En ny Torula-Art og to nye Saccharomyces — Arter. Vidensk.

Medd. fra den Naturhist. Foren. Copenhagen, 1892.
GUILLEBEAU, A. — Beitrage zur Lehre von den Ursachen der fadenzieh-

enden Milch. Landw. Jahrbiicher der Schweiz. 1891.
HABERLANDT. — Das Vorkommen und die Entwickelung der sogenannten

Milchsaurehefe. — Wissensch.-prakt. Untersuchungen auf dem

Gebiete des Pflanzenbaues. 1875.
IlAGEN-ScHOW. — Pure yeast and fermentation. — Transact, of the Lab.

Club, p. 122, Vol. III. 1890.
HANSEN, E. CHR. — Les champignons stercoraires du Danemark. —

Resume d'un m&moire publi^ dans les "Videnskbl. Meddelelser " de

la Societ^ d'histoire naturelle de Copenhague. 1876.

BIBLIOGRAPHY. 235

HANSEN, E. CHH. — Contributions a la connaissance des organismes qui
peuvent se trouver dans la biere et le mout de biere et y vivre. —
Compt. rend, des : Meddel. fra Carlsberg Laboratoriet. Copen-
hagen. I. Bd., 2. II. 1879.*
— Oidium lactis.— Compt. Kend. des Medd. fra Carlsb. Lab. I. B.,

2. H. 1879.

— Saccharomyces colored en rouge et cellules rouges ressemblant a des
Saccharomyces.— Compt. rend, des Medd. fra Carlsb. Lab. I. B.;

2. H. 1879.

— Sur 1'influence que 1'introduction de Fair atmospherique dans le moiit
qui fermente exerce sur la fermentation. — Compt. rend, des
Medd. fra Carlsb. Lab. I. B., 2. H. 1879.

Hypothese de Horvath. — Compt. rend, des Medd. fra Carlsb. Lab.

I. B., 2. H. 1879.
— — Mycoderma aceti (Kiitz.) Pasteur et Myc. Pasteurianum nov. sp. —

Compt. rend, des Medd. fra Carlsb. Lab. I. B., 2. H. 1879.
— Charnbre humide pour la culture des organismes microscopiques. —
Compt. rend, des Meddel. fra Carlsberg Laboratoriet. I. Bd.,

3. H. 1881.

— Kecherches sur la physiologic et la morphologie des ' ferments
alcooliques. I. Sur le Sacch. apiculatus et sa circulation dans la
nature. — Compt. rend, des Meddel. fra Carlsberg Laboratoriet.

I. Bd., 3. H. 1881.

— Kecherches sur les organismes qui, a diffe'rentes epoques de
1'annee, se trouvent dans 1'air, a Carlsberg et aux alentours,
et qui peuvent se de>elopper dans le mout de biere (II). —
Compt. rend, des Meddel. fra Carlsb. Laborat. I. Bd., 4. H.
1882.
Recherches sur la physiol. et la morpkol. des ferments alcooliques.

II. Les ascospores chez le genre Saccharomyces. III. Sur les
Torulas de M. Pasteur. IV. Maladies provoque"es dans la biere
par des ferments alcooliques. — Compt. rend, des Meddel. fra
Carlsb. Lab. II. Bd., 2. H. 1883.

Bemerkungen iiber Hefenpilze. — Allgem. Zeitschr. fur Bierbrauerei

und Malzfabrikation. 1883.
— Neue Untersuchungen iiber Alkohplgarungspilze (Monilia). —

Berichte d. deutschen botan. Gesellsch. 1884.
— Uber Wiesners neue Priifungsmethode der Presshefe. — Dinglers

Polytechn. Journal, 1884, S. 419.
— Einige kritische Bemerkungen iiber Dr. Hueppes Buch : " Die

Methoden der Bakterienforschung." — Zeitschr. f. wissensch.

Mikrosk. und mikroskopische Technik. Bd. II. 1885.

* The "Meddelelser fra Carlsberg Laboratoriet "—Danish and French-
can be procured at the library of ffagerup, Copenhagen.

236 BIBLIOGRAPHY.

HANSEN, E. CHR.— Vorlaufige Mitteilungen uber Garungspilze. (Gelatin.
Netzwerk. — Scheidewandbildung. — Sacch. apiculatus.) — Botan.
Centralblatt. Bd. XXI., Nr. 6. 1885.

— Recherches sur la physiologie et la morphologie des ferments
alcooliques. V. Methodes pour obtenir des cultures pures de
Saccharomyces et de microorganismes analogues. VI. Les voiles
chez le genre Saccharomyces. — Compt. rend, des Meddel. fra
Carlsb. Laboratoriet. II. Bd., 4. H. 1886.

— Uber rot und schwarz gefarbte Sprosspilze. — Allg. Brauer- und
Hopfenzeitung. Nr. 95. Nuremberg, 1887.

— Noch ein Wort liber den Einfluss der Kohlensaure auf Garung und
Hefebildung. — Zeitschr. f . d. ges. Brauw. Nr. 13, S. .304.
Munich, 1887.

— Neue Bemerkungen zu Foths Abbandlung : " Einfluss der Kohlen-
saure auf Garung und Hefebildung." — Wochenschr. f. Brauerei.
S. 378. Berlin, 1887.

— Methods zur Analyse des Brauwassers in Riicksicht auf Mikro-
organismen. — Zeitschr. f. d. ges. Brauwesen. Nr. 1. Munich,
1888.

— Uber die zymotechnische Analyse der Mikroorganismen der
Luft. — Prager Brauer- und Hopfenzeitung. Nr. 19. Prague,
1888.

Untersuchungen aus der Praxis der Garungsiudustrie. — I. Heft.

Munich (Oldenbourgs Verlag), 1888. Zweite Ausg., 1890.

— Recherches sur la physiologie et la morphologie des ferments
alcooliques. VII. Action des ferments alcooliques sur les diverses
especes de sucre. — Compt. Rend. des. Meddel. fra Carlsb. Laborat.
II. Bd., 5. H. 1888.

— Recherches faites dans la pratique de Tindustrie de la fermentation.
Contributions a la biologie des microorganismes. I. Introduction.
II. Culture pure de la levure au service de 1'industrie. III. Obser-
vations faites sur les levures de biere. IV. Sur Texamen pratique,
au point de vue de la conservation, de la biere contenue dans les
tonneaux des caves de garde. — Compt. rend, des Meddel. fra
Carlsb. Labor. II. Bd., 5. H. 1888.

Uber die im Schleimflusse le bender. Baume beobachteten Mikro-
organismen.— Centralbl. f. Bakteriologie u. Parasitenk. V. Bd.,
p. 632 fl. 1889.

— Some points in connection with practical brewing : I. On the bac-
teriological analysis of the water and the air for brewing puposes.
II. On my system of pure yeast culture and its application
in breweries with top fermentation. — (Transact, of the Laboratory
Club. London, 1889.)

• Production des varie'te's chez les Saccharomyces. — Ann. de micro-
graphic. P. Miquel. T. II., Nr. 5., p. 214. Paris, 1890.

BIBLIOGRAPHY. 237

HANSKN, E. CHR. — Ueber die Pilzstudien in der Zymotechnik, mit
besonderer Riicksicht auf Prof. Dr. Zopfs neues Werk : " Die Pilze
in morphologischer, physiologischer, biologischer und systematis-
cher Beziehung." — Breslau, 1890. Zeits. f. d. ges. Brauwesen.
Nr. 16. Munich, 1890.

— Nouvelles recherchea sur la circulation du Saccharomyces apiculatus
dans la nature. — Ann. d. sc. nat. Botanique. VII. ser. T. XL,
Nr. 3, p. 185. 1890. Ann. de Microgr. Paris, 1890.

Qu'est-ce que la levure pure de M. Pasteur ? Une recherche expe"ri-

mentale. — Compt. rend. d. Meddel. fra Carlsb. Lab. III. B.,
1. H. 1891.

— Recherches sur la physiologie et la morphologie des ferments
alcooliques. VIII. Sur la germination des spores chez les
Saccharomyces. — Compt. rend. d. Meddel. f. Carlsb. Lab. III.
B.. 1. H. 1891.

— Neue Untersuchungen iiber den Einfluss, welchen eine Behandlung
mit Weinsaure auf die Brauereihefe ausiibt. — Zeitschr. f. d. ges.
Brauwesen. Nr. 1. Munich, 1892.

— Kritische Untersuchungen iiber einige von Ludwig und Brefeld
beschrtiebene Oidium-und Hefeformen.— Botan. Zeit. Nr.19. 1892.

— Recherches faites dans la pratique de I'industrie de la ferment-
ation. (Contribution a la biologie des microorganismes). V. Sur
1'analyse zymotechnique des microorganismes de 1'air et de
1'eau. VI. Nouvelles recherches sur les maladies provoque"es
dans la biere par des ferments alcooliques (Deuxieme memoire).
VII. Sur 1'exteusion actuelle de mon systeme de culture
pure de la levure.— Compt. rend. d. Medd. fra Carlsb. Lab.
III. B., 2. H. 1892.

— Untersuchungen aus der Praxis der Garungsindustrie (Beitrage zur
Lebensgeschichte der Mikroorganismen. — II. Heft. Munich.
Oldenbourgs Verl. 1892.

— Ueber die neuen Versuche, das Genus Saccharomyces zu streichen. —

Centralbl. f. Bakt. u. Paras. XIII. B. Nr. 1. 1893.
HARZ. — Untersuchungen iiber die Alkohol- und Milchsauregarung. —
Z. d. Allg.— Oester. Apotheker-Vereins. 1870-71.

— Grundziige der alkoholischen Garung.— Munich, 1877.
HAYDUCK. — Ueber die Existenz von Hefenrassen mit konstant sich
erhaltenden Eigenschaften. — Wochenschr. f. Brauerei, Nr. 20.
Berlin, 1885.

—Ueber Milchsauregarung.— Wochenschr. f. Br., Nr. 17. Berlin, 1887.
HEDIN, S. G. — Om bestamning af drufsocker genom Forjasning och
uppmatning af kolsyrans volym. — Lunds Univers. Arskrif t.
T. XXVII. 1891.

HEINZELMANN, G. — Die Reinzuchthefe in der Brennerei. — Zeitschr. fur
Spiritusindustrie. XV. Jahrg. Nr. 25. 1892.

238 BIBLIOGRAPHY.

HKSSE. — Ueber quantitative Bestimmung der in der Luft enthaltenen

Mikroorganismen. — Mitteil. d. K. Gesundheitsamtes. Bd. II. 1884.
HITSCHCOCK. — Studies of atmospheric dust. — Americ. monthly micro-
scop. Journal, I. 1880.
HOLM, J. CHR. — Aeltere und neuere Ansichten iiber Oberhefe und

Unterhefe. — Deutscher Brauer- und Malzer-Kalender. 1886-87.
Die Vorrichtungen in der Brauerei zur Kiihlung und Liiftung der

Wiirze.— Zeitschr. f . d. ges. Brauw. Nr. 20. 1887.
Bemerkung zu Foths Abhandlung iiber den Einfluss der Kohlen-

saure auf die Garth atigkeit der Hefe.— Zeitschr. f. d. ges. Brau-

wesen. p. 301. Nr. 15. Munich, 1889.
Sur les me"thodes de culture pure et spe"cialement sur la

culture de plaques de Koch et la limite des erreurs de cette

me"thode. — Compt. rend. Meddelels. fra Oarlsb. Laborat. Bd.

III., H. 1. 1891.
Ueber die Methoden der Reinkultur und im besonderen iiber die

Plattenkultur von Koch und iiber die Fehlergrenze dieser

Methode.— Zeitschr. f. d. ges. Brauw. p. 458 flg. Munich, 1891.
Analyses biologiques et zymotechniques de 1'eau destined aux

brasseries. — Compt. rend. des. Meddelel. fra Carlsb. Laborat.

III. Bd., 2. H. 1892.

HOLM and JORGKNSEN. — Le proce'de' de M. Effront, etc. (see Jorgensen).
HOLM et POTJLSEN. — Jusqu'a quelle limite peut-on, par la methode de

M. Hansen, constater une infection de "levure sauvage " dans

une masse de levure basse de Saccharomyces cerevisiae ? — Oompt.

rend, des Meddelels. fra Carlsb. Laborat. II. Bd., 4. H. 1886.
— Jusqu'a quelle limite, etc. Deuxieme Communicat. — Compt. rend.

des Meddelels. fra Carlsb. Laborat. II. Bd., 5. H. 1888.
HOLST, A. — Oversigt over Bakteriologien for laeger og studerende. —

Christiania, 1890.
HOLZNEB. — Ueber Triibung des Bieres. — Der Bayerische Bierbrauer.

p. 14. 1871.
— Zymotechnische Riickblicke auf das Jahr 1877. — Zeitschr. f. d. ges.

Brauwesen. p. 142. 1878.
HORVATH. — Ueber den Einfluss der Ruhe und der Bewegung auf das

Leben. — Pfliigers Archiv fur die gesamte Phjsiologie, Bd. 17.

1878.
HUEPPE, F. — Untersuchungen iiber die Zersetzungen der Milch durch

Mikroorganismen. Zur Geschichte der Milchsauregarung. — Mitt.

a. d. K. Gesundheitsamte. Bd. II. 1884.

Die Methoden der Bakterienforschung. 1891.

HULLE, L. v. D. und LAER, H. v. — Nouvelles recherches sur les bieres

Bruxelloises a fermentation dite spontanee. — M&n. cour. et autres

M6m. publics par 1'Ac. Royale de Belgique. Brussels, 1891.
IKUTA, M. — Die Sake"-Brauerei in Japan. — Chemikerzeit. Nr. 27. 1890.

BIBLIOGRAPHY. 239

IJRMISCH, M. — Der Vergarungsgrad, zugleich Studien iiber zwei Hefe-
charaktere. — Wocbenschr. f. Brauerei. Nr. 39 — 46. Berlin,
1891.

JACOBSEN, J. C. — Ueber die Carlsberger reingeziichtete Hefe. — Wocben-
schr. fiir Brauerei Nr. 10. Berlin, 1885.
JANSSENS, F. A.— Beitrage zu der Frage iiber den Kern des Hefezelle.

— Centralbl. f. Bakt. u. Paras. XIII. B., Nr. 20. 1893.
JAQUEMIN, G. — Du Saccbaromyces ellipsoideus et de ses applications
industrielles a la fabrication d'un vin d'orge. — Corapt. rend. T.
CVI. Nr. 10. 1888.

JENSEN, C. O. — Bakteriologiske Undersogelser over visse Mselke- og
Smorfejl. — 22. Beretn. fra den kgl. Veterinaer-og Landbohojsk.
Laborat. Copenhagen, 1891.

JODLBAUER, M. — Ueber die Anwendbarkeit der alkoholiscben Garung
zur Zuckerbestimmung. — Z. f. Eiibenzuckerindustrie. Nr. 15.
1888.
JORGENSEN, ALFRED. — Zur Analyse der Presshefe. — Dinglers polytechn.

Journal. Bd. 252, S. 424." 1884.

— Ueber das Verhaltniss der Alkohol-Fermentorganismen gegeniiber
der Saccbarose. — Allg. Brauer- und Hopfenzeitung, Nuremberg.
Nr. 20. 1885.
— Giebt es verscbiedene Rassen der sogenannten Saccharomyces cere-

visiae ? — Ibid., 1885, Festnummer.

— Vorlaufiger Bericht iiber Versucbe im grossen mit absolut reiner
Oberbefe, nach Hansens Methode dargestellt. — Allg. Zeitscbr. fiir
Bierbr. und Malzfabr. Nr. 36. Vienna, 1885.
— Reingeziichtete Oberhefe. — Allgem. Brauer- und Hopfenztg. Nr. 8.

Nuremberg, 1886.
— Ueber den Unterscbied zwischen Pasteurs und Hansens Standpunkt

in der Hefenfrage. — Zeitschr. f. d. ges. Brauw. Municb, 1888.
— Neue Bemerkungen iiber die Kulturmetboden und die Analyse der

Hefen. — Zeitschr. f. d. ges. Brauw. Munich, 1888.
— Brefelds neue Hefenuntersuchungen. — Allg. Brauer- und Hopfenztg.

Nr. 74. Nuremberg, 1888.

— Die zymotecbnische Wasser- Analyse in Hueppes Buch : Die
Methoden der Bakterienforschung.— 4. Aufl. 1889.— Centralblatt.
fiir Bakteriologie. V. Bd., Nr. 22. 1889.

Die Anwendung der nach Hansens Methode reingeziichteten ober-

garigen Hefe in der Praxis. — Brauer- und Malzer-Kalender fiir
Deutschland und Oesterreich. 1889-90.
— Recension von : Duclaux, Sur la conservation des levures. — Botan-

isches Centralblatt. X. Jahrg., Nr. 49. 1889.
— Die Aufbewahrung der ausgewahlten Hefenrasse. — Allg. Zeitschr.

f. Bierbr. und Malzf., Nr. 46. Vienna, 1890.
Sarcina.— Allg. Br. und Hopfenztg. Nr. 115. Nuremberg, 1890.

240 BIBLIOGRAPHY.

JORGENSEN, ALFRED. — Zur Analyse der obergarigen Hefe in Brauereien
und Brennereien nach Ilansens Methode. — Zeitschr. f. d. ges.
Brauwesen. Nr. 3. 1891.
— Ueber die Analyse der Brauereihefe nach Hansens Methode. — Der

amerik. Bierbr. 1891.

— Les me"thodes de Pasteur et de Ilansen pour obtenir des cultures
pures de levure. Une re"ponse a M. Velten. — La Gazette du
brasseur. Nr. 215. Brussels, 1891. — (German in Allg. Brauer-
und Hopfenzeitung. Nr. 142. Nuremberg, 1891).

Die Feinde des Brauereibetriebes. Vortrag, gehalten bei der

schwedischen Brauerversammlung in Gothenburg. — Allgem.
Brauer- und Hopfenzeitung. Nr. 97-98. Nuremberg, 1891.
— et HOLM, JUST CHR. — Le precede de M. Effront pour la purifica-
tion et la conservation de la levure a 1'aide de 1'acide fluorhy-
drique, et des fluorures. — Moniteur scientif. du Dr. Quesneville.
4 ser., T. VII. Mars. Paris, 1893. (Chemiker Zeitung. Nr. 23-
1893.)

— On the application of Hansen's system in breweries with top-
fermentation. — Written for the International Brewers' Congress
in Chicago, 1893. — Proceed, of the Intern. Br. Congr. New
York, 1893.

KABRHEL, G. — Ueber das Ferment der Milchsaure<>arung in der Milch.
—Allgem. Wien. med. Ztg. Nr. 52 u. 53. 1889.

KAYSER, E. — Action de la chaleur sur les levures. — Ann. de 1'Inst.

f Pasteur. Nr. 10. 1889.
— Etudes sur la fermentation du cidre. — Ann. de 1'Inst. Pasteur.

Nr. 6. 1890.
— Note sur les ferments de 1'ananas. — Ann. de 1'Inst. Pasteur. 1891.

Contribution a l'6tude physiologique des levures alcooliques du

lactose. — Ann. de I'lnstitut Pasteur. Nr. 6. 1891.
— Contribution a I'e'tude des levures de vin. — Ann. de 1'Inst. Pasteur.
T. VI. 1892.

KELLNER, MORI und NAGAOKA. — Beitrage zur Kenntniss der invertier-
enden Fermente. — Zeitschr. fur physiolog. Chemie. XIV. Bd.,
3. H. 1889.

KERN. — Ueber ein Milchferment des Kaukasus. — Botanische Zeitg.
1882. Biolog. Centralblatt., 1882.

KLEBS, J. — Ueber fraktionierte Kultur. — Archiv. f. experiment. Path-
ologie. Bd. I.

KLEIN, C. — Beitrag zur Kenntniss des roten Malzschimmels. — Mitt. a.
d. oesterr. Versuchsstation f. Brauerei u. Malzerei. V. Heft.
Vienna, 1892.

KLETN, E. — Micro-organisms and disease. — 3rd Edition, London, 1886.

KLEIN, L. — Botan. Bakterienstudien I. — Centralblatt fur Bakteri-
ologie und Parasitenkunde. Bd. VI. Nr. 12-14. 1889.

BIBLIOGRAPHY. 241

KLEIN, L. — Botan. Bakterienstudien II. — Berichte der deutschen botan.

Gesellschaft. Bd. VII. Berlin, 1889.
KNIERIM, W. v. und AD. MAYER. — Ueber die Ursache der Essig-

garung.— Die landw. Versuchsstat. Bd. XVI. 1873.
KOCH, ALFRED. — Jahresbericht iiber die Fortschritte in der Lehre
von den Garungsorganismen. I. (1890). 1891. II. (1891).
1892.
KOCH, R. — Verfahren zur Untersuchung, zum Konservieren und Photo-

graphieren der Bakterien. — Beitr. z. Biol. II.
— Ueber Desinfektion. — Mitt. d. K. Gesundheitsamtes. 1881.
— Zur Untersuchung von pathogenen Organismen. — Mitt. d. K.

Gesundheitsamtes. Bd. I. Berlin, 1881.

KOEHLER, J. — Saccharomyces membranaefaciens Hansen. — Mitt. a. d.
oesterr. Versuchsstation f. Brauerei u. Malzerei. V. Heft.
Vienna, 1892.
KOKOSINSKY, ED. — Levure pure de fermentation haute. — Gaz. du

brasseur. Nr. 115. 1889.
— A propos de levure pure. — La brasserie du Nord (de la France).

Nr. 169. 1889.

—La levure pure.— Gaz. du brass. Nr. 142. 1890.
— Application industrielle de la methode Hansen a la fermentation
haute dans le Nord de la France. — Compt. rend, de la Station
scientifique de brasserie. Ghent, T. I. 1890.
KRAMER, ERNST. — Studien iiber die schleimige Garung. — Sitzb. d. K.

Akad. d. "Wise. Vienna. Monatshefte fiir Chemie. X. 1889.
— Einwirkung von Saccharomyces Mycoderma auf Wein. — Agramer

botan. phys. Inst. 1889.
— Bakteriologische Untersuchungen iiber das " Umschlagen " des

Weines.— Landwirtschaftl. ^7ersuchsstationen. Bd. XXXVII.
— Ueber einen rotgefarbten, bei der Vergarung des Mostes mitwirk-

enden Sprosspilz. — Oesterr. landw. Centralblatt. I. 1891.
KRASSER. — Ueber das angebliche Vorkommen eines Zellkerns in den

Hefenzellen. — Oesterr. botan. Zeitschr. Nr. 11. 1885.
KRATSCHMER und NIEMILOWICZ. — Ueber eine eigentiimliche Brot-

krankheit.— Wiener klin. Woch. Nr. 30. 1889.

KUKLA. — Die heurigen triiben Biere. — Bericht der Versuchsanstalt fiir
Brauindustrie in Bohmen. Prager Brauer- und Hopfenztg.
Prague, 1889.
KUTZING. — Ueber Hefe und Essigmutter. — Journal fiir prakt. Chemie.

XL, p. 385. 1837.

— Phycologia generalis. — S. 148. 1843.
— Grundziige der philosophischen Botanik. 1851.
LASCHE, A. — Die amerikanische Brauereihefe. — Der Braumeister. Nr. 6.

Chicago, 1891.
— Mycoderma-Arten. — Der Braumeister. Nr. 7. Chicago, 1891.

R

242 BIBLIOGRAPHY.

LASCHE, A. — Das Mycoderma und die Praxis. — Der Braumeister.

Nr. 10. Chicago, 1891.
— Saccharomyces Joergensenii. — Der Braumeister. Nr. 8. Chicago,

1892.
—Two species of red Mycoderma. — Der Braumeister. Nr. 9.

Chicago, 1892.
— The action of certain species of pure yeast in practice. — Der Brau-

nieister, pp. 270 and 308. Chicago, 1892.

Influence of certain temperatures upon different yeast forms, con-
tained in acid and alkaline nutrient media. — Der Braumeister.

Nr. 15 and 17. Chicago, 1892.
LAER, H. VAN. — Note sur les fermentations visqueuses. — (Memoires

couronnes et autres me"moires publ. par 1' Academic royale de

Belgique. T. XLIII., 1889).
— Application industrielle de la me'thode Hansen a la fermentation

haute. — Station scientifique de brasserie. Compt. rend. T. I.

Ghent, 1890.
— Contribution a 1'histoire des ferments des hydrates de carbone.

Bacille des bieres tournees. — Mem. publ. par 1'Acad. royale de

Belg. 1892.
— et v. D. HULLE. — Nouvelles recherches sur les bieres Bruxelloises a

fermentation dite spontanee. — Mem. cour. et autres mem. publics

par 1'Acad. Royale de Belgique. Brussels, 1891.
LAFAR, FRZ. — Physiologische Studien iiber Essiggarung und Schnell-

essigfabrikation. I. — Centralbl. f. Bakt. u. Paras. XIII.

Nr. 21-22. 1893.
LANGER, TH. — Grundriss der Chemie fur Brauer und Malzer. — Leipzig,

1890.
LAURENT, C. — Le Bact^rie de la fermentation panaire. — Bull, de 1'Acad.

roy. de Belgique. T. X., Nr. 12. 1885.
LECHARTIER et BELLAMY. — IStude sur les gaz produits par les fruits.

Note sur la fermentation des fruits. — Cornpt. rend. LXIX.,

1869 ; LXXV., 1872 ; LXXIX., 1874.
LIBORIUS, P. — Beitrage zur Kenntniss des Sauerstoffbediirfnisses der

Bakterien.— Z. f. Hygiene. I. Bd., 1. H. Berlin, 1886.
LINDET, L. — Influence de la temperature de fermentation sur la produc-
tion des alcools superieurs. — Compt. Ptend. de 1'Ac. des sc. de

Paris. T. CVII., Nr. 3. 1888.
LINDNER, P. — Ueber ein neues, in Malzmaischen vorkommendes,

Milchsaure bildendes Ferment. — Wochenschr. f. Br. Nr. 23.

1887.
— Die Askosporen und ihre Beziehungen zur Konstanz der Hefen-

rassen.— Wochenschr. f. Br. Nr. 39. 1887.
— Ueber rot und schwarz gefarbte Sprosspiize. — Wochenschr. f. Br.

Nr. 44. Berlin, 1887.

BIBLIOGRAPHY. 243

LINDNER, P. — Nachweis von Mikroorganismen in der Luf t von

Garungsbetrieben. - Wochenschr. f. Br. 1887.

— Verandert sich der Charakter einer Brauereihefe bei fortgesetzter
Kultur unter veranderten Ernahrungsbedingungen ? — Woclienschr.
f. Br. Nr. 3. 1888.
— Ueber eimge Garversuche mit verschiedenen Hefen. — Wochenschr.

f. Br. Nr. 14. 1888.
— Das Langwerden der Wiirze durch Dematium pullulans. — Wochen-

schrift f . Br. Nr. 15. 1888.

— Die Sarcina-Organismen der Garungsgewerbe. Berlin, 1888.
— Die Ursache des langen Weissbieres. — Woclienschr. f. Br. Nr. 9.

1889.
— Ruft Sarcina im untergarigen Biere Krankheitserscheinungen

hervor oder nicht ? — Wochenschr. f. Br., p. 161. 1890.
— ^-Bemerkungen zu Jorgensens Aufsatz iiber Sarcina. — Wrochenschr.

f. Br. Nr. 41. 1890.

LINOSSIER, G., et Roux, G. — Sur la fermentation alcoolique et la
transformation de 1'alcool en aldehyde provoquees par le cham.
pignon du muguet. — Compt. rend, de 1'Acad. des sc. de Paris
T. CX. 1890.

LINOSSIER, G. — Action de 1'acide sulfureux sur quelques Champignons
infe"rieurs et en particulier sur les levures alcooliques. — Ann. d.
I'lnst. Past. T.V. 1891.
LINTNER, C. — Ueber Biertriibung. — Der Bayrische Bierbrauer, p. 64.

1871.
— Ueber einige Resultate zyrnotechnischer Untersuchungen. — Zeitschr.

fiir das ges. Brauwesen, p. 221. 1876.
— Lehrbuch der Bierbrauerei. 1878.
— Ueber Malz und dessen Einfluss auf die Haltbarkeit und Giite des

Bieres. — Zeitschr. f . d. ges. Brauwesen, p. 384. 1880.
— Altes und Neues iiber Bierbrauerei. — Zeitschr. fiir das ges. Brauw.
p. 153. 1881.

Zymotechnische Riickblicke auf das Jahr 1884. — Zeitsch. f. d. ges.

Brauw. S. 399. Munich, 1885.

LINTNER, C. J. — Ueber Isomaltose und deren Bedeutung fiir die Bier-
brauerei.— Zeitschr. f. d. ges. Brauw. S. 6. 1892.
— Ueber die Vergarbarkeit der Isomoltose. — Ibid., S. 106. 1892.
— Handbuch der landwirtschaf tlichen Gewerbe. Berlin, 1893.
LISTER. — On the nature of fermentation. — Quarterly Journal of micro-
scopical science. Vol. 18. 1878.
— On the lactic fermentation and its bearing on pathology. — Transact.

of the Pathological Society of London. Vol. 29. 1878.
LOEFFLER. — Vorlesungen iiber die geschichtliche Entwickelung der

Lehre von den Bakterien. 1887.
LOEW, E. — Ueber Dematium pollulans. — Pringsh. Jahrb. Bd. VI.

244 BIBLIOGRAPHY.

LOEW, 0. — Die chemischen Verhaltnisse des Bakterienlebens. — Centralbl.

f. Bakt, und Parasitenkunde. IX. Bd. 1891.
LUDWJG, F. — Weitere Mitteilungen iiber Alkoholgarung und die

Schleimfliisse lebender Baume. — Centr. f . Bakt. und Paras.

VI. Bd. Nr. 5-6. 1889.
MACCARTIE, J. C., und DE BAVAY.— On the application of Hansen's

system in top-fermentation breweries. — Austral. Br. Journal,

20 Dec., 1886, and 20 Jan., 1889. The Brewers' Journal

(London), Nr. 291. 1889.
MACH, E., und PORTELE, K. — 1. Ueber die Garung von Trauben- und

Aepfelmost mit verschiedenen reingeziichteten Hefenarten.

2. Ueber das Verhaltniss, in welcheni sich Alkohol und Hefe

wahrend der Gahrung bilden. 3. Ueber den Gehalt an stickstoff-

haltigen Substanzen in Traubenmosten aus dem Anstaltsgute in

S. Michele. 4. Uber die Veranderungen im Gehalt von Gesamt-

saure und Glycerin wahrend der Garung und Lagerung der

Weine. 5. Versuch iiber die Abnahme des Farbstoffgehaltes

beim Lagern der Weine. 6. Ueber die Zusammensetzung einer

Anzahl Aepfel- und Birnsorten aus dem Anstaltsgute. — Die Land-

wirtsch. Versuchsstationen, herausg. v. Nobbe. B. XLI. Heft. 4.

Berlin, 1892.
MARCANO. — Fermentation de la fecule, presence d'un vibrion dans les

grains du mai's qui germe. — Compt. rend. p. 345. 1882.
MAERCKER, M. — Handbuch der Spiritusfabrikation. — 5. Auflage.

Berlin, 1890.
MAERCKER, CLUSS und SCHUPPAN. — Das Flusssaureverfahren in der

Spiritusfabrikation. Berlin, 1891.
MARSHALL WARD, H. — The ginger beer plant, and the organisms

composing^ it. Philos. Trans. Roy. Soc., Vol. 183 B. p. 125. 1892.
MARTINAND. — Etude sur 1'analyse des levures de brasserie. — Comp.

rend, de 1'Ac. d. sc. de Paris. T. CVIL, Nr. 19. 1888.
— et RIETSCH. — Des micro-organismes que Ton rencontre sur les

raisins murs et de leur developpement pendant la fermentation. —

Compt, rend, de 1'Acad. d. sc. Paris. T. CXII.
MARX, L. — La levure pure de brasserie. — Revue universelle de la

brasserie et la malterie. Nr. 622. 1885.
— Le laboratoire du brasseur.— 2me ed. Paris, 1889.
— Les leviires des vins. — Moniteur scientifique du Dr. Quesneville.

Paris, 1888.
MATTHEWS. — On the red mould of Barley. — Journal of the Roy. Micr.

Soc. London, 1883.
MIFLET. — Uutersuchungen iiber die in der Luft suspendierten Bakterien.

— Beitr. z. Biol. d. Pflanzen. III. Bd. 1879.
MIQUEL. — Etudes sur les poussieres organise'es de I'atmosphere. —

Annuaire de 1'observ. de Montsouris pour 1'an 1879.

BIBLIOGRAPHY. 245

MIQUEL. — Nouvelles Recherches sur les poussieres organ, de 1'atmosph. —

Ann. de 1'obs. Montsouris. 1880.
— Etude ge"ne"rale sur les bact6ries de 1'atmosphere. — Extrait de

1'annuaire de Montsouris. 1881.
— Eludes generates des bacteMes de 1'atmosphere. — Ann. de 1'observ.

Montsouris. 1882.

— Des organisines vivants de 1'atmosphere. Paris, 1883.
— Dixieme memoire sur les poussieres organisees de 1'air et des eaux. —

Ann. de Montsouris pour 1888.
— De 1'analyse microscopique de 1'air au moyen de filtres solubles. —

Ann. de micrographie. S. 153. Paris, 1889.

— Manuel pratique d'analyse bact<§riologique des eaux. Paris, 1891.
MITSCHERLICH. — Lehrbuch der Chemie. 1834.

NATHAN. — Die Bedeutung der Hefenreinzucht fur die Obstweinberei-
tung.— Der Obstbau. Nr. 2. Stuttgart, 1891. Also printed
in Wittmacks Gartenflora. Berlin, 1891.
— Neuere Fortschritte auf dein Gebiete der Fruchtweinbereitung. —

Der Obstbau. Nr. 3-4. Stuttgart, 1892.
NAGELI. — Die niederen Pilze in ihren Beziehungen zu den Infektions-

krankheiten. Munich, 1877.
— und LOEW. — Ueber die chemische Zusammensetzung der Hefe. —

Sitzb. der bayer. Akad. d. Wissens., p. 161. 1878.
— Theorie der Garung. 1879.
—Ueber die Beweguugen kleinster Korperchen. — Sitzb. d. math.-phys.

Klasse d. Akad. d. Wissench. Munich, 1879.
— Untersuchungen iiber niedere Pilze. Munich, 1882.
NENCKI, M. — Die isomeren Milchsauren als Erkennungsmittel einiger
Spaltpilzarten.— Centralbl. f . Bakt und Parasitenkunde. IX. Bd.
1891.

NEUMAYER, JOH. — Untersuchungen iiber die Wirkung der verschie-
denen Hefearten, welche bei der Bereitung weingeistiger Getranke
vorkommen, auf den menschlichen und tierischen Organismus. —
Inaug.-Diss. Munich, 1890.

OLSEN, JOHAN-. — Er Sublimat i en Fortynding af 1 pro Mille et
paalideligt Desinfektionsmiddel ? — Nyt Magasin for Lseger,
XIV. B. Christiania, 1884.

Gjaring og Gjaringsorganismer. — Christiania, 1890.

PASTEUR, L. — Memoire sur la fermentation appelee lactique. — Ann. de
chim. et phys. LIL, p. 404. 1858.

Memoire sur la fermentation alcoolique. — Ann. de chim. et de phys.

LVIIL, p. 359. 1860.

Fermentation butyrique. Animalcules infusoires vivant sans oxygene

libre et determinant des fermentations . — Oompt. rend . , T . LIL 1 86 1 .

Memoire sur la fermentation ace"tique. — Ann. scientif. de 1'Ecole

normale superieure. T. I. 1864.

246 BIBLIOGRAPHY.

PASTEUR, L. — Etudes sur le vin. 1866.
— Etudes sur le vinaigre. 1868.
— if tudes sur la biere. 1876.
— Note sur la fermentation des fruits et sur la diffusion des germes

des levures alcooliques.— Compt. rend. T. 83, S. 173. 1876.
— Examen critique d'un 6crit posthume de Claude Bernard sur la

fermentation alcoolique. — Lecture faite a 1'acadeinie de Medecine

le 26 nov. 1878.
PEDERSEN, R. — Recherches sur quelques facteurs qui ont de 1'influence

sur la propagation de la levure basse du Sacch. cerevisise. — Compt.

rend, des Meddel. fra Carlsb. Labor. I. Ed., 1. II. 1878.
— Sur 1'influence que 1'introduction de 1'air atmospherique dans le

mout qui fermente exerce sur la fermentation. — Compt. rend, des

Meddel. fra Carlsb. Labor. I. Bd. 1. H. 1878.
PETERSEN, A. — Einige Bemerkungen iiber das Liiften der Wiirze. —

Zeitschr. f. d. ges. Brauw Munich, 1888.
Sarcina ini Biere ohne irgend eine Krankheitserscheinung. — Zeitschr.

f. d. ges. Brauw. Nr. 1. 1890.
PETRI. — Zusammenfassender Bericht iiber Nachweis und Bestimmung

der pflanzlichen Mikroorganismen in der Luft. — Centralbl. fiir

Bakt. und Parasitenk. II. Bd., Nr. 5, 6. 1887.
PICHI, P. — Sopra 1'azione dei sali di rame nel mosto di uva sul Saccharo-

myces ellipsoideus. — Nuova rassegna di viticoltura ed enologia.

Fasc., Nr. 5. 1891.
— Ricerche morfologiche e fisiologiche sopra due nuove specie di

Saccharomyces prossime al S. membranaefaciens di Hansen. — la

memoria con 4 tav. Estr. d. Ann. d. R.S. di viticolt. Conegliano.

Ser. III., An. I. Fasc. 2. 1892.
— Sulla fermentazione del mosto di uva con fermenti selezionati. — Ibid.

Ser. III., An. I. F. 1. 1892.
— e MARESCALCHI. — Sulla ferm. del mosto di uva con fermenti selez.

—Not. 2a Ibid. F. 3. 1892.
Ricerche fisiopatologiche sulla vite in relazione al parassitismo della

peronospora. — Ibid. F. 1. 1892.
POPOFF. — Sur un bacille anaerobie de la fermentation panaire. — Ann. de

1'Inst. Pasteur, p. 674. 1890.
PRAZMOWSKI. — Untersuchungen iiber die Entwickelungsgeschichte und

Fermentwirkung einiger Bakterienarten (Clostridium butyricum).

—Leipzig, 1880.
PRIOR. — Ueber die Erfahrungen beim Arbeiten mit Niirnberger Rein-

hefen. — Bayer. Brauer- Journal. Niirnberg, 1891.
RATZ, ST. v. — Ueber die schleimige Milch — Archiv. fiir Tierheilkunde.

Bd. XVI. S. 87. 1890.
RAUM, JOH. — Zur Morphologic und Biologie der Sprosspilze. — Zeitschr.

f. Hygiene. X. Bd. 1891.

BIBLIOGRAPHY. 247

RAYMAN, B., und KRUIS, K. — Chemisch-biologische Studien (Saccha-

romyces und Mycoderma). — Prague, 1891.

REESS — Botaii. Untersuchungen iiber die Alkoholgarungspilze. 1870.
— Ueber den Soorpilz. — Sitzungsber. der physikalisch-med. Sozietat

Erlangen. Botan. Ztg. 1878.

REINKE.— Ueber Sarcina.— Wochenschr. f . Brauerei. 1885, 1886.
RICHET. — De la fermentation lactique du sucre de lait. — Compt. rend.

LXXXVI. 1878.
— De quelques conditions de la fermentation lactique. — Compt. rend

LXXXVIII. 1879.
RIETSCH. — De la possibilit6 de comnmniquer divers bouquets aux

vins par 1'addition dans la cuve a fermentation de levures

cultivees. — Bull, de la Societe d'agriculture, stance 8 fe"vr«

1890.
— et MARTINAND. — Des micro-organismes que Ton rencontre sur les

raisins murs et de leur developpement pendant la fermentation. —

Compt. rend, de 1'Acad. d. sc. Paris. T. CXII.
ROMMIER, A. — Sur la possibility de communiquer le bouquet d'un vin

de qualite a un vin commun en changeant la levure qui le fait

fermenter. — Bull, de la soc. chimique de Paris. Se*r III.,

Tom. II. 1889.
Roux — Sur une levure cellulaire qui ne secrete pas de ferment invertif .

Bull, de la Soc. chim., XXXV., p. 371. 1881.
SALAMON, GORDON. — Cantor Lectures on Yeast. London, 1888.
SALKOWSKY. — Ueber Zuckerbildung und andere Fermentationen in der

Hefe.— Z. f. phys. Chem. Bd. XIII. 1889.
SCHEELE, C. W. — Anmarkningar om sattet att conservera attika. —

Kungl. Vetenskaps-Academiens nya Handlingar. T. III., p. 120.

Stockholm, 1782. Also in Memoires de Chimie. Dijon, 1785.
SCHIFFERER, A. — Uber die nichtkrystallisirbaren Produkte der Ein-

wirkung der Diastase auf Starke. — Inaug. Diss. Kiel. 1892.
SCHLEGEL, H. — Ein Versueh mit geziichteter Weinhefe in der Praxis. —

Weinbau und Weinhandel. 9. Jahrg. Nr. 44. 1891.
SCHLOESING, TH. — Sur la fermentation en masses du tabac pour poudre.

Mem. des Manufactures de 1'Etat. T. II., Livr. 1. 1889.
SCHMITZ. — Resultate der Untersuchungen iiber die Zellkerne der

Thallophyten. — Separat-Abdruck d. Sitzungsb. d. niederrhein.

Ges. f . Natur- und Heilkunde. S. 18. Bonn, 1879.
SCHOLL, II. — Beitrage zur Kenntnis der Milchzersetzungen durch

Mikroorganismen. — II : Ueber Milchsauregarung. Fortschr. d.

Medicin, Nr. 2. 1890.
SCHROTER. — Ueber einige durch Bakterien gebildete Pigmente. — Cohns

Beitrage z. Biologie der Pflanzen. I. Bd., 2. H. 1872.
SCHULZE, FR. — Lehrbuch der Chemie fiir Landwirte. — (II. Bd. 2. Abt.

1860.)

248 BIBLIOGRAPHY.

SCHUMACHER. — Beitrago zur Morphologic und Biologie der Hefe. —

Sitzungsbericht d. K. K. Akad. d. Wiss. Vienna, 1874.
SCHUURMANS STECKHOVEN. — Saccharomyces Kefyr. — Inaug. Diss.

Utrecht, 1891.
SCHUTZENBERGER — Die Garungserscheinungen. Leipzig, 1876.

— Les fermentations. Paris, 1879.
SCHWACKHOFER, F. — Ueber reingeziichtete Hefe. — Allgem. Zeitschr. f.

Bierbrauerei und Malzfabrikation. S. 1040. Vienna, 1890.
Beitrag zur Beurteilung des Wassers fiir die Zwecke der Brauerei

und Malzerei. — Mitt. d. osterr. Versuchsstation fiir Brauerei u.

Malzerei in Wien, Heft III. 1890.
SCHWANN. — Vorlaufige Mitteilung betreffend Versuche iiber die Wein-

garung und Faulnis — Poggend. Ann. XLL, p. 184. 1837.
SEYNES, J. de.— Sur le mycoderma vim.— Compt. rend. T, LXVII.

1868. Ann. d. sc. nat. Botanique V., Serie X. 1869. Recherches

sur les vegetaux inferieurs, 3e fasc. Paris, 1885.
SOYKA. — Ueber den Uebergang von Spaltpilzen in die Luft. — Sitzungs-

ber. der math.-phys. Klasse der Akademie der Wissensch. zu

Miinchen. Bd. IX. 1879.
STENGLEIN. — Zusammenstellung der Erfahrungen mit Reinzuchthefe. —

" Alkohol."— Berlin, 1893.
STORCH, V. — Nogle Undersogelser over Flodens Syrning. — 18. Beretn.

fra den kgl. Veterinaer-og Landbohojsk. Laborat. Copenhagen,

1890.
— Untersuchungen iiber Butterfehler und Sauerung des Rahmes. —

Milchztg. 1890.
SUCHSLAND, E. — Ueber das Wesen der Tabakfermentation und iiber die

sich daraus ergebende Moglichkeit, den Fermentationsprozess

behufs Veredelung der Tabake zu beeinflussen. — Period. Mitteil.

des Tabak-Vereins. Nr. 38. Mannheim, 1892.
THAUSING, J. — Einfluss der Hefegabe auf Hauptgarung, Hefe und

Bier. — 14. Jahresbericht der ersten osterr. Brauerschule in

Modling. 1884.
Ueber reingeziichtete Hefe. — Allg. Zeitschr. f. Bierbr. und Malz-

fabrik. S. 755. Vienna, 1885.
Die Theorie und Praxis der Malzbereitung und Bierfabrikation.

Leipzig, 1893.
— Ueber die Bildung des " Bruches " bei der Hauptgarung. — Brauer-

und Malzer-Kalender. II. Teil. Stuttgart, 1889-90. Zeitschr.

f . d. ges. Brauwesen, p. 61. Munich, 1890.
TIEGHEM, PH., VAN. — Sur le Bacillus Amylobacter et son role dans la

putrefaction des tissus vegetaux. — Bull, de la Soc. bot. de France.

1877.
— et LE MONNIER. — Recherches sur les Mucorinees. — Ann. de sc.

nat. Bot. 5. ser T. XVII. 1873.

BIBLIOGRAPHY. 249

TIEGHEM, PH., VAN. — Nouvelles recherches sur les Mucorinees. — Ann.

de sc. nat. Bot. 6. ser. T. I. 1875.
— Troisieme memoire sur les Mucorinees. — Ann. de sc. nat. Bot.

6. ser. T. IV. 1878.

— Leuconostoc mesenterioides. — Ann. de sc. nat. 6 ser. T. VII.
TOLLENS und STONE. — Ueber die Garung der Galaktose. — Ber. d. d.

chem. Ges. XXI. p. 1572. 1888.
TRAUBE. — Theorie der Ferment wirkungen. — Berlin, 1858. Ber. d. d.

chem. Ges. 1874, 1876.

TULASNE. — Selecta fungorum carpologia. Paris, 1861.
TURPIN. — Memoire sur la cause et les effets de la fermentation alcoo-

lique et aceteuse.— Compt. rend. VII., p. 379. 1838.
TYNDALL. — Further researches on the deportment and vital persistence

of putrefactive and infective organism*. London, 1877-78.
-- — Essays on the floating matter in the air in relation to putrefaction

and infection. London, 1881.
UDRANSKY, L. — Studien iiber den Stoffwechsel der Bierhefe. — I. Beitr.

zur Kenntniss der Bildung des Glycerins bei der alkoholischen

Garung.— Zeitschrift fur physiologische Chemie. Bd. XIII. 1889.
UFFELMANN, J. — Verdorbenes Brot. — Centralbl. f. Bakt. und Parasitenk.

VIII. Bd. Nr. 16. 1890.
VELTEN. — Conference. — Revue universelle de la brasserie et malterie.

Nr. 742 et 743. 1888.
VUYLSTEKE. — Contribution a 1'etude des Saccharomyces fermentant en

concurrence. — Ann. de micrographie. — Ann. II., Nr. 5, 1889. —

German in Zeitschr. fur das ges. Brauw. Nr. 24, 1888, und Nr.

1, 1889.
WEIGMANN, H. — Der Zweck und die Aufgaben der bakteriologischen

Abteilung der milchwirtschaftlichen Versuchsstation in Kiel.—

Milchzeitung, p. 793 u. 813. 1889.

—Ueber bittere Milch.— Milchzeitung Bd. XIX., p. 881. 1890.
Zur Sauerung des Rahmes mittelst Bakterienreinkulturen.— Landw.

Woch. f. Schlesw.-Holst., Nr. 29. 1890.
— Neue Mitteilungen iiber Rahmsauerung mittelst Reinkulturen von

Saurebakterien.— Milchzeitung, Bd. XIX., p. 944. 1890.
— Erfahrungen iiber die Rahmsauerung mit Bakterienreinkulturen. —

Landw. Woch. f. Schlesw.-Holst., Nr. 16. 1892.

WICHMANN, H. — Die Hefereirikultur und die Bakterienfrage. — Mit-
teilungen der osterreichischen Versuchs-Station fur Brauerei und

Malzerei in Wien. I. H. S. 64. 1888.

WICHMANN, H. und ROHN S.— Ueber Bierfiltration.— Ibid. S. 79.
—Ueber Wasser filtration. —Mitt. d. oesterr. Vers. Stat. f. Brauerei

und Malzerei. V. Heft. Vienna, 1892.
— Biolog. Unters. d. Wassers f. Brauereizwecke. — Mitt, d, oest. V. S.

f. Br. a. Malz. V. Heft. Vienna, 1892.

250 BIBLIOGRAPHY.

WIESNER. — Bedeutung der technischen Rohstofflehre (techn. Waren-

kunde) als selbststandiger Disziplin und iiber deren Behandlung

als Lehrgegenstand an techn. Hochschulen. — Dinglers poly-

technisches Journal. Bd. 237. 1880.
— Untersuctmngen iiber den Einfluss, welchen Zufuhr und Entziehung

von Wasser auf die Hefezellen aussern. — Sitzb. der Wiener

Akademie. Bd. 59. 1889.
WIJSMANN, H. P. — Ueber den Stickstoffgeh alt von Saccharomyces

ellipsoideus. Vortrag. — Utrecht. Zeitschr. f. d. ges. Brauwesen.

Nr. 17. 1891.

WILL, H. — Wie wird reine Hefe geziichtet ? — Zeitschr. f. d. ges Brau-
wesen. Nr. 9. Munich, 1885.
—Ueber einige fur die Brauindustrie wichtige Heferiarten und deren

Unterscheidungsmerkmale. Allg. Brauer- und Hopfenztg. 1885.

Festnummer.
— Ueber Sporen- und Kahmhautbildung bei Unterhefe. Zeitschr. f . d.

ges. Brauw. Nr. 16. 1887.
— Ueber das natiirliche Vorkommen von Sporenbildung in Brauereien.

Z. f . d. ges. Brauw. Nr. 17. 1887.

— Untersuchungen iiber die Mikroorganismen der Tropfsacke. Bor-
der Wiss. Stat. Munich, 1887.
— Ueber eine wilde Hefe, welche dem Biere einen bitteren Geschmack

giebt. In Ber. d. wissensch. Stat. Munich, 1889.
— Beschreibung von zwei Krankheitshefen. Zeitschr. f. d. ges. Brau-
wesen. Nr. 12. Munich, 1890.
— Zwei Hefearten, welche abnorme Veranderungen im Bier veran-

lassen. Zeitschr. f. d. ges Brauw. Nr. 7 — 8. Munich, 1891.
— Untersuchungen iiber die Verunreinigung gebrauchter Trubsacke.

Mitt. d. wissenschaftl. Station f. Brauerei. Munich. Zeitschr. f-

d. g. Brauwesen. Nr. 9. 1892.
WILSON, W. R. — Pure Yeast Culture in London. — " The Brewers'

Journal," p. 527. London, 1892.
WINOGKRADSKY. — Ueber die Wirkung ausserer Einfliisse auf die Entwicke-

lung von Mycoderma vini. Bot. Centralbl. Bd. XX., S. 165. 1884.
WOODHEAD. — Bacteria and their Products. London, 1891.
WORTH ANN. — Untersuch. iiber das diastat. Ferment der Bakterien. —

Zeitschr. fur physiol. Chemie. Bd. VI. 1882.
— Einige Bemerkungen iiber die Yergarung von Mosten mit reinge-

ziichteter Hefe. Weinbau und Weinhandel. N. 23. 1892.
— — Weiteres iiber die Vergarung von Mosten mit reingeziichteten

Hefen. Weinbau und Weinhandel. N. 42. 1892.
— Unterguchungen iiber reine Hefen. Landwirtschaftl. Jahrb.

Herausg. v. Thiel. p. 90. Berlin, 1892.
YUNG. — Des poussieres organisees de 1'atmosphere. — Arch, des sc. phys.

e.t nat. T. IV. Geneva, 1880.

BIBLIOGRAPHY. 251

ZALEWSKY — Ueber Sporenbildung in Hefezellen. — Verh. d. Krakauer

Akad. d. Wiss. Matn.-naturw. Sektion I., Bd. XII. 1885. Rel

Bot. Centr. Bd. XXV.
ZEIDLER, A. — Beitrage zur Kenntniss einiger in Wiirze und Bier vorkom-

menden Bakterien. — Wochenschr. f. Brauerei. 1890.
ZIMMEEMANN, O. E. R. — • Bakteriologische Diagnostik. — I. Teil.

Chemnitz.
ZOPF, W. — Entwickelungsgeschichtl. Untersuchungen iiber Crenothrix

polyspora. Berlin, 1879.
— Ueber den genetisclien Zusammenhang der Spaltpilzformen. — Sitzb.

d. Berl. Akad. d. Wiss. 1881.

— Zur Morphologie der Spaltpnanzen. Leipzig, 1882.
— Ueber Bacterium merismopedioides. — Sitzb. des bot. Ver. d. Prov.

Brandenburg. Juni, 1882.
—Die Spaltpilze. Breslau. 3. Ausg., 1885.
— Oxalsauregarung (an Stelle von Alkoholgarung) bei einem typischen

(endosporen) Saccharomyceten, Sacch. Hansenii n. spec. — Bericht

d. d. botan. Gesellsch. Bd. VII., H. 2. 1889.
— Die Pilze in morphologischer, physiologischer, biologischer und

systematischer Beziehung. — [Encyclopedic der Naturwiss. I Abt.

1. Teil. Handb. der Botanik, herausg. v. Schenk. Bd. IV. p-

271 — 781.] Breslau, 1890. Has also appeared as a separate

work.
— und LIESENBEBG. — Ueber den sogenannten Froschlaichpilz (Leu-

conostoc) der europaischen Riibenzucker- und der javanischen

Rohrzuckerfabriken. Beitr. zur Phys. und Morph. niederer

Organismen, herausg. v. Zopf. Heft. I. Leipzig, 1892.

INDEX.

Acetic acid bacteria, 56

Acetic acid fermentation, 57

Adametz, 66, 149, 193

Aerobic species, 56

Aeroscope, 38

Air analysis, Hansen's zymotechnic, 47

analysis, Koch and Hesse, 40

analysis, Miquel, 37 — 40

examination of, 36

examination of air and water,
36

filtration of, 41

sterilisation of, 14
Alcoholic ferments, 111
Amthor's investigations, 150, 151
Anaerobic species, 56
Analysis (Hansen's methods), 124

Hansen's, of brewers' yeast, 134

of the yeast in propagating
apparatus, 137

withdrawal of samples from

fermenting- vessel for, 134
Anatomical structure of bacteria, 53
Appert, 10, 204, 205
Application of the results of scientific

research in practice, 204
Arthrosporous bacteria, 55
Ascospores, anatomical structure of,
126, 134

budding of, 128

formation of, 125

germination of, 127

temperature limits for the

formation of, 131, 160—173
Aspergillus Oryzae, 92
Aubry, 16, 217

Bacillus butyricus, 67

ethaceticus, 60
Bacteria, 51

anatomical structure of, 53

arthrosporous, 55

endosporous, 55

Bacteria, exercising invertive, diastatic,
and peptonising action, 76

growth forms of, 51 — 53

involution forms of, 53

multiplication of, 54

slime-forming, 73

staining of, 2

zoogloea, formation of, 56
Bacterium aceti, 57 — 60

Pasteurianum, 57 — 60

vermiforme, 72
Bail, 97, 120
Bavay, 146, 223
Beer, filtration of, 12
Bellamy, 117
Belohoubek, 188, 202, 219
Beyerinck, 149, 194
Biernacki, 17
Bisulphite of lime, 16
Boettcher's moist chamber, 8
Borgniann's investigations, 149
Botrytis cinerea, 83
Bottom-fermentation yeast, 183

species of, 186
Bourquelot, 194
Boutroux, 181
Bread-fermentation, 180
Brefeld, 23, 112, 116

Brown's, 119

Nageli's, 118

Pasteur's, 117

Traube's, 116

Brewers' bottom-fermentation and top-
fermentation yeast, practical
analysis of, 134
Brown, A. J.," 56, 119
Buchner's aeration experiments, 118
Budding of spores, 128
Budding fungi, gelatinous formation,
156 [221

Bungener (on pure cultivated yeast),
Burton yeast, 223
Butyric acid ferments, 66

INDEX.

253

Cagniard-Latour, 10, 113

Cane-sugar solution, 214

Carlsberg vessels, 20

low-fermentation yeast, No. 1,
No. 2, 184

Cartie, Mac (on pure cultivated yeast),
223

Cellulose fermentation, 70

Chalara Mycoderma, 106

Chamberland flasks, 19

Chevreul, 18

Chicandart, 181

Chloride of lime for cleansing pur-
poses, 16

Cierikowski, 107

Cladosporium herbarum, 109

Classification of genus Saccharomyces,
159

Clostridium butyricum, 67—69

Cohn, 24, 51

Control of yeast in practice, 134

Cooling of wort, 213

Counting of yeast-cells, 32 — 35

Crenothrix Kiihniana, 80

Culture experiments, 1
fractional, 26

of Saccharomycetes on solid
medium, 143

Dairy, 227

De Bary, 23, 71, 107

Delbriick, 32, 63, 226

Dematium pullulans, 107

Dilution methods, 26—32

Diseases in beer, caused by yeast, 25,

145, 163, 167, 173, 174, 206,

207

in milk, 6-5
Disinfection, 14 — 17
Dispora caucasica, 70
Distillers' yeast, 187
Draff, 45

Drying-machines, 46
Duciaux, 56, 64, 66, 115, 192
Diinnenberger, 181
Durst, 32
Dusch, 11, 205

Effront, 25—26
Ehrlich, 3
Ehrenberg, 23

Endomyces decipiens, 131

Endosporous bacteria, 55

Engel, 112

Eriksson, 110

Eurotium Aspergillus glaucus, 89

Ferment, alcoholic, 111

acetic acid, 56

butyric acid, 66

lactic acid, 61
Fermentation, alcoholic, 111

acetic acid, 56

butyric acid, 66

cellulose, 70

kephir, 70

lactic acid, 61

rice-wine, 92

viscous, 73

products, chemical examination
of, 149

theory of, Bref eld's 116
Fermentative action of -the Mucor

species, 99

Fermenting-rooms, Hansen's examina-
tion of the air in, 47
Film formation, 137
Filter-bags, 16
Filtration of beer, 12

of air, 41

Fitz, 27, 67, 69, 86
Flasks, Carlsberg, 20

Chamberland's, 19

for forwarding small and large
cultures, 213

Freudenreich's, 19

Hansen's, 19

Pasteur's, 1 8

vacuum, 37, 43
Fliihler (on pure cultivated yeast),

221, 226
Fokker, 61
Forti, 151, 226
Fractional cultivation, 26

sterilisation, 13
Frank, 110

Frankland, 41, 60, 222
Fresenius, 105
Freudenreich's flasks, 19
Frogs' spawn fungus, 75
Fruit-wine fermentation, 226
Fumago, 110
Fusarium grammearum, 106

254

INDEX.

Gelatine cultures, Hansen's, 30
Koch's 29

Gelatinous formation secreted by the
budding fungi, 156

Gemmae, 97

Generation, spontaneous, 10

Germination of spores, 127 — 130

Ginger-beer plant, 71

Glycerine, proportion of, in the fer-
mentation products of various
Saccharomycetes, 149 — 150

Grains (draff), 45

Griessmayer, 2^6

Grotenfelt, 64, 71, 149, 180

Growth forms of bacteria, 51 — 53

Gruber, 15, 16, 69

butyric acid bacteria, 69

Gronlund, 146, 175, 202

Guillebeau, 66

Haematimeter, 33
Hansen's acetic acid bacterium, 57
aeration experiments, 118
air analysis, 47

analysis of brewery yeast, 134
analytical methods, 124
counting of yeast-cells, 32
cultivation of Saccharomycetes

on solid medium, 143
examination of the air, 42
flasks, 19
Koch's plate cultures examined

by, 31

method for zymotechnical an-
alysis of air and water, 47
preparation of pure cultures,

28-31

reform in scientific and practical
fermentation brought about
by, 206

Saccharomyces species, 159
water analysis, 49
and Kiihle's yeast-propagating

apparatus, 209
Hayduck, 32
Hesse, 40
High-fermentation yeast, 183, 186,

221

Hoffmann, 18
Holm, 32, 50, 123

and Poulsen's investigations,
135

Hueppe, 41, 62, 64, 68, 119
Hydrofluoric acid, 17, 25

Introduction to alcoholic ferments, 111
Involution forms of bacteria, 53
Irrnisch, 187
Isomaltose, 149

Jacquemin, 151
Janssens, 159
Jensen, 66
Jorgensen, 211, 221

Kayser, 143, 149, 193 -194, 226

Kephir, 70

Kern, 70

Koch, 3, 15, 22, 29, 50, 205

Koehler, 176

Kokosinski, 146, 221, 224

Kramer, 192

Krause, 63

Krieger, 146

Kruis, 120

Kukla, 183, 202

Kiitzing, 23, 57

Lactic-acid bacteria, 61
fermentation, 61

Lactose, yeasts fermenting, 192 — 193
Laer, Van, 64, 73, 225
Langer, 226

Lasche, 146, 147, 177, 202—203
Laurent, 181
Leaven, 180—181
Lechartier, 117

Leuconostoc mesenterioides, 75
Liebig1, 114
Lime, bisulphite of, 16
Lindner, 63, 73, 78, 144, 146
Lintuer, juur., 149, 222
Lintner, senr. (on pure cultivated

yeast), 216
Lister, 27
Loffler, 66

Low-fermentation yeast, 183, 186
Ludwig, 179

Mac Car tie, 223, 224
Mach, 151, 226
Maercker, 62, 226
Marcano, 181
Marpmann, 64

INDEX.

255

Marshall Ward, 71
Martinand, 151
Marx, 151, 176, 221, 226
Mass-cultures, pure cultures for physio-
logical experiments with, 24
Matthews, C. G., 106
Mayer, 58

Micrococcus prodigiosus, 63
Micro-organisms of air and water, 36
Microchemical examination, 1

reagents, 5
Microscope, 1

Microscopic appearance of sedimentary
yeast, 124

preparations, 1

and physiological examination,!
Milk, diseases in, 65

" -sugar, yeasts fermenting, 192

-193
Miquel's air analysis, 37 — 40

Koch's plate cultures examined

by, 31

Mitscherlich, 23

Mixtures of Saccharomycetes, 136
Moist chamber, Boettcher's, 8

Ranvier's, 7
Monilia Candida, 100
Morris, 149
Mould-fungi, 82

Mucor species, action on the various
sugars, 99

fermentative action of, 99

circinelloides, 97

erectus, 97

Mucedo, 95

racemosus, 96

spinosus, 97

stolonifer, 98

yeast, 97
Multiplication of bacteria, 54

yeast, 158

Multiplying capacity of yeast cells, 32
Miiller-Thurgau, 151, 226
Mycoderma cerevisiae and vini, 200

Nageli's theory of fermentation, 118
Nathan, 151, 226
Net-work, gelatinous, 156
Nucleus in yeast cell, 159
Nutritive substrata, 21

medium, cultivation of Saccha-
romycetes on solid, 143

Oidium, 103
Orleans process, 57

Panum, 33

Pasteur, 25, 27, 37, 56, 116, 205

acetic acid bacteria, 57

" Etudes sur la biere," 62, 113—
114, 205

theory of fermentation, 117

flasks, 18

practical results, 205
Pasteurisation of beer, 12
Pedersen's aeration experiments, 118
Pediococcus acidi lactici, 63

cerevisise, 78, 80

causing ropiness in white beer,

73

Penicillium glaucum, 86
Peptonising bacillus, 77
Peters, 60, 63, 76, 181
Petersen (Sarcina), 80, 202
Petri (Filter), 41
Physiological methods, 24 [1

and microscopical examination,
Pichi, 146, 151, 226
Plate cultures, determination of errors

made in, 31 — 32
Portele, 151, 226
Poulsen, 135
Practice, application of the results of

scientific investigation in, 204

control of yeast in, 134
Prazmowski, 67
Preparations, microscopical, 1
Preservation of pure cultures, 214
Pressed yeast, 180

Propagating apparatus, Hansen and
Kiihle's, 209

Bergh and Jorgensen's, 211

analysis of yeast in, 137
Pure culture, preparation of, 22, 122

Hansen's methods, 28 — 31

Koch's method, 29

Pasteur's methods, 24, 27

preservation of, 214
Pure cultures for morphological in-
vestigations, 23

for physiological experiments
with mass cultures, 24

sending of, 213

Quick vinegar process, 58
Qvist's lactic acid bacterium, 65

256

INDEX.

Ranvier's moist chamber, 7

Ratz, 65

Rayman and Kruis' examination of

beers, 120
Raum, 158

Reagents, micro-chemical, 5
Reess, 86, 112, 120

Reinke (on pure cultivated yeast), 218
Resistance to infection of the different

yeast types, 208

Rest, state of, of mould-fungi, 85, 88
Resting spores, 55
Rietsch, 151
Rommier, 151

Saccharomyces acidi lactici, 180

anomalus, 181

apiculatus, 147, 195

Aquifolii, 175

Saccharobacillus Pastorianus, 64
Saccharomyces cerevisiae, I., 127, 159
—161

conglomeratus, 182

ellipsoi'deus, I., 170

ellipso'ideus, II., 173

exiguus, 176

Hansenii, 178

Ilicis, 175

Jorgensenii, 177

Kephyr, 194

Ludwigii, 129, 179

Marxiamis, 146, 176

membranoefaciens, 146, 177

minor, 180

Pastorianus, I., 161
II., 165
III., 167

pyriformis, 72, 175

Tyrocola, 194

cell of, described, 158

variations in the species, 151
Saccharomycetes, analysis of, 124

ascospores, 125

behaviour towards the different
sugars and the other con-
stituents of the nutritive
liquid, 146

classification of, 159

culture on solid nutritive me-
dium, 143

diseases in beer caused by, 145

film formation, 137

germination of spores, 127

Saccharomycetes, microscopic appear-
ance of sedmentary yeasts, 124

pure cultures, 122

temperature limits, 142
Sake, 92

Samples, forwarding of, in Hansen's
flasks, 213

in filter-paper, 214

withdrawal of, from ferment-
ing-vessel for analysis of
yeasts, 134
Sarcina forms, 77
Scheele, 10, 204, 205
Schlen, 41
Schloesing, 227
Schmidt-Mulheim, 65
Schmitz, 159
Schroder, 11, 205
Schroeter, 29
Schulze, 10
Schumacher, 188
Schwackhofer, 226
Schwann, 10. Ill, 204
Sclerotium, 85
Sedimentary yeast, 124
Slime-forming bacteria, 73
Soda solution for cleansing purposes, 16
Solid nutritive medium, cultivation of

Saccharomycetes on, 143
Spallanzani, 10, 36, 204
Spherical yeast, 97
Spores, budding of, 128

germination of, 127

conditions regulating formation
of, 126

structure of, 126, 134

temperature limits of. forma-
tion, 131
Stahl, 114, 120
Staining method, 2

Ehrlich's, 3

tubercle bacillus. 3
Starch-resolving bacterium, 76
Sterilisation, 9 — 14

of glass and metal articles, 1 1

of nutritive liquids and solid
nutritive substrata, 11

of air, 14

Storch's lactic acid bacteria, 65, 66
Sublimate, disinfection by means of, 16
Suchsland, 227

Sugar, behaviour of Mucor species to-
wards the various kinds of, 99

INDEX.

257

Sugar, behaviour of Saccharomycetes,
etc., towards the various
kinds of (review), 146 — 149

Tartaric acid, 24—25
Temperature limits for the Saccharo-
mycetes, 142
Thausing, 226
Tobacco fermentation, 227
Top-fermentation yeast, 186, 221
Torula, 189
Traube, 116

Tubercle bacillus, staining, 3
Turpin, 57, 113
Tyndall, 13, 37

Vacuum flasks, 37, 43

Variations in the species of Saecharo-

myces, 151
Velten, 116
Vuylsteke, 136

AVard, Marshall, 71

Water analysis, Hansen's zy mo techni-
cal, 47
examination of air and, 36 — 50

Weigmann, 65, 66

Wichmann, 50

AViesner, 188

AVill, 17, 144, 146, 174, 218

AVilson, W. R, 223—224

AVindisch, 146

Wine yeast, investigations by Marx,
Amthor, and others, 151

Winogradsky, 203

AV inter, 75

AVort, introduced into fermenting-
vesselsin a state of sterility, 13

Wortmann, 76, 151

Yeast, analysis of, 124
ascopores, 125
Burton, 223

bottom-fermentation, 186
cell, microscopic appearance of,

158

cells, counting of, 32 — 35
cells, partition wall formation,

127
culture on solid nutritive

medium, 143
diseases in beer caused by, 25,

145, 163, 167, 173, 174, 206—

207

film-formation of, 137
high-fermentation, 186
low-fermentation, 186
Mucor, 97

network formation of, 156
pure cultivation, by Hansen's

method, 26—31, 122
purifying by Pasteur's method,

24, 25

pure cultivation in practice, 214
grouping of the species for

practical purposes, 186
pressed, 180
propagating apparatus, Hansen

and Kiihle, 209
propagating apparatus, Bergh

and Jorgensen, 211
properties of the different

species in practice, 185
sedimentary, 124
spherical, 97
top-fermentation, 186
treatment of, with hydrofluoric

acid, 25
varieties of, 151

Zoogloea, formation of bacteria, 56
Zopf, 63, 80, 110, 120, 178
Zygospores, 96

GEORGE WILSON, PRINTER, 676, TURNMILL STREET, LONDOX, E.G.

CARTON'S
PATENT BREWING SUGARS.

CARTON, HILL & CO.,

Sole licensees an£> flDanufacturers,

GARTON'S L^E.VO-SACCHARUM is a refined inverted Sugar, specially pre-
pared for Brewing, which since 1861 has been most successfully used by
Brewers in different parts of the world for the production of Export and
Stock Ales, running Ales and Porter.

Unlike ordinary Sugar, it is free from impurities detrimental to a
keeping Beer, and being perfectly inverted, it undergoes vinous fermentation
simultaneously with the Malt wort.

It supplies a purely saccharine basis in the proportion by which it
displaces Malt, and in like ratio lessens the amount of inherent nitrogenous
matter; it .also aids the elimination of the remaining gluten, and gives the
balance of Sugar required to support after fermentation.

By the use of GARTON'S SACCHARUM, Brewers are rendered less
dependent upon the constituents of their water, and can brew Beer surpass-
ing even Burton Ales in brightness and endurance ; they are less liable to
climatic influences in summer, arid the risks of brewing are thereby
minimised.

Beer thus brewed is well suited for transit, and is specially adapted for
bottling, by reason of its freedom from sediment and from liability to
burst bottles.

By the use of GARTON'S SACCHARUM a relatively larger quantity of
Beer may be brewed, and in addition to its economy, as compared with
Malt and Hops, A GREAT SAVING (SAY TEN PER
CENT.) IS EFFECTED IN THE QUANTITY OF HOPS
REQUIRED. ^^ ^

Prices and further particulars supplied upon application to —

GARTON, HILL & CO.,

Southampton Wharf,

BATTERSEA, S.W.

IReade Srothers'

YEASTFORT

-"*•

OR

YEAST NUTRIMENT.

A FOOD FOR BARM.

Gives vigour to Yeast, and supplies those elements in which it is lacking,

thus obviating the necessity for frequent changes of yeast, and minimising

the risk of diseased ferments. It thus produces a full and vigorous

fermentation, and a sound and healthy Beer.

IReade Srothers'

™- E C U M I N ™*

OR

FROTHING POWDER.

For giving a foaming head to Ale and Porter.

Imparts neither flavour nor smell and does not impair the brightness of

the liquor. It puts a creamy head on the liquor which remains

to the last glass drawn from the cask.

Price 2s. 6d. per Ib.
• »»»»*»»»»»»»»»»»»»»»»»»»»»»»•»•»•«•»»•»»»»•»»«•»»»»»»•

IReade IBrothers'

ODOURLESS

LIQUID BEER PRESERVATIVE.

A tasteless and odourless preventive of the acetous and lactic fermentations.

Far superior to Bisulphite of Lime, Salicylic Acid, and other

ordinary preservatives.

Prices on application —

READE BROTHERS & CO.,
flDanufacturing Gbemists, Wolverbampton.

GEORGE CLARK & SON,

Manufacturers of

CARAMEL,*^

Giving heading and palate fulness.

CARAMEL CRYSTALS,

The highest Tinctorial power in the market.

SACCHROSITE,

For Priming Ales.

SOLE MANUFACTURERS

OF

CARAMELINE,

For Stout Priming. Giving to Black Beers a lasting luscious
flavour. Easy of manipulation, and thoroughly reliable.

SAMPLES &. QUOTATIONS ON APPLICATION.

6, Trinity Square,

Tower Hill, London, E.G.

TELEGRAMS :— " EXTRACT," LONDON.

lEyamination.

Oar Biological Laboratory being now extended and fitted
with the most modern apparatus, we are prepared to carry
out Bacteriological examinations of Yeast with increased
promptitude, and to advise as to the best means of cor-
recting faults that may arise in the process of brewing having
their origin in fermentation, and also as to the best means
of insuring healthy fermentation.

This Laboratory is under the direction of Mr. E. J. Bo AKE, B. A.,
who has had considerable experience in the examination and
cultivation of Yeasts.

The wide field over which our experience extends gives
peculiar facilities for dealing with this difficult but most
important subject.

The sterilised tubes which we introduced about two
years ago have proved of very great utility. By the use
of these sterilised tubes it is now possible to transport a
sample of Yeast from the brewery, no matter how distant,
into our Laboratory in exactly the same condition as it
was when first drawn. Tubes together with printed in-
structions will be forwarded on application, as will terms
and other necessary information.

A. BOAKE, ROBERTS and Co.,

Stratford, London, E.

Telegrams:— "Boahe," London.

THIS BOOK IS DUE ON THE LAST DATE
STAMPED BELOW

BOOKS REQUESTED BY ANOTHER BORROWER
ARE SUBJECT TO RECALL AFTER ONE WEEK.
RENEWED BOOKS ARE SUBJECT TO
IMMEDIATE RECALL

LIBRARY, UNIVERSITY OF CALIFORNIA, DAVIS

D4613 (12/76)

3 1 i 75 0 1072