Fermentation Organisms

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FERMENTATION ORGANISMS
FERMENTATION
ORGANISMS
A LABORATORY HANDBOOK
ALB.
KLOCKER
ASSISTANT IN THE CARLSBERG LABORATORY, COPENHAGEN-
TRANSLATED FROM THE GERMAN BY
G. E.
LECTURER
ALLAN,
B.Sc.
J.
THE UNIVERSITY OF
BIRMINGHAM
H.
MILLAR,
F.LC.
FORMERLY LECTURER IN THE BRITISI1

SCHOOL OF MALTING AND BREWING
IN
AND REVISED BY THE AUTHOR
WITH
146
ILl.VSr RATIONS IN
THE TEXT
LONGMANS, GREEN, AND

39
PATERNOSTER ROW, LONDON

NEW YORK AND BOMBAY
1903
CO.
2)e&icateJ
HONOUR AND GRATITUDE
PROF. EMIL CHR. HANSEN,
THE AUTHOR.
Ph.D.,
FROM
THE AUTHOE'S PEEFACE

TO THE GERMAN EDITION
In the course
of a
number
of years, during
which
had the honour to act as assistant to Prof. Emil

Chr. Hansen at the Carlsberg Laboratory, and to
assist him in the practical courses of instruction
conducted there from time to time, the wish was
I
frequently expressed by students to have at their
handbook which would contain an adedescription of the fittings, apparatus and

methods of a fermentation laboratory, as well as
disposal a
quate
of the biology of the organisms of fermentation

short, a guide
ments
in
by means of which the usual experi-
of such a laboratory can be carried out.
similar request reaching
publisher,
Max Waag,
me from
the well-known
of Stuttgart, I could but con-
clude that a real necessity existed for such a book.
work are divided

The first of these contains
a description of the manner in which the science
The contents
of the present
into three sections.
of the
organisms of fermentation has gradually
developed
at
the
same
time,
an indication
is
given of the most important steps which have
marked the progress
of our science.
PREFACE
viii
The second
section describes the fitting
the laboratory and
ducting
special
necessary for con-
methods
Laboratory
work.
explained,
all that is
up of
are
then
being given to the
attention
preparation of pure yeast cultures in large quantiFinally, the third section treats of the
ties.
most
important micro-organisms of the alcoholic
fer-
The book thus deals with

which Hansen has opened up so
mentation industry.
that domain in
many new
paths.
For each section there is a bibliography which

embraces the most important researches, and conexplanatory
tains
The
notes.
literature
after
January, 1900, could not be included.
In
have made experiments for the
sake of confirmation, and have quoted some re1st
certain
cases
sults not hitherto published.
The branch
which
of the fermentation industries with
my
book is chiefly concerned is that of

brewing, which was the first to make use of Hansen's pure culture system, and hence to adopt a
rational mode of working.
Brewing has thus, to
a certain extent, become the model for the other
branches of the alcoholic fermentation industry.
The
science of the organisms of fermentation as
set forth in this
book
deals,
however, not only
with practical applications, but also with important theoretical aspects of chemistry and botany.
ALB. KLOCKER.
Copenhagen, January
1900.
PREFACE TO THE ENGLISH TRANSLATION

BY
PEOPESSOE ADEIAN
J.
BEOWN,
M.Sc,
P.I.C.
A VERY considerable and rapidly increasing amount

of attention
to
now
is
being given in this country
Technical Microbiology in
fermentation industries, and

is
its
relation to the
consequently there
a growing demand for sound textbooks on the
study of the " fermentation organisms

use of students
branch
of
who
work.
"
for the
are taking up this special
But
it
is
continually
forced on the notice of the writer, whose

is
intimately
connected
Microbiology, that
this
with
quately satisfied at present.
of
very inade-
is
Whilst
work
teaching
the
demand
being
we
are almost
too well supplied with textbooks on Bacteriology

as related to the organisms of disease, the
ber of books
in our
own language
the subject of "fermentation organisms"

limited,
and
this
is
especially
num-
dealing with
the
is
case
very
with
works describing the more modern developments

of experimental method connected with the
study of these organisms.
For this reason the
PREFACE
X
publication
K locker's
an
of
English
translation
Gdrungsorganisrmn,
work
Herr
of
specially
devoted to the treatment of laboratoiy methods
employed
study of "fermentation organ-
in the
be welcomed by
isms," should
all
students of Technical Microbiology, for
now
and
they have
teachers
placed in their hands a book which cannot
to be of great assistance to them.
The
fail
special
merit of this book requires no recommendation

here, for
written by one who is a specialist in

and whose name is well known, not
it is
his subject,
only for his
own
valuable researches, but also as a
distinguished assistant of the illustrious Dr. Emil
whom
Hansen, to
C.
every one connected with
technical fermentation in this country

is
The
so deeply indebted.
and abroad
translators of the work,
Mr.
J. H. Millar and Mr. G. E. Allan, have been

most successful in the performance of their task,,
and we congratulate them on a volume which
should
be
the
laboratory
companion
of
every
student of Technical Microbiology in this country.
We
beheve also that
book
this
will
be a useful
addition to the library of the pathological bacteriologist.
Pathological
phenomenal growth,
past history and lose
its
oldei-
is
all
development
We
in
think
it
owing to
inclined to forget it&
connection with
branch of microbiology from which
ally sprang.
in
bacteriology,
will
it
the
origin-
be found that the
method of experiment and research
connection with
the
study of "fermentation
PREFACE
organisms"
still
deserves
the
xi
careful attention
of bacteriologists, and confidently
book to
recommend
this
their notice.
ADRIAN
School of Malting and Brewing,
The University, Birmingham,
16th October, 1902.
J.
BEOWN.
The
to
translators desire to express their best thanks
Mr. T. H. Pope
for
his
great assistance in
reading the manuscript and proofs of this translation during its progress through the press.
CONTENTS.
SECTION
SECTION
II.
INTRODUCTION.
I.
THE LABORATORY.
(Pp. 1-15.)
(Pp. 16-169.)
PAGE
FITTINGS AND APPARATUS

1.
General Principles for fitting up the Laboratory

Preparation of Bench Surfaces
Solutions for Washing the Bench during Work
The Microscope Table
The
2.
3.
Sterile
Room
...
...
21
22
....
The Tube
23
24
24
24
26
Correction Objectives
The Condenser
Immersion Objectives
The Stage
25
Changing the Objectives
28
28
Testing the Microscope
28
and Cover Glasses

The Micrometer
Counting Apparatus
Klonne and Miiller's Object Marker
29
Slips
31
32
33
Cover-Glass Gauge
4.
18
19
20
20
Hansen's Sterile Cupboard

The Microscope and its Accessories
Spherical and Chromatic Aberration
Achromatic and Apochromatic Objectives
17
34
Apparatus for Artificial Illumination

Small Auxiliary Apparatus
Bottles for Reagents and Immersion Oil
Thermostats and their Accessories
Rohrbeck's Thermostat
Panum's Thermostat
34
35
.
.35
........
36
36
39
CONTENTS
XIV
Sohribaux's Thermostat
Large
Warm Chamber
....
.....
Thermostat for Low Temperatures

Pfeifier's Microscope Heating Apparatus
Reichert's Regulator
Muencke's Form of Lothar Meyer's Regulator
Roux's Regulator
A. Petersen's
FACE
42
......
Soxhlet's Regulator
Koch's
Lamp
5.
54
....
its
Modifications
Petri Dishes
Apparatus
Gypsum
for Spore Cultivation

Blocks and their Containing Vessels
Water Holder
Moist Chambers
Hollow Glass Slips
Sterile
8.
Chamber
Chamber
Moist Chambers
Ranvier's Moist
Bottcher's Moist
Stand
9.
II.
for
51
54
The Chamberland Flask and
7.
50
52
The Pasteur Flask

The Carlsberg Vessel
Flasks
46
48
49
49
51
.
Culture Vessels
Prior's Vessel
45
50
Apparatus
Sterilising
With Dry Heat

Steam Sterilisation
6.
...
Thermometers
43
44
Additional Apparatus
56
59
59
62
64
64
64
67
68
68
69
69
70
Pipettes
70
70
Rubber Tubing for Flasks

Platinum Brush
71
71
NUTRIENT MEDIA
1.
Liquid Media
Beer Wort
Water
71
71
78
Yeast Water
Meat Extract
Fruit Syrups
79
.
....
Solutions of Saccharose and Dextrose
Beer
Discontinuous Sterilisation
Sterilisation by Filtration
2.
Media
Gelatine and Agar-Agar
Other Solid Media
Solid Culture
79
79
80
80
81
81
81
81
86
CONTENTS
III.
1.
METHODS
Microscopical Investigation o Micro-Organisms
86
.86
Preparation Making
Removal of Grease from Cover Glasses

Fixing and Staining of Yeast Cells
Staining of Yeast Spores
Staining of the Yeast Cell Nucleus
Fixing and Staining of Bacteria
87
.88
....
.89
....
....
...
....
.
90
90
Staining of Bacteria Spores
Staining of Flagella according to Loffler
2.
91
Reagents
Development in Moist Chambers
Experiments with various Flasks.
Inoculation
Solid Culture Media
The Manipulation of Nutrient Liquids
Experiments with Solid Culture Media
Cultures of Anaerobic Organisms
Suppression of Bacteria in Yeast Growths
Preparation of Pure Cultures
Liquid and
.93
93
97
....
Hansen's Dilution Method

Dilution in and on Solid Substrata
Koch's Plate Culture
.
99
Method
103
106
110
Fractionated Culture after Klebs and others
Method
....
.......
...
....
........
Preservation of Ordinary Brewery Yeast
5.
Yeast
7.
Spore Cultures of Moulds

Preparation of Film Cultures of Saccharomyces
Counting of Yeast Cells and Seeding with a Definite
of Cells
Preparation of Spore Cultures

Spore Cultures of Sacoharomyces
Spore Cultures of Bacteria

6.
113
113
114
114
117
117
Preservation of Bacteria
of
Ill
112
Pure Cultures of Bacteria

Pure Cultures of Mould Fungi
Methods of Preservation
Hansen's Saccharose Method for the Preservation of Yeasts
and Moulds
Preservation on Cotton Wool or Filter Paper (after Hansen)
Transmission
101
103
106
Hansen's Second Pure Culture Method

Lindner's Droplet Culture
4.
...
....
...
.111
Surface Plate Cultures
Pasteur's
99
100
Bref eld's Glass Slip Cultures
Schiinfeld's
92
92
of
3.
88
118
119
121
121
124
124
125
Number
126
CONTENTS
xvi
PAGE
8.
The
Biological Analysis of Yeast
....
Preliminary Investigation
133
Separation of the Various Forms in the Sample

Hansen's Spore Method for the Analysis of Brewery Bottom
Yeast for Wild Yeast
.
Method
Acid Method
Application of the Spore
The Tartaric
to
Top Yeasts
Analysis for Bacteria
Testing the Contents of the Pure Culture Apparatus

.Testing the Contents of Fermenting Vessels
Lindner's Drop Culture
Hansen's Test
9.
10.
The
of
the Stability of Beer in Cask
Biological Analysis of Water, Air
and
and
Soil
The Hygienic Analysis of Water (after Koch)

Water Analysis for Brewery Purposes (after Hansen)
Sohwackhofer's Standard of Fitness of a Brewery Water
.
Holm's Results
Hansen's Analyses
Hansen's Results
Soil
11.
Analyses
of
Air
145
154
Hansen's Pure Culture System in Fermentation Industries

The Pure Culture System in Bottom Fermentation Breweries
The Hansen-Kiihle Pure Culture Apparatus
The Pure Culture System in Top Fermentation Breweries
The Pure Culture System in Wine Manufacture
The Pure Culture System in the Manufacture of Cider
The Pure Culture System in the Manufacture of Spirit and
Pressed Yeast
.
149
149
150
13&
187
137
138
143
143
145
Principles of the Technical Analysis of Water, Air
134
135
135
140
Soil
13S
133
155
156
156
158
163
164
168
168
III.
THE MICRO-ORGANISMS OF MOST IMPORTANCE IN THE FERMENTATION INDUSTRY.
SECTION
(Pp. 170-345.)
I.
TRUE FUNGI (EUMYCETES)

1.
Fungi (Phyoomycbtes)
Zygomycetes
ALG.JE
...
General Characteristics
Mucor
M. Mucedo,
...
(1)
171
Mucoracese
171
...
171
171
171
174
M. racemosus, p. 179 M. erectua, p. 180

M. oryzae, p. 181
M. (Amylomyces)
Rouxii, p. 181 M. spinosus, p. 183 M. alternans,
M. corymbifer, p. 183.
p. 183
p.
177
....
Rhizopus
Bh. nigricans,
(2)
p.
184
Rh.
oryzee, p. 185.
...
184
CONTENTS
xvii
I'AGE
The Higheb Fungi (Mycomycetbs)

A.
1st
Abcomycetbs
Obdek
....
Gymnoasce^
True Yeast Fungi (Saccharomycetes)

General
1. The Saccharomycetes Distinct Fungi
2. Structure and Shape of Yeast Cells
The
The
8.
Cell Contents
Cell
Wall and
its
Shape
of
the Cells
Modes
of
Propagation
Gelatinous Formation
.
186
186
186
186
187
187
189
186
186
190
191
Budding
.191
Spore Formation
The Chemical Constituents of the Cell
211
Fermentation Phenomena
212
Ferments
212
Influence of Air and Temperature on Fermenta.
4.
5.
tion
214
Energy
Power
of
and
....
Fermentation
...
Permeability of the Cell
Membrane
Products of Fermentation
Auto-Fermentation
Yeast Types
6.
....
....
....
Vitality of Yeast in Nutrient
the
Dry
Influence of Physical Factors
217
218
219
219
.......
Diseases caused by Fermentation Products
Variation
Temporary Varieties
Permanent Varieties
Practical Results and Variation
10. Circulation in
Nature
222
224
227
Disease Yeasts and Mixed Fermentations
Competitive Relations
222
Solutions and in
State
Diseases caused by Turbidity
9.
217
Injurious and Stimulating Influence of Chemical
and Physical Factors
8.
Influence of Chemical Factors
7.
Fermenting
228
228
229
22^
233;
23$
236-
in Practice
...
241
246'
Systematic
249'
Saccharomyces
General Characteristics, p. 249 Grouping according
to Action on Sugars, p. 249 S. cerevisiGe I., p. 251
Carlsberg
Carlsberg Bottom Yeast No. 1, p. 252
Bottom Yeast No. 2, p. 252 Four Species of Brewery
1.
249i
CONTENTS
xviii
Bottom Yeast Described by Will, p. 252 Beer Top

Distillery Top Yeast, p. 253
S. Pas;
Yeast, p. 253
torianus
254; S. Pastorianus
I., p.
PastoriaDus
II.,
ellipsoideus
S.
256; S. ellipsoideus
III., p.
Johannisberg
258
p.
II.,
S.
259
S. Ilicis, p.
257
258
Disease Yeasts
S.
Aqui-
S.
tilaginosus, p. 260
Mazun,
2.
Two
p.
Vordermanni, p. 260 S. pyriformis,

261 S. Marxianus, p. 261 S. exiguus, p. 262
anomalus, p. 262; S. membranaefaciens, p. 264
Bailii, p. 265
S. mali Duolauxi, p. 265; S. car260
folii, p.
S.
255; S.
I., p.
Walporzheim,
258
p.
Isolated by Will, p. 259
p.
II., p.
267
p.
S. fragilis, p.
S.
266
Kefir, p. 266
Ludwigii, p. 267.
Schizosaecharomyces
Sch. Pombe,
mellacei, p. 271.
2nd Order
269; Sch. octosporus,
p.
Pebispobace^
General Characteristics,
...
p.
271
269
270; Sch.
p.
.271
Anixiopsis stercoraria,
The Belation between Vegetable Growth

272
and Perithecium Formation, p. 272 Damage in the
p.
Brewery,
273
p.
Preservation in the Laboratory,
p. 273.
Aspergilleae
1.
Aspergillus
277
A. oryzit, p. 277
fumigatus,
A.
p.
277.
2.
Penicillium
274
274
A. glaucus, p. 274
Sake, p.
Manufacture
277
A.
niger,
of
p.
...
278
P. glaucum, p. 278.
3ed Obder
Sph-eeiace.i:
Sphaeriese
1.
Sphifirella
Sph. Tulasnei,
283
porium herbarum,
p.
Discomycetes
Cup Fungi,
The Gonidial Form Clados283 Hormodendron clados;
S.
...
PezizaceiP
Sclerotinia
Fuokeliana,
The Gonidial Form
285
286
286
286
p.
282
porioides, p. 285.
4th Order
282
p.
282
Botrytis
cinerea, p. 286.
B.
Fungi Impeefkcti
The Torula Species
Hansen's Seven Torula Species, p. 290

ner's Eleven Slime Yeasts, p. 292.
Saccharomyces apiculatus
...
289
.289
Rich. Meiss-
294
CONTENTS
xix
PAGE
Mycoderma
M.
296
Species
296
cerevisiae, p.
Monilia Candida
M.
vini
I.
...
and
II., p. 297.
...
...
...
Javauica, p. 301.
Chalara mycoderma
Oidium
....
lactis
Dematium
pullulaus
Sphaerulina
intermixta
and Dothidea
ribesia,
298
301
303
305
p.
311.
Cladosporium herbarum
II.
311
FISSION FUNGI (SCHIZOMYCETES)
Gekebal
1.
Structure and
The
The
Form
Wall and
Cell
its
Oelatiuous Formation
.
Shape of the Cell

Methods of Reproduction
.
312
312
312
.813
.315
316
Spore Formation
...
...
.
3.
Variation
4.
Disease Bacteria in the Alcoholic Fermentation Industries
5.
Application of Bacteria in the Alcoholic Fermentation In-
dustries
Systematic
Coooacea;
1.
viscosus, p. 331
M. saprogenes
319
324
328
330
Micrococcus
316
317
329
M.
312
312
...
Cell Fission
of Bacterial Cells
Cell Contents
Flagella
2.
vini
and
I.
331
II., p.
331.
Diplococcus
2.
and
I.
II., p. 331.
Pediococcus
P. cerevisise, p. 331
formis, p. 331
3.
P. viscosus, p. 331
maxima, p. 332
and S. alba, p 332.
S.
BactebiaceJ!:
Bacterium and Bacillus
331
aurantiaca,
p.
332
S.
fiava
...
.
Acetic Acid Bacteria
p.
333
333
Vitality in Nutritive
333
336 Bact. Pasteurianum, p. 337 Bact. Kiitzingianum, p. 338 Bact. oxydans, p. 340 Beijerinck's
aceti, p.
Bact. aceti, p. 340
acetigenum,
p.
340
332
332
The Reaction of Mucilage, p. 333

Media and in the Dry State, p.
The Formation of Acetic acid, p. 334
Bact.
Swarming State,
331
P. sarcinse-
P. acidi lactici, p. 331.
...
...
Sarcina
S.
Xyliniun, p. 340
Bact.
Bact. industrium, p. 340 TermoBact.
CONTENTS
XX
PAOK
bacterium aoeti, p. 840

T. luteBoens, p. S40
vermitorme, p. 841
Baot. termo, p. 841.
Slime-forming Species
;
Baot.
341
Bac. viscoBus
342
and
I.
II.,
Bac. visoosus vini,
p.
341
Bac. viaoomiH
III., p.
p. 842.
Lactic Acid Bacteria
34iJ
Bac. acidificans longissimua,

p.
842
342
p.
p.
....
843
Bac. butyrious,
Granulobaoter Bacoharobutyricum,
porda, p. 844
I.
To Section
To Section
II.
III.
p.
Bac. piluliformans,
844
p.
344
p.
344
343
Bac. lupuli-
Bac. Hub-
344.
LlTBKATURK Bbvibw
To Section
la('ti<:i,
Saocharobac. PastorianuB, p. 348.
Butyric Acid Bacteria

Oloatridium butyricum,
tilis, p.
Bac. acidi
(pp. 347-381).
368
Indbx to Subjects and Authors
347
357
'
(pp. 883-392).
SECTION
I.
INTEODDCTION.i
In
this text
book the author has endeavoured
to give a
review of the biology of fermentation organisms in relation

to the use of these organisms in fermentation industries,
and
especially in the manufacture of beer.
In spite of
this limitation, however, the contents are of very varied
character,
is
and branch
off in different directions.
The book
not a text-book of the chemistry of fermentation or of
technical fermentation in the ordinary sense of the term.
For the high degree of development to which our

knowledge of the fermentation organisms has attained we
are indebted to a large number of investigators whose
work has been
steadily progressing for
many
years.
To
understand the development of our science up to the

present day, let us, in what follows, glance back along
the path traversed, and note
its
turning points, each one
of which has been productive of practical

of which
is
recognised at the present day.
was, of course,
use.
first
made when
results, the value
The beginning
the microscope came into
This apparatus, so indispensable for the examination
of fermentation organisms, was invented in the year 1590,
but Leeuwenhoek, in Eolland (1632-1723), was the
first
to
The bracketed numbers given in this section relate to the bibliography

To the latter we have appended explanatory
notes bringing in many amplifications and explanations which could not
>
at the end of the book.
find a place in this short introduction.
making a close study of these forms of life.

He was followed by a number of distinguished microscopists who all, more or less, added to our knowledge of
All these were
the natural history of micro-organisms.
employ
in
it
and systematic morphologists and not experiOf the more distinguished microseopists who
followed Leeuwenhoek we may mention the names of Otto
Friedrich Mliller (1730-85), in Denmark, and Ehrenberg
descriptive
menters.
(1795-1876), in Germany.
In the year 1822 Persoon gave to yeast the systematic
name Mycoderma,
that he regarded
it
a designation which seems to indicate
fungus (mycoderma
as a
signifies
fungoid
film).
About the same time

Cagniard Latour (V.
in
1, 2),
the middle of the thirties
Schwann
(VII.) stated expressly that yeast
is
(VI.
1,
a plant.
2)
and
Kiitzing-
Meyen agreed
with this view and gave to the new genus the systematic
name
of Saccharomyces
(i.e.,
sugar fungus) which
has
it
since retained.
Considering the state of knowledge at that time, verjvaluable
contributions
to
the natural
history
fungus were made by Eilhard Mitscherlich.
from a paper published by him
of
It is
yeast
evident
in 1841 (IX. 1), that this
investigator recognised the substance invertin.
In 1843
(IX. 2) he read a paper on the multiplication of yeast
he
had observed under the microscope the phenomenon of

budding, and had followed the development from a single
cell.
Schwann, Cagniard Latour and Kutzing expressed the

opinion
that
it
is
the living yeast
alcoholic fermentation.
istic
theory, Justus
v.-
cell
which excites
In direct opposition to this vital-
Liebig (1839-40) came forward with
his theory of mechanical decomposition (VIII.
1).
Accord-
ing to Liebig, every fermentation consists of molecular
INTRODUCTION
motion which
transmitted from a substance in a state
is
of chemical motion, that
is,
of decomposition, to other sub-
stances the elements of which are loosely
In his
work on fermentation
last
(VIII.
bound together.
2),
he sought to
bring this theory into agreement with the observations of
Louis Pasteur on auto-fermentation.
Liebig's explanation
of the latter is that the cells contain a
stance which
decomposing sub-
produces sugar for the auto- fermentation.
Although he at first looked upon yeast as a lifeless mass,

an albuminoid compound, yet he came gradually to the
view that
it
consists of living cells.
But, in his opinion,
there could be no question of fermentation being a physioprocess
logical
in this respect he held to his chemical
conception.
At
that
time a vigorous dispute was taking' place
between the followers and the opponents of the doctrine

of generatio
sequivoca,
i.e.,
spontaneous
of
Let us look somewhat closer at this doctrine.

taneous
generation
organisms from
doctrine,
to prove
lifeless
Needham
embryos.
was the
For
it.
we understand
material
generation.
By
spon-
development of
the
without eggs, seeds or
(1745), an energetic supporter of this
tirst
this
to
make experiments endeavouring
purpose he heated meat extract in
closed flasks, and, on organisms appearing in the flasks, he
assumed that they had been
produced by spontaneous
generation.
Spallanzani (1765) showed, however (I), that certain

errors
were made in these experiments
flasks
hermetically and boiled them for an hour, after
he sealed his
which treatment no development of micro-organisms could

be observed.
"
" o^
From
his experiments
he concluded that the
t^^ micro-organisms are present in the air and
^SS^
only develop after they have found their
On
way into the liquid.
these experiments the foundation of the technique
1*
of sterilisation
was
laid,
and a substantial addition made to
the methods of cultivation.
The
Swedish
and
chemist
Scheele
apothecary
put
Spallanzani's experiments to practical use in the sterilisation of vinegar
by heating
Appert, in France (1810),
(II.)-
went a step further and used the method
for preserving
soup, beer, wine, etc. (HI.).
In 1836-37 Franz Schulze (IV.) and Theodor Schwann

(VI. 1) published the results of researches in which they
sought to prove that

is,
rendered
sterile,
when
air,
freed from
its
germs, that
can come into contact with a sterilised
nutritive liquid without micro-organisms developing in the
The experiments
latter.
were made in the following

liquid
of the last-named investigators
way
were closed with plugs
through which sterilised

the air from
its
acid whilst
Schwann
air
flasks containing nutritive
fitted
with bent glass tubes
was sucked.
germs, Schulze passed

subjected
it
it
In order to free
through sulphuric
to a high temperature.
Their opponents, however, would not accept such proofs,
but asserted that, in these experiments, the air had been

violently treated, and, as a consequence, had suflfered such
a change that the inert matter could no longer be vitalised
by contact with it.

Then in 1854 H. Schroeder and Th. v. Dusch (X.) showed
that air can be freed from germs by filtration through
thus the above-mentioned contention was
cotton wool
disposed of. In fact, this method is still employed when
;
we wish
to sterilise air.
Belief
die out.
4),
by
in
Not
generatio sequivoca, however, did not yet

until
who exposed
his
1860 was the victory won by Pasteur (XI.
all
the failings of the experiments
made
opponents to prove the existence of spontaneous
generation
in every case without exception he could prove
that either an omission or an error had been made.
In
INTRODUCTION
consequence of these brilliant
generatio sequivoca
Up
to
researches the theory
more and more

the present time no single case
into
fell
ill
of
repute.
of spontaneous
generation has been experimentally proved.

It has been
lisation
and
remarked above that the principles of
also a substantial part of our culture
steri-
methods
are the result of the experiments
made
doctrine of generatio sequivoca.
For the recent develop-
in relation to the
ment in this direction we are indebted chiefly to Pasteur

and his school. The appearance of Pasteur marks a very
great and important epoch.
Besides the above researches relating to the doctrine of
spontaneous generation,
we might
also refer to another of
Pasteur's important researches which interests us here, viz.,

his investigation
on
lactic acid bacteria
and
scribes lactic acid fermentation
(1857).
He
finds microbes, which,
he assumes, cause this fermentation in milk (XI.
as
later
1)
on he mentions the same fermentation in beer and
He
worts.
mentation
further proved (1861) that butyric acid fer-
is
brought about by a special micro-organism
In addition we might mention his researches on
(XI. 5).
acetic acid fermentation (1864
shown
in
first
and
1868).
1837 that this fermentation
bacterium (VII.)
was
de-
is
Kiitzing had
caused by a
but important progress in this direction
made when Pasteur published
his experimental
studies on the subject (XI. 7).
Pasteur also made (1861) the discovery that certain
micro-organisms thrive in the absence of free oxygen (XI.

5).
He
calls
such forms anaerobic to distinguish them
from those organisms to which
free
oxygen
is
necessary
and which he terms aerobic.

In 1807 Chaptal had announced that the formation of
a film on the surface of wine always precedes the souring
of the wine and, as already stated, Kiitzing had described
;
the acetic acid bacteria (VII.), which form acetic acid in
beer and wine
but Pasteur was the
idea that bacteria
diseases
excite
to spread the
first
fermented liquids
in
(XI. 8).
The method
Appert
is,
of
His name has also been connected with
practical value.
method
as the
preservation invented by Scheele and
thanks to Pasteur's works, becoming of increasing
is
known
as "
Pasteurisation
it,
".
Pasteur's doctrine that bacteria are responsible for the
fermented liquids gave
diseases of
rise
demand
to a
Pasteur communicated (XI. 8) a
the use of pure yeast.
process for the purification of brewer's yeast,
ing that
it
for
recommend-
should be cultivated either in sugar solution
with the addition of tartaric acid or in wort containing a

small quantity of carbolic acid.
He
end by chemical means.
desired
seeks thus to gain the

(In
most
cases indeed
means of purifying the yeast

was then unknown that some of the
this process is successful as a
from
bacteria.)
It
most dangerous diseases of fermented liquids are brought

about by foreign,
"
wild
"
yeasts,
and that
process
this
favours these particular forms at the cost of the good

yeast, as
2,
6).
3,
was shown
later
by Emil Chr. Hansen (XIX.
In fact the Pasteur process led in a direction
exactly opposite to that in which the desired end lay.
practical
consequence of the doctrine of bacterial
diseases
was the construction
which
it
was attempted
in air
away from the brewery
any
bacterial infection
way.
For
this
to
of apparatus
by means
of
keep the living germs present

worts,
and
so to
ward
which might be contracted
oft"
in this
purpose Velten, a co-worker of Pasteur's,
constructed closed cooling apparatus for the aeration and

cooling of worts.
into general use
as
That
this apparatus did not then
was a simple consequence
come
of the fact that,
mentioned above, the yeast which was added to the
INTRODUCTION
was not pure
carefully cooled worts
we, in the light of Hansen's work,
The replacement
in the sense in
now
use this expression.
the old cooling
vessel
apparatus could only be of real use
when
of
which
by the new
yeast was
obtained which could be depended on.
Pasteur had (XI.
2, 3)
from the beginning of
his career
combated the mechanical decomposition theory of Liebig.

His investigations had led him,
like
Schwann,
present and that
it is
by the mere use of a

by means of chemical
" is life
want
of
are
cells
not possible to bring about fermentaconstituent of the yeast or even
tion
Pasteur,
to the result
when yeast
that alcoholic fermentation only begins
without
"
agents.
air,"
Fermentation,"
and he believed that
oxygen that makes the yeast
said
it is
exciters
cells
the
of
fermentation, these .then taking oxygen from the sugar and
thereby producing the peculiar decomposition.

theory has not, as
NageH
we
Pasteur's
shall see later, stood the test of time.
(1879) in the
main supports Liebig (XVIII.)
He
expresses his molecular-physical theory in the following
words
''
:
Fermentation
is
the transference of the conditions
of motion of the molecules, atomic groups and atoms of
the various compounds constituting the living plasma, to
the fermenting material, in consequence of which, equili-
brium
in the molecules of the latter
being their disintegration
Traube's enzyme theory (1858)

(XII.).
is
destroyed, the result
".
may
be referred to here
According to this theory fermentation
as an effect due to
is
the various enzymes contained
and not to the yeast
cell
itself.
explained
in yeast
This theory has lately
been confirmed by the discoveries made by Emil Fischer
and Ed. Buchner
in the chemistry of fermentation.
E. Fischer's investigations (XXIII.
1,
2)
on enzymes
have not only brought to light new and important facts,

but have also pointed to quite new views as to the nature
of the processes concerned,
and Ed. Buchner (XXV.) by
submitting
high
yeast
cells
to
pressure,
succeeded
in
obtaining an extract capable of producing fermentation
Thus the actual processes

enzyme action, included in the
in solutions containing sugar.
of fermentation are now, like
domain of organic chemistry.

Pasteur closed his studies in fermentation with his
book, Etudes sur la Biere (1876), and proceeded to other
fields of investigation
still
where, as
is
well
known, he gained
greater renown.
A few
years previously (1870) the descriptive botanist
and microscopist,
Max
Reess,
had carried out a research
(XIV.), which, considering the then state of the science,
must be regarded
as of importance.
different species.
He
The spore formation

discovered (VI. 2) by Schwann (1839), and observed later
(1868) by Jules de Seynes (XIII.) in some of the fungi of
alcoholic fermentation, was found by him to occur in many
regarded this as the most important
distinguishing characteristic of the genus Saccharomyces.
Later investigations have confirmed the correctness of this
On
view.
the other hand his statements of the conditions
of this spore
He
formation must be regarded as erroneous.
distinguished the species according to the appearance
of the
cells.
He
did not recognise the pure culture and
could not therefore deal experimentally with the question

of species.
He
used the form
of
the
cell
as the dis-
tinguishing character of the species, calling the ellipsoidal

cells
"
Sacch.
ellipsoideus,"
Pastorianus," etc.
It
the
was proved
later
different forms,
and
Sacch.
by Hansen that one
and the same species of yeast can occur
cell
"
sausage-shaped,
in
all
these
that, consequently, the shape of the
cannot be applied in this way.
The Reess
species
have therefore not found acceptance in modern experimental

science.
INTRODUCTION
We
have
in the
lost
now
reached that point where interest was.
theoretical
of the question.
9'
The
well
as
as in
the
practical
side
technologists felt themselves deceived
by the expectations aroused by the above researches on

yeast.
The facts taught them by science did not hold good
in practice, and often, indeed, their position became precarious.
Large sums of money were
lost in the breweries-
on account of accidents during fermentation, accidents
the-
causes of which could not be explained, and against whicb
The yeast was spoken
precautions could not be taken.

as something mystical.
The view
of
of the science at that
time (1884) was depicted in the following expression of

Thausing's (XXII.)
"
Science has given us fine researches
on fermentation organisms and on the nature of fermentation, but it has yielded almost
the brewery
far as practical application
darkness.
nothing of direct value to
now, as before, the process of fermentation,
The
is
concerned,
investigations of
is
veiled
by
Hansen on the
pure yeast entitle us to great hopes
if
so-
a mystic
culture of
they do not
lie
we
are near the attainment of an end the importance of which
cannot be sufficiently valued.
In the
we
have to reckon with the state of
at
present."
first place,
aflairs as
however,,
they stand
Similar pronouncements had already been
made by Holzner (XVII.) and Lintner (XX.).

Some years before this Hansen had published some of his
investigations but only now was attention directed to him.
As botanist Hansen began and completed the reform which
inaugurated the new era in the biology of the fungi of
;
alcoholic fermentation,
and
also in fermentation technique
as a consequence of the practical results achieved.
In 1880 and 1881, he conducted experiments on the
micro-organisms occurring in air at various times of the
During these researches he observed a characteristic

which enabled him to decide whether a flask contains a pure
year.
10
culture of a yeast fungus or not, and on this he founded his
At the same time he began
pure culture method.
first
his
experiments on the diseases produced in beer by yeast

fungi,
and expressed the
forms
belief that the wild yeast
sometimes produce as great disturbances in fermentation

industries as bacteria do.
little later
he arrived at
points of view for the investigation of species,
the outlines of spore analysis.
The
new
and indicated
results of these pioneer
investigations are to be found as short notes interspersed
throughout his second treatise on the micro-organisms of the

air (XIX. 2)
which appeared at the beginning of 1882, but
remained unnoticed at the time.
His treatises published
1883 formed, however, the real turning point {XIX.
had probed these questions
this time he
to
in
At
3).
such a depth,
that he was able to inaugurate a reform in theoretical as

well as in practical relations.
In connection with pure culture methods,
we have men-
tioned in the foregoing that Mitscherlich (IX. 2) had, in

1843, observed the budding of single yeast cells under the
Of
microscope.
ticular
perfection this
life
his successors Brefeld (1874) deserves par-
mention as the one
method
who brought
to a high degree of
for studying the
history of diflferent fungi (XV.
1, 2).
morphology and
But the procedure
followed by these investigators did not suffice
when
abso-
lutely pure cultures of micro-organisms were required in
large quantities such as
experiments
are
necessary for physiological
the requirements are then
and accordingly the
efforts of the
quite
different,
subsequent investigators
were specially aimed at working out a process to
suit this
case.
Here we must place
in the front
sought to prepare pure cultures of
rank Lister (1878), who

lactic acid bacteria
He
by
distributing
them
down
only some of the culture flasks contained a
until
in the culture liquid (XVI.).
diluted
INTRODUCTION
growth, and from
this
1]
he then infers that the flasks which
show development each contain a pure culture.

But this method affords no security, and Hansen
fore, in 1880-81,
worked out
his first method,
there-
which has
been referred to above.
He made
the important observa-
tion that the yeast
after they
have been well shaken
cells,
Tip in the flask containing nutrient liquid, sink to the bottom,
and form there
distinct
and well-separated spots of
Examination showed, as was

flasks, in
yeast.
to be expected, that
those
which only a single yeast spot had developed,
This observation was a considerWith this method Hansen combined

cell-counting by means of a cover glass divided into squares.
This rendered it possible to sow a single cell in each flask,
and an exact method of preparing pure cultures in large
contained a pure culture.

able step forward.
was thus obtained.

At the same time Robert Koch published
quantities
his investi-
gations on pathogenic bacteria, and, like Hansen, he
felt
the need of a satisfactory pure culture method for the pre-
paration of mass cultures.
by him
first
Nutrient gelatine was brought
into extensive use in bacteriology (XXI.
method
in nutrient gelatine.
pletely set,
it
1).
His
(1881) for pure culture consisted in dilution
Before the viscous gelatine had com-
was stroked with the point
of an inoculation
needle which had previously been in contact with the

growth from which the required pure culture was to be
prepared. The last streak made in this way may contain
The method was, as one can see, a very
isolated colonies.
imperfect one, and
Koch soon introduced
that of plate cultures (1883) (XXI.
2).
another, viz.,
In this method the
germs are distributed in liquefied gelatine, and are, by this

means, more thoroughly dispersed.
Like Hansen, Koch
also observed the single spot but the last-named method
;
is
not so sure as that of Hansen, in which the
cells
can be
12
more uniformly distributed owing

nutrient medium.
frequently showed
than
one
cell
several species
to the use of a liquid
In Koch's plate culture a colony very

itself to
have been derived from more
the possibility consequently arises that
may
Shortly after
be mixed together in one colony.
Koch had communicated
his plate culture
method, Hansen published his second pure culture method
(XIX.
4,
The
5).
dilution, in this case, is carried out in
nutrient gelatine, but the starting point

cell
which
is
is
from the single
With respect
two methods are equally good,
gelatine being made as it appreciably
controlled under the microscope.
to their accuracy, Hansen's
the substitution of
lightens the
work connected with the preparation
of the
pure culture.
From
the above
it is
plain that Koch's
conform with the requirements

starting point, as does that of
starts
from a single
cell.
more
Hansen, since the latter
With
cannot be entirely carried out.
method does not

a
strictly necessary for
bacteria,
however,
Koch's method
is
suitable for separating the various elements of
cultures, so that
one does not
isolate
mixed
only that kind which
occurs most frequently, but also most of the others.
method
this,
much
His.
also acquired the distinction of bringing nutrient
gelatine into general use as a culture
medium
from the
appearance which the growths have on this medium, the

characters of the species can to some extent be
In course of time the number of species

various divisions of the fungus system
made
out.
belonging to
which were treated
in Hansen's investigations on the organisms occurring in
beer and beer worts became very large.
(XIX.
1)
His
first treatise
on fermentation organisms was published in 1879.
Among bacteria, he made a special study of those of vinegar.

He explains hitherto unknown differences in form, and shows
how to recognise the conditions causing these variations.
INTRODUCTION
What we know of
13
the morphology of these species
is
due
him (XIX. 9). He further studied mould fungi

and the fungi of alcoholic fermeutation generally, but more
chiefly to
especially the Mucors
and the Saccharomycetes.

His experimental researches on this subject are contributions to the
general biology of the whole of the fungi (XIX. 8). As an
example we might cite his researches on the circulation in
nature, on the life cycle and on the conditions for spore and
film formation.
He
gives definite methods for bringing into
play the last-named functions, so that what was formerly

entirely
beyond control can now be brought about with

There might be named, in addition, his researches
certainty.
on the germination
of spores, on the relation between the

and the conditions of culture, on the limits of
form of the
cell
life of cells,
using different methods of preservation, on the
Ijehaviour of species towards the sugars, etc.
These inves-
became likewise of immediate importance for the

recognition of species new points of view were here brought
tigations
to light, and this question

attained.
was sifted to a depth not hitherto

The above-mentioned group of investigations
showed that the Saccharomycetes, under
certain
methods of
treatment, appear with constant characteristics, and that
can here, as with other fungi, make a separation into
we
species,
a point which was doubted by several investigators at the time

when Hansen began his work. Another part of Hansen's
work treats of variation (XIX. 8). He shows how, under
certain conditions of culture, the characteristics can vary, that
these variations are either temporary (variation of the
cell
form, variation of alcohol production") or permanent, and
how
the latter retain their characteristics through endless
generations and under

less
and
filmless
all
methods of treatment (spore-
varieties).
Theoretically these inves-
tigations are of special interest as
showing
that,
in the
apparent irregularity of the variations, conformability to
14
law
prevails.
portance
For the fermentation industries their im-
lies in
show how new and
the fact that they
permanent races can be prepared with certainty.
Having mentioned, in the foregoing, chiefly the theoworks of Hansen, we will now give a review of his
retical
But, in fact,
practical investigations.
sharp
hand
line,
since here theory
we can draw no
and practice constantly go
Practical difliculties, with
in hand.
which the two
Carlsberg breweries and also the Tuborg brewery in Copen-
hagen had to contend, induced Hansen, confident of

to strive
The
with
all his
energy to
effect a
success,
fundamental reform.
practical consequence of his theoretical investigations
was, on the one hand, the elimination of the disease yeasts
on the other, the separation of the culture yeast (the Sac-
charomyces
species
latter.
and
cerevisise of
former investigators) into several
races, and, finally, systematic choice
This choice forms the
from the
most substantial part of
Hansen's pure culture system, and was based on the study of
new points of view. Finally, he worked

new method for the analysis of brewery yeast (XIX. 10).
It was then made clear how very different these species and
races are, and how each gives a special character to the
The great differences of the yeast
liquids fermented by it.
showed themselves in a surprising manner, especially when
the so-called wine yeast, Saccharomyces ellipsoideus, was
these species from
out a
separated into
its
systematic units.
Hansen published,
siderations, but, at the
1883, not only theoretical con-
in
same time, the
results of experiments
which he had carried out in the old and new Carlsberg

His new system was worked
breweries in Copenhagen.
out to the smallest details, and had also been tested in

practice, so that it could
be applied at once without any
preliminary experimenting.
above,
As one may gather from the
Hansen himself introduced
his
system for the bot-
INTRODUCTION
torn fermentation breweries.
into different countries
This system spread quickly

and found an entrance hot only
into the breweries, but gradually also into the spirit
and
pressed yeast industries, and the manufacture of wine.
The
between Pasteur's and Hansen's woi-k in

and forcibly expressed by Delbriick
in a lecture (XXIV.) delivered in Berlin in 1895 " Looking
back on the last twenty-five years, there are two great
relation
this respect
was
clearly
epochs marking the scientific development of brewing;

Pasteur's work,
which was done after 1870, and which is

when we nowadays strive, by the
adopted in principle
setting
up
forms
one
of cooling vessels, to
epoch
ward
Hansen's, the
off external infection,
other.
attempts could not lead to a fruitful
But Pasteur's
because one
issue,
was missing which was furnished by Hansen in his

systematic choice of pure yeast. These two men and their
discoveries have been the moving forces of the last decade,
link
and have brought brewing
By
to
what
it is
to-day."
Hansen's discoveries, the subject of micro-biology
and fermentation technique here treated was given new

and an impulse was imparted to the formation of a
life,
rich literature.
From
that time onwards, the interest of
technical fermentation laboratories
the Saccharomycetes
is
claimed especially by
and now new laboratories
for
promot-
ing the industry are being erected in which biologists

side
by
side with chemists,
polised the
whole
field of
where formerly the
work.
An
latter
work
mono-
ever increasing body
up the subject, and

names and work will be given in the following
sections, where the subject, as reviewed in this introduction, will now be examined more closely and treated in
of distinguished investigators has taken
their
fuller detail.
SECTION
II.
THE LABOEATOEY.i
Micro-biology
has,
during
its
work
in the service of the
alcoholic fermentation industries, developed a special tech-
nique and elaborated special methods;
assumed
a character of
its
own, as
may
research has
its
indeed be seen in
the fitting up and in the apparatus of the laboratories which

are
now
to be
found in
many
places,
sometimes as private
Some
laboratories, sometimes as state institutions.
purely for research,
as, for
example, that at Carlsberg, and
work is to promote the science

organisms by scientific investigation in
their
practical direction
class, are
them with
others,
fermentation
of
a theoretical and
and the most belong
designed to serve practical

analyses,
men by
and
to
this
furnishing
and providing them with pure
tions of selected species
cultiva-
Laboratories
varieties of yeast.
study of fermentation organisms are
for the
are
now
also to
be found attached to a number of the technical colleges

(Hochschulen).
after
it
These were set up for educational purposes
had been recognised what an important influence
the study has on the scientific instruction and on the industrial activity of
As
the
the manufacturer.
number
of laboratories increased,
and according
as they were constructed for the study of special branches

of the fermentation industry, the outfit which
'
The bibliography
will be
found at the end

16
of
had been
the book.
FITTING UP THE LABORATORY
17
formerly characteristic of these laboratories naturally experienced changes in regard to

of the individual opinion of
chemical,
we have taken
up
influence
also
biological
part
and not the
as a pattern sometimes the Carls-
sometimes certain brewery laboratories
berg laboratory,
fitted
The
managements
For the following description,
contributed to these changes.
which comprises only the
fittings, etc.
diiferent
which the author has
for purely practical purposes,
had an opportunity of inspecting; brewing
chosen by
is
preference to exemplify the application of the methods in
In general
practice.
may
this description applies also, as
be supposed, to those laboratories which are associated with

other branches of the fermentation industry.
I.
and Apparatus.
General Principles for fitting up the Laboratory.
1.
In
Fittings
many
when
cases,
a laboratory of the physiology of
fermentation has to be fitted up, the

fixed so that there
is
no choice
done with the space at one's
If there is
disposal.
in the matter, a site facing the north
sunlight
is
have been
site will
the best has therefore to be
is
any
choice
to be preferred, as
not only very troublesome in microscopical work,
most micro-organisms.
Further,
but
is
also
very much to be recommended in cases where there
also fatal to
sufficient room, that the space be divided in such a
there
is
a small room to be used only for
it is
way
work with
is
that
yeast
and a larger one in which are placed the

microscope table, cupboards for cultures and apparatus, a
working bench, etc. work on moulds, for example, might
and
bacteria,
be performed on the last named.
The
conidia of moulds,
being developed on the exposed surface of the nutrient
medium, cannot be retained by the
liquid, and,
of their lightness, are very easily carried about
on account
by the
air
18
from place
Special
place.
to
precautions are therefore
when working with moulds

before, not to work with moulds
necessary
said
the best
in the
way
is,
as
same room
where yeast and bacteria are being studied.

It is therefore necessary to
dust and germs as
po.ssible.
For dust always contains
of
bacteria as well as of moulds
germs of micro-organisms
and
The surface
yeasts.
provide a place as free from
of all fittings in the laboratory
should therefore be as smooth as possible, without project-
ing corners or hollows in which dust can
settle.
ought to be at hand uo more apparatus than
is
There
absolutely
necessary; cultures and apparatus should therefore usually
be kept in cupboards; only those objects should stand on

the tables which are being used at the time, and these
away
Only in this way is

it possible to keep everything dust-free and clean.
The
laboratory should appear as if nothing were being carried
on oven at the time when most is being done. If such a
system is once introduced into the working of a laboratory
should be put
time
is
again after use.
economised and security
is
ensured during the pro-
gress of experiments.
Cupboards and drawers ought therefore

so that dust cannot force
its
way
in
to close tightly
this is attained
by
providing the cupboard doors and the drawers with over-
lapping edges.
suitable height for the
97 centimetres (about 38
Preparation of Bench Surfaces
must be prepared
as
many
is
The working
tables
so that they can stand washing with spirit,
experiments have to be performed on a wet bench.
This can be done in the following

prepared,
(1)
and
way two
:
solutions are
an almost saturated solution of aniline hydro-
chloride in water,
chlorate
working table
in.).
distilled water.
and
(2)
a solution of
part of potassium
part of copper sulphate in 120 parts of

Solution
(1) is first
rubbed into the wood
FITTING UP THE LABORATORY

and then
solution
(2),
19
the solutions being used alternately
wood has become sufificiently

must have soaked into the wood
The one
until the
black.
solution
before the other
is
applied.
If
the aniline salt partially crystallises, the
bench must be moistened with warm water before solution

(2) is applied.
If it is
found from the colour of the wood
that one of the solutions has been used in excess, the other
solution
applied twice successively.
is
not to use too
much of
solution
(2),
When
becomes green instead of black.

the bench
complete the wood
is
with linseed
water
oil
As a
rule
it is
better
wood
as in this event the
this treatment of
well rubbed for some time
is
Repeated washings with lukewarm
varnish.
will often be necessary, especially if the colour rubs
off.
The above described preparation was much used formerly,

but there are also other means for producing such a surface.
From experiments made by

.seems better than the
the author the following recipe
Two
solutions are also used in
grams of
aniline hydrochloride are
first.
this method, viz.: (!) 600
dissolved in 4 litres of water, and (2) 86 grams of cupric
grams of potassium
chloride, 67
ammonium
chloride are dissolved in 1
mediately
before
mixed
is
with
use,
volume
and 33 grams of
litre of water.
Im-
chlorate,
volumes of
4
of
solution
solution
(2),
(1)
are
and the wood
treated with this mixture once a day for four or five
Afterwards the bench receives an application of
days.
the linseed oil varnish.
The black colour develops more
quickly than by the former process.

Solutions
Washing
for
The mixture
66
parts
of concentrated
spirit.
of
consists
table
is
of
per
Bench during Work
boiled
washing the bench
water
The sponge used
kept in the mixture
laboratories
gram
the
of spirit to be used for
when not
and
in
33
parts
washing the
in use.
In some
an aqueous solution of mercuric chloride
litre)
is
used instead of
2*
spirit.
(1
20
The Microscope Table. The microscope table is most conwhen facing the north. The proper height
veniently situated
of the table
is
about 86 centimetres (about 34
the stool belonging to
The
Sterile
above, to
Room.
it
in.),
that of
62 to 63 centimetres (about 24
It
is
in.).
especially desirable, as stated
up a small room where only experiments with
fit
yeast and bacteria are performed.
has two such "sterile" rooms.
The Carlsberg laboratory

The windows, which are
double, are well sealed so that no draught can set the air of
Where
the room in motion.
the
windows do not look to
the north, the panes are of frosted glass or are painted over.
The walls and the
Curtains are absolutely to be avoided.

ceilings are painted
smooth surface and

ance
when
room
germs
is
to be
have to be taken,
made very damp
this is of import-
washed down or
these surfaces have to be
special precautions
the
with enamel which gives a perfectly

also resists moisture
as, e.g.,
when
if
the air in
in order to purify
floating in the air being then precipitated.
it,
the
This
is
accomplished by keeping the room

some time by means of a small sprayer, after which the
room is left quiet before it is used. The floor is covered
with linoleum so that all cracks and clefts are covered.
full of
Pipe systems in the room are avoided
water spray for
the gas pipe just
passes through the wall and ends in a stopcock to which rub-
ber tubing
working
is
attached leading to the Bunsen burner on the
table.
In addition to the latter there
table furnished with drawers above
is
a small
and cupboard below
containing spatula, forceps, inoculating needles (brass rods

or pieces of platinum wire fused into glass rods),
selection of the nutrient liquids
and
also a
most used and empty sterile
flasks.
In working with flasks a Bunsen burner

can be
made luminous
small flame.
or non-luminous
is
used which
and gives a large or
HANSEN'S STERILE CUPBOARD

To complete the equipment
of tinned copper
is
required.
working with a Pasteur
of the room, a shallow dish
This
flask,
21
is
used sometimes when
sometimes for holding the
sterilised spatulas or inoculating needles
which are
set to
cool here after sterilising in the flame, the dish being covered
with a glass plate
2.
sterilised in the
same manner.
Hansen's Sterile Cupboard.
If the circumstances are such that a sterile
be obtained,
we must
Fig.
avail ourselves of the
1.
room caimot
Hansen
" sterile
Hausen's Sterile Cupboard.
and perform the finer kinds of work in it.

This cupboard was the model for fitting up the sterile room,
cupboard
and
is
"
It is obvious that
really a miniature of the latter.
the cupboard ought also to be used

particularly delicate
work
when
it is
desired in
to take special precautions against
infection.
This cupboard (Fig.
framework and
and
is
floor
polished with
1) consists chiefly of glass,
being mahogany.
The
linseed oil varnish,
only the
latter is smooth,
and bears washing
22
with dilute
spirit.
The dimensions are about
as follows
height, 56 centimetres (22 in.); length, 63 centimetres (25 in.):
breadth, 50 centimetres (20

a sliding door
The front
in.).
side consists of
which can be kept open at any desired height.
Before the cupboard
used
is
it
washed
is
and
inside
out,
either with boiled water alone, with yV per cent, solution

of mercui-ic chloride, or with dilute spirit
important
to brush and then to
first
up and down,
the door slides
which
settle in
damp
it
specially
is
the surface
in order to prevent
where
germs
the groove from penetrating into the cup-
board.
The cupboard
is
then closed and allowed to remain
(usually for an hour),

still,
and the water
carried the
till
the air in.side has become quite
particles
with which
germs present down
tc the
it is
damp
saturated have
floor.
The cupboard must be kept sufficiently damp duringexperiments, as otherwise the germs are apt to be stirred up
again.
In experiments where infected solutions or the like are

liable to
be
spilt, it is
an advantage to cover the
floor of the
cupboard with a zinc tray which can be easily removed and
which can be cleaned and

3.
sterilised before
The Microscope and
The microscope
is
its
and after
use.
Accessories.
one of the most important adjuncts
in investigations connected with the physiology of fermentation.
It will be shortly described here, and, in addition,
references will be
made
detailed information
The
to special literature
may
compound microscope
systems of glass
investigation
lenses,
where more
be obtained.
(Fig.
the one
2)
consists
nearer
the
being called the objective, and
nearer the eye, the eye-piece.

into a brass tube.
The
two
of
object
the
of
other
All these lenses are fitted
objective forms a real, enlarged
and
THE MICROSCOPE
inverted image of the object which
this
image
is
being examined
again magnified by the eye-piece.
image that we see in a microscope
However good
may
the lenses
images, and the error
is
and
an inverted one.
by the
eye-piece.
Microscope.
Chromatic
caused by the objective arise
chromatic aberration.
is
Thus the
be they never form exact
increased
Flo. 2.
Spherical
is
23
Aberration.
The
errors
mainly from spherical and
Spherical aberration
to the fact that, of the rays of light
is
to be ascribed
which, diverging from
24
a
pass
point,
t'ocussed at the
is
through a
lens,
same point
as the outer ones.
therefore hazy in outline.
known,
is
composed of
the central ones are not
The image
Further, white light, as
different coloured constituents,
is
and
on passing through the lens these are separated so that a

coloured image
is
produced and the outline contains the
familiar rainbow colours.
To reduce
spherical aberration
various diaphragms are inserted in the tube which cut off
the peripheral rays, and to eliminate chromatic aberration

the lenses are composed of a biconvex and a plano-concave
made respectively of crown and flint glass.

This
method gets rid of chromatic aberration almost entirely.
lens
In order
there
is
to further correct for this
and spherical aberration
placed between the objective and the eye-piece a
so-called collective lens.
Achromatic
and
Apochromatic
we have
objectives as
described
Objectives.
styled
are
Such
achromatic
recently so-called apochromatic objectives have also been
These are made of special kinds of glass
constructed.
(borate, phosphate, baryta
and
fluoride glass),
which more perfect colour correction

are far more expensive than the
is
first
by means
of
Thej'
attained.
named, which are
quite good enough for the ordinary demands of fermentation
work.
The Tube,
The
tube
is
so arranged that
it
can be
elongated and can thus increase the magnification
quently
it is
fre-
provided with a scale of divisions by which
the lengthening can be determined.
on a brass stand which
carries,
The tube
among
is
supported
other things,
two
screws, one for coarse and one for fine adjustment.
Correction
fications
Objectives,
Objectives
for
high magni-
have in some cases an additional adjustment for
the varying thickness of cover glasses.
In these there
is
ring on the objective provided with a scale, the numbers of
THE MICROSCOPE
25
Avhich correspond with cover glass thicknesses expressed in
tenths of a millimetre
mark
the ring
is
turned until the proper
coincides with a fixed index.
The Condenser,
A centrally perforated stage, on
which the preparation to be examined is laid, is also
fixed
to
the stand; under this stage there
which serves to project the rays of
light
is
a mirror
through the
aperture in the stage, through the preparation, and so
along the tube to the eye, thus providing the necessary
The mirror
light for observation.
is
double, being plane
on the one side and concave on the other; the concave
and
side gives the strongest light
Of
the higher magnifications.
therefore used for
is
late years other condensers
have been used, especially that designed by Abbe.

apparatus, which
may
be seen in Fig.
much more
of the microscope, causes a
The
pass through the microscope.
means
make
of a diaphragm, for
it
magnification more light

is
forward in Fig.
allow more or
condenser
is
that
2),
is
intense light to
be so intense
as the iris
With increasing
form
diaphragm (brought
which can be easily adjusted so
less light
by
as to
A very suitable
required.
known
This
lying in front
light is regulated
may
the preparation indistinguishable.
of diaphragm
2,
to pass through.
If
the
as to
Abbe
not used there are circular diaphragms with
openings of different
sizes
which can be brought under the
The condenser
aperture of the stage.
is
especially advan-
tageous in the investigation of stained preparations of

bacteria.
Immersion Objectives.
and
in order to get
objectives are used.
which
definition,
ver}- small since their
and are immersed

lies
very high magnifications,
good
immersion
In these the objective lenses are very
powerful (and therefore

great),
For
specially
on the cover
in a
glass.
curvature
drop of liquid (water or
Water immersion was
is
oil)
intro-
26
duced by Amici, whilst homogeneous or

first
oil
immersion was
suggested by Stephenson.
In order to understand the advantage of the immersion
system over the dry
it
is
necessary to
know what
the
angular aperture and the numerical aperture of the lens
The angular aperture is the greatest angle formed by

two lines drawn from the focus to the edge of the lens.
The numerical aperture is the product of the refractive
are.
Fig.
3.
Diagram
of a section through the front lens of the objective
and the
cover glass, showing the direction of the different rays of light with and with-
out immersion liquid.
index of the
and the
medium between
cover glass and objective
sine of half the angular aperture (Fig. 3).
The
greater the numerical aperture the more rays pass from

the object through the objective.
numerical aperture
index of air
is
is
always
equal to
1,
less
In the dry system the

than
1,
for the refractive
and half the angle of aperture
must, of course, be always smaller than 90, and
its
sine
THE MICROSCOPE
consequently
less
than
The
1.
27
refractive index of water
but in practice the aperture for water immersion
is
1'33,
is
never greater than 1'25
the index
is
the same,
viz.,
for cedar- wood oil
and glass
but the aperture
1-52,
not
is
more than 1'40. The difference between the immersion

and dry systems may be understood from the accompanying sketch (Fig.
This gives a section through the front
3).
lens, L, of the objective, the cover glass,
and the interven-
ing medium, that to the right through air (refractive index

/i
EO
1),
that to the left through
oil (/a
and GO, proceeding from the
Two
1'5).
rays,
object are refracted in
OP;
passing through the cover glass towards the normal
GO
EO
in the direction D,
ray,
GOD,
leaves the cover glass
sion liquid, in this case
through the
oil in
the
passes into the immer-
it
the ray therefore will continue
the same direction as through the glass
will pass into the objective at A.
hand, the dry system

will
When
which has the same refractive
oil,
index as the cover glass
and
in the direction J.
is
on the other
If,
used instead of the immersion there
be air between the cover glass and objective with
i-efractive
index /a=1, so that a ray, EO, which leaves the
cover glass at J does not pass into the objective at
Only those rays which

such as
FO
are
less
all.
oblique than some ray
will be able to strike the objective.
It
is
thus
seen that far more rays take part in the production of an
image when the immersion system

quence of this a much better image
The strength of
cated by a fraction,
the
oil
e.g.,
used,
is
is
immersion lenses
yV.
tV, etc.
and
in conse-
obtained.
By
is
usually indi-
this is
meant the
equivalent focal distance of the respective lenses, expressed

in inches.
Immersion lenses ought

use
by means
to be cleaned
immediately after
of an absolutely dust-free linen rag
should be kept in a tightly closed box.
which
quite dust-free
28
material should also be used for drying the lenses, for dust
often contains particles which are capable of scratching
glass.
However, the lenses themselves should be moved
and rubbed as little as possible. The liquid can be removed
from the edge of the glass, when necessary, by means of
blotting paper, and under certain circumstances the glass
may also be cleaned with benzene or alcohol. The other
parts of the microscope can be dusted
by means
of a soft
brush.
The Stage
it
is
up
fitted
is
in various ways,
These are, however,
positions.
so that
e.g.,
capable of rotation and of adjustment in
diflferent
details, the description of
which may be omitted.

There
is
sometimes a scale and vernier on the stage, the
vernier being an arrangement which allows a finer reading of

the scale to be made.
with the fixed
scale,
It
is
a smaller scale running parallel

its
divisions are exactly
latter.
That division of the
and ten of
equal to nine divisions of the
vernier which coincides with a division of the stage scale
gives the required fraction in tenths.
Changing the Objectives.
There
are various
The
of attaching the objective to the tube.
which
in
some
cases are simply screwed into the tube,
also be adjusted
ways
objectives,
may
by means of a revolving arrangement (nose-
piece),
allowing several objectives to be attached to the tube
at the
same time
by simple
brought into the required
rotation
any
The
position.
objective
may
be
objectives can also
be changed by a sliding arrangement or with the aid of

clips.
These different arrangements enable one to change
the objectives more easily and quickly.
Some
general rules for the testing of a microscope will
be given in what follows.

Testing the iWicroscope.
each microscope several
There are usually given with
test objects
most frequently these
THE MICROSCOPE
from the wings
consist of scales
janira)
scope
2&
of a butterfly
and a diatom {Pleurosigma awjulatum).
may
(EpvuepheU
The micro-
then be tested by examining these under different
magnifications.
The
be found in the table
latter are to
The
always supplied with the microscope.
first
preparation
examined with a low magnifying power (60 to 150 times) ;

the transverse marks on the scales ought then to be quite
is
distinct.
The
markings
to be plainly visible
and using oblique
To obtain
diatom has
silicious shell of the
fine crossed
with a magnification of 400 to 500 these ought

;
with a magnification of 150 to 200
light they
ought to be distinguishable.
oblique light the concave mirror
is
turned so that
With very strong immer-
the light enters from one side.
sion lenses the markings are resolved into a mass of small

six-sided figures.
lines
ought
In such a test the sharpness of the out-
also to be noted.
whether the screws,
When
protect
it
should be ascertained
etc., fit well.
the microscope
up so as to
commended
Lastly,
it
is
from
not in use,
dust.
it
for this purpose, that half of
the light being painted with
oil
must be covered
be re-
bell jar is to
it
turned towards
colour to protect the micro-
scope at the same time from sunlight which, in course of

time, affects the fine lenses, the mirror and the stand.
Slips
and Cover Glasses.
In the microscopical
investi-
gation of micro-organisms glass slips and cover glasses are

used.
The growth
is
placed on the slip in a liquid and the
cover glass laid on the top.

in general 7 5 cm. long,
The
25 cm.
slips are rectangular,
broad,
and
1'5
mm.
and
thick.
Cover glasses are of various thicknesses, thin ones being

used for finer work. The use of thick glasses, e.g., 020 mm.
If mixed
thick, is to be recommended for ordinary work.
cover glasses are bought, they ought to be sorted according
to their thickness,
which can be determined by means of
the apparatus described on page y4.
The cover
glasses
-so
mm.
used most are 18
square; but round and rectangular
ones of various sizes are also employed.
For several kinds of
which are etched
vsrith
being numbered.
cover glasses should be used
squares, the squares in
some
cases
Fig. 4 represents a squared cover glass,
used by Hansen in his
Fig.
vsrork
4.
first
The
pure culture method.
Hansen's Squared Cover Glass.
squares were used for determining the total number of
which were present in a drop of any
liquid.
was contained within the boundaries of the whole

Hence the small size of the latter.
The most frequently employed squared cover
have larger squares
numbers the squares
(see Fig.
in
the
and
top
Fig.
6).
row and the
z.
cells
This drop
square.
glasses
Will only
left-hand
THE MICROSCOPE
pointed forceps;
it
taken out quickly and as much as
is
wax
possible of the melted
either side a thin even
By
to solidify.
allowed to run
acid.
aid of a very fine needle
leaving on
is
allowed
and a small
wax
then scratched on the
immersed for a moment in hydrofluoric
glass
If there is
off'
layer of wax, which
ruler the required lines are
and the cover
31
no silver or platinum crucible obtainable
for holding the acid, a
common
porcelain crucible or dish
or a watch glass can be used after being coated with

paraffin or beeswax.
the acid
it
After taking the cover glass out of
washed with water and then
is
water to melt
wax
off the
it
laid in
warm
afterwards dried and
is
placed in chloroform in order to remove any traces of

grease.
have a stock of these squared
It is convenient to
cover glasses, which are
used
investigating the
in
life
history of development and for preparing pure cultures,
as will be described later on.
The Micrometer.
It will
be necessary in
to be able to measure those objects which

the microscope.
It is usually in the
micrometer
is
many
cases
we examine
in
used for this purpose.
form of a thin glass plate situated
in
the eye-piece and on which a certain number of equal

divisions are etched.
When
the micrometer
piece the size of the object can be
how many micrometer
divisions the
is
in the eye-
measured by
finding-
object covers.
On
the table of magnifications supplied with the microscope
are usually to be found those niimbers with which direct

readings must be multiplied in order to get the true length
of the object;
*-^'>
TTrVff
of ^
the length
millimetre,
is
given in micro-millimetres,
magnitude denoted by
fj,.
This factor can be determined independently by using a

stage micrometer, that
is,
a glass strip on which 21 divi-
sions are etched, their distance apart being
metre or 10
/i
-^
of a milli-
with this micrometer on the stage and the
32
other in the eye-piece
it
may
be then noted
how many
number
divisions of the micrometer are equal to a certain
on the stage micrometer.

the stage micrometer
If,
= 20
/a)
for example, 2 divisions of
correspond with 7 divisions,
of the micrometer of the eye-piece, the apparent size ex-
pressed in divisions of the ejê-piece micrometer have to be
by ^S = 2'857 in order to get the true length

expressed in fi. The higher the magnification, the smaller
multiplied
is
this factor.
Counting Apparatus.
yeast
cells
"
"
net
In
eye-piece
which
piece of glass on
is
order to be able to count

used, consisting of a circular
Fig.
7.
Haematimeter.
The object
()
on to
it.
glass
{b)
the cut-out cover glass fastened
(After Hayeni-Nachet.)
into sixteen or twenty-five smaller squares

in the
same way
used in conjunction with a haematimeter.
is
a glass slip
(a),
which
This (Fig. 7)
fastened a cover glass
(b),
which a circular piece has been cut
out.
to
from the middle of
is
drop of liquid containing the
cells
to
be counted
placed in the shallow space thus formed and enclosed
ordinary cover
The thickness
glass.
glass is usually 0"1 or 0'2
Thoma's haematimeter
micro-organisms.
is
up
it is fitted
as the micrometer in the eye-piece and
is
up
etched a square, divided
is
A is
is
by an
of the perforated cover
mm.
(Fig. 8) is also
a glass
slip,
used for counting
on which a cover glass
(a)
fastened which has a circular hole in the middle and is
02 mm.
thick.
circular cover glass
(c),
01 mm.
thick,
THE MICROSCOPE
is fitted
centrally in this hole
glass slip
thus,
middle of
two
(c),
and
an annular space
sets
of
is
33
also fastened to the

is
(d)
twenty -one
In the
formed.
parallel
etched which cut each other at right angles.
lines are
There are
thus formed a large square with a side of 1 mm., and small

squares with a side of 0'05
examined
glass
(b),
is
placed on this
mm. B
The method
8.
The drop of liquid to be

square and enclosed by the cover
the depth of the liquid layer
ing to O'l
Flo.
mm.
Thoma's
cover glass.
Klonne
(e)
gives a vertical section of the chamber.
of counting
Chamber.
is
described later.
{A) View from above; {B) from the side;
(The significance of the remaining
and
marker (Fig.
9)
thus formed amount-
Muller's
Object
(6)
letters is given in the text.)
Marker.
made by Klonne and
The
object
Miiller (Berlin) is
often used in the course of the preparation of pure cultures
and during observations on growth.

This instrument
is
used to
mark
a certain spot in a pre-
by impressing a coloured ring round it on the cover

The apparatus is fitted on the microscope in place of
the objective. There is an opening where the front lens of
the objective would otherwise be. The lower half of the
apparatus is capable of vertical movement, being fitted with
paration
glass.
34
somewhat weak spring. The opening at the point must

be quite flat and ought not to have a greater diameter than
0'75 mm.
The method of using it will be referred to
a
later.
Cover-Glass Gauge.
tioned that
In
the above
it
are
sometimes
provided
with a
any thickness
of cover
objectives
correction so as to be adjustable for

glass.
It is occasionally
important to be able to measure
The instrument represented
the thickness of cover glasses.

in Fig. 10
Fig.
is
9.
has been men-
Klonne and MuUer's
Fig.
When
the upper part
10. Cover-glass Gauge.
Object Marker.
is
screwed
down
as far as possible, the zero of the scale
the screw-head coincides with a
mark on
the stem.
measuring the thickness of a cover glass the screw
is
on
In
screwed
back, the cover glass inserted edgeways in the space thus
made, and the screw again brought down until

the cover glass.
The number which
is
now
gives the thickness in ^h) io-
Apparatus for
cal
Artificial
work one must often
lamp
is
necessary on the
Illumination.
it
In
has the advantage of coolness
mark
microscopi-
resort to artificial light
microscope table.
touches
at the
and so
Electric light
otherwise a gas or
oil
lamp
THE MICROSCOPE
must be
As
used.
35
the continued use of a strong, yellow
light is injurious to the eyes, it is better to let the rays
from the lamp pass through a blue liquid, by means of which

an agreeable greenish or bluish light is obtained which does
not
affect the
Such a
eyes.
liquid
may
be prepared by
adding ammonia to an aqueous solution of copper sulphate
The
contained in a glass globe.
best illumination
is
then
obtained by a suitable adjustment of mirror, globe and lamp.
The positions of the microscope, globe and lamp can then be

marked on the table so that these can easily be put into
place at any time.
In addition
as well to fix a screen between the
it is
microscope and the lamp, partly to protect the eyes from the
direct light of the lamp,
If a gas
lamp
is
and partly on account of the
heat.
damp
cloth
used as the source of
light, a
should be hung on the screen to keep the air moist.
Small Auxiliary Apparatus,

been described, various small
In
addition to
articles
are
what has
required,
e.g.,
glass spatula, platinum wire, forceps, preparation needles,
Two
etc.
glasses
may also
dilute sulphuric acid
in one, soiled glass slips are put, in
the other, cover glasses
on the
be kept on the table for holding
in this
way
the micro-organisms
and thus prevented from passing

after the glass has dried and before it is cleaned
glass are killed
into the air
again.
Bottles for Reagents and Immersion

scopical
work
Oil.
For micro-
certain chemical reagents are indispensable.
These are preferably kept in bottles provided with glass

stoppers which are drawn out into the form of a rod the
;
latter reaches almost to the
some
bottom of the bottle and in
cases has a thickened end.
Figs. 11
sent two of the commonest forms of these

Fig.
13 represents a bottle which
successors
is
and 12 reprebottles.
made by Ley hold's
(Cologne) according to the design
3*
of
Arthur
mill
M(!y((r,
wliicli
iliiH iidvatil.airc,
(tuniiol,
lii^
riinily
Uwil/
Im.s
of Uir niidilh^
HupdriliioiiH
riiMiii'.l
ri'iiiii
\)nck
TIki
iiîiiii
I'lill,
iiH,
ruiiiiiii^r iiil/O Uii'.
'jH
'"'ft
'
'
iicci'|iI,iimc,i<,
Ih
i'iicIohoiI
Ih Imiil, iiiwiir'dH
II,
in
iiil.o
ii
n,
IJii'
I'iiii
li(|iiid
iiii'd
Uiii
ir iipMi'l.,
i'ii'|i.
iMd,()rnal/i('(i,lly
wliicJi
Iml.l.ht;
l/lii!
I'iiii
oiii.
rcinoviii^
rniiiiivi^d
ihi;
Iirh
<'iir|j,
riiiiiicl
(i|Minil,(ir In dr'iiw Uiii În.HH rixl
(if iJii^ liiiMJi',
i|iiiirl:itr
^'(iikh'iiI
wliicli
I'iin,
Hoihid hIiicc UiIh
li<|iiid.
niiiH
kopt
loiiiiil
Uir
Hhii|)(> wlii(:li i'nni|)(ilH l.lid
III',
OUOANISMS
h'lOIMVIMN'I'ATION
Mi
liiU.cr
jii'i'vi'iiLh l.lir
ihi
Uir.
hIkiuM
lii|uid
THERMOSTATS
and on the
sides.
It is divided into
above, the smaller below
Fig.
37
two
each section
parts,
is
tlie
larger
provided with
14. Kohrl>eck's Thermostat
only used
when moist
air is required in the thermostat, a saucer of
water being
double doors.
The lower chamber
is
38
made
Tlie doors are
placed inside.
being covered with a piece of
The space between

water
distilled
thermostat
of glass, the outer one
which can be removed.
felt
the walls of the box
is filled
with
The
water being used to prevent furring.
provided at one side with a water gauge
is
carrying a two-way cock, which allows the water either to

enter the gauge or to be
drawn
in connection with the space
the box
opening
this
is
In the top
off.
is
an opening
between the double walls of
used for
the space with
filling
water, and, after this has been done, for holding a ther-
mometer which passes through a cork and has

dipping into the water.
in the sketch) there
is
On
bulb
its
the opposite side (to the right
a similar opening to
There
which a thermo-
regulator
is
which
connected with the inside of the thermostat; a
is
thermometer
fitted
is
(see p. 46).
placed in this
is
by which
the chamber containing the cultures
a third opening,
the temperature of
may
There
be read.
two other openings also leading to this chamber they

act as valves and are provided with caps which can be
are
closed
Lastly there
either completely or partially.
opening to which a hygrometer
may
be
fitted.
is
an
In the
thermostat there are several movable shelves, which are

perforated to allow of free circulation of
Thermometers
air.
are required here also.
The heating
is
done by means of gas flames which are
placed under the apparatus, as
The water
is
may
be seen from the figure.
thus warmed, but in order to keep the tem-
perature constant the therm oregulator
is
Quite a large quantity of water
indispensable.
is
used in such a
thermostat, about 43 litres being required for a thermostat
40 cm. high, 50 cm. broad, and 25 cm.
Two
gas
deep.
flames are used so as to reach the desired
temperature quickly, but this process

shorter
if
the thermostat
is
filled
may
be made
still
with water previously
THERMOSTATS
The regulator
heated.
39
adjusted by being
is
water bath of the desired temperature before
warmed
in a
to
it is fitted
the thermostat.
Besides the
Muencke
above form there are numerous others.
(Berlin)
and Altmann (Berlin)
also
make good
thermostats.
Panum's Thermostat,
In
many
investigations
is
it
important to have at the same time a large number of
chambers at different temperatures.

(see Fig. 15),
which has a
Panum's thermostat
series of constant temperatures,
realises the required condition.
We
will describe in
what
follows its construction along with the improvements which

this apparatus has
undergone in the Carlsberg laboratory.
This thermostat consists of three cupboards soldered together which are designated in the accompanying Fig. 15
with the letters A, B-C, and D.
The reason that it is not made
of one cupboard divided into three parts
is
that
A and D
re-
more repairing than the middle one B-C. The former

have therefore to be made separate from the latter. The
inner dimensions of each of the compartments A, B-C, and
D is about 63 cm. in length, breadth and depth. The first
compartment, A, is double-walled, and is made of tinned
copper the jacket, c, is filled with distilled water, which is
quire
kept at the required temperature by a gas lamp, a

flame
is
by means of a
controlled
regulator,
into the water through a tubular opening.

is
placed under a projecting wing,
case
this
wing
is also
d',
b,
the gas
which dips
This gas lamp
of the external copper
tinned and in addition covered with
asbestos paper, excepting the part immediate!}' above the
lamp which
plate.
is
As
is
thickened by the addition of another copper
the wing
is
burnt through in course of time
it
not put into permanent connection with the outer copper
case but joined to
packing so that
it
by screws and flanges with rubber

can be replaced when necessary.
it
40
There
is
also
another tubular opening (not shown in the
figure) at the top of the
compartment A, and
in front of
that holding the regulator, and through this a thermometer

dips into the water to register
its
temperature.
Water
is
THERMOSTATS
run
41
through one of the above openings, and can be run

off at the stopcock, d", situated on the wing.
in
The space in the compartment, A, is divided into two

and 2, by a partition ledges, on which loose shelves
parts, 1
can be
There
are fixed
laid,
the walls at various heights.
to
an opening in the roof for a thermometer,
is
e,
to
give the temperature of the air in the space A.
The other two compartments, B-C and D, are made of

D and C8 being care-
tinned sheet iron, the compartments
damp due
to the
divided into two compartments
B and
fully painted all over with red lead to resist
B-C
cooling in D.
is
each of these being again divided into three spaces
and
6,
7,
8),
up
ledges for fitting
tinned sheet iron.
metal
These are
several stages.
all
In compartment C8 there
strip, /, soldered
next to
(3, 4, 5,
the walls of which are also provided with
made
is
of
a bent
along the bottom against the wall
so that the condensation water
and run
this wall can pass over the strip
which forms on
into a long
narrow
box placed below.
The
compartment, D,
last
is
an
ice
box consisting of an
outer and inner receptacle, the latter of these being cooled by
water trickling
down over it from
a mass of ice resting on a
strong grating, g. To distribute the water the inner receptacle

has
its
run
roof sloping to the sides and to the back.
off
through the opening, h
through which there
water
is
caught in a
is
a tube,
i,
The water is
in this is a cork passing
forming a water trap
vessel, k, placed beneath.
the
The cork must
be taken out every day in order to remove dirt coming from

the
in
ice.
There
is
a movable trap,
which condensation water
The whole apparatus

layer of
box,
felt,
is
I,
collects
fitted to the top of
and
is
D9,
removed.
completely surrounded by a
m, 8 cm. thick, and enclosed in a tight wooden
o.
The
ice
holder
is
closed
by an
iron
lid,
above which
is
42
a
wooden
lid
lid, p,
provided with an 8 cm. layer of
felt.
The
can be opened and shut easily by means of a counter-
poise weight
hung over a pulley fastened
to the wall of the
room behind the thermostat.

Each of the spaces 1 to 8 is provided with
glass door,
and doors
a tightly fitting
of sheet iron are fitted
four large compartments
on each of the
and D, which are closed
A, B, C,
by pressing against rubber strips fitted on to the

Four corresponding doors, also shutting tightly,
are attached to the wooden case, their inner sides being
tightly
partitions.
with woollen pads.
coated
below, and,
All
when opened and
tion on adjustable brackets,
these
doors are hinged
resting in a horizontal posi-
may
be used as tables.
The space under the thermostat is used as a cupboard.

working and the regulator set, for
example, at 40 C, compartment 1 will be at this temperature
If the apparatus is
while the temperature in 9 will be only a few degrees
above
In the intervening compartments the tempera-
0.
ture varies between these
two extremes.
In individual
compartments the temperature varies from wall to wall and

also
from top
If the
is
to bottom.
temperature of the room containing the thermostat
somewhat high,
sometimes
as
diflBcult to
may happen
ice
summer,
it
will be
reach a low enough temperature in
the cold part of the thermostat.

15, 10) with an
in
holder
is
small iron box (Fig.
then placed in
the tempera-
ture of the whole thermostat
is thus lowered and at the

same time a specially low temperature compartment is
obtained.
Schrlbaux's
used
is
Thermostat.
that of Schribaux which
consists of a
thermostat
is
frequently
seen in Fig. 16.
wooden cupboard with a copper
floor;
It
the
heated gases of the burner pass through brass tubes fixed

to the walls.
special regulator,
mentioned on page 49,
THERMOSTATS
ia
usually employed along with
tions
are greater
in
it.
43
The temperature
thermostat than
this
in
varia-
those of
Rohrbeck and Panum, as the large doors when opened

allow a considerable cooling to take place.
Large
Warm
ments, in which
In
Chamber,
many
more extensive
experi-
cultures are dealt with simultaneously
Fig. 16.
Schribaux's Thermostat.
same temperature, it would, of course, be difficult to

room for them in an ordinary thermostat.
circumstances allow, a small room can sometimes be
at the
find sufficient
If
made
into a thermostat.
same
principles as an ordinary thermostat,
It
is
the same on a larger scale.
fitted
up according
and
is,
to the
in fact,
The Carlsberg laboratory
44
"warm chamber"
contains a
which
of this kind,
kept at 25 C, and which has proved eminentlj^
The room
250 cm. high, 160 cm. long, and 160 cm. broad.
is
Movable shelves are

the
roof
usually
is
satisfactory'.
fitted
composed
are
The walls and
along the walls.
two layers
of
between which animal charcoal
placed
is
hollow stones
of
:
the door
also
is
filled with kieselguhr.

The heating is effected
by means of warm water wdiich passes from a vessel
outside which is heated by a gas flame, circulates througfi
copper pipings along the walls and so returns to the vessel.
The piping is about 960 cm. long, 4 cm. in diameter, and is
double and
fixed about 55 cm. above the floor.

is
the wall and
fixed in
room;
at another
outside
into
may
latter
the
Reichert regulator
in communication with the
is
place a thermometer passes
room
from the
that the temperature
so
be observed from without.
the
of
There are also two
openings in the wall closed with cotton wool which act as

ventilators.
passed
Through
of yeast.
If a
15 C.
Panum
thermostat for 15 C.
is
often necessary in the analysis
thermostat
must be
is
not available, a special
many
In
fitted up.
cases a
might be used instead of the above, although
cellar
be
in.
A temperature of
temperature
A.
The
may
these, such things as tubing
is
its
of course not particularljr constant.
Petersen's
Thermostat
for
Low Temperatures.
principle of a thermostat for low temperatures differs
from that of the thermostats described where the temperature
above that of
is
process
is
reversed.
its
surroundings
in this case the
Anton Peterson has constructed such
a thermostat for use in the laboratory of the old Carlsberg
brewery
in
Copenfiagen.
The thermostat
consists
of a
cylindrical double-walled copper box with a loose lid also
double walled.
.space
The outside
between the walls
covered with
is
filled
with water.
felt,
and the
Above on one
THERMOSTATS
Hide there
an
is
inlet for the water,
45
and on the opposite side
Near the inlet there are also perforations for

regulator and thermometer.
In the lid there are, in
addition, two openings, one for a thermometer which proan outlet.
which the cultures are
jects into the interior of the space in
Tap water
placed, the other acting as ventilator.
is
used
for obtaining the desired temperature, its temperature being
generally lower than 15" C.
The water passes from the tap
through a tube to a small con.stant-level
cistern,
and from
there through another tube to the regulator (see page 49).

Pfeiffer's
Microscope Heating Apparatus.
When
it is
desired to folloM' out the development of a micro-organism

at a certain constant temperature
under the microscope, this
can be done by using the small thermostat designed by L.

Pfeift'er,
which
is
shown
The arrangement
in Fig. 17.
consists of a
mahogany
completely surrounds the stand, and which

tight
when
Its front wall
closed.
flap
the observer) have
door so that the preparation
In order to
make
box, whicli
almost
air-
has a glass window to
admit sufEcient light for observation

walls (seen from
is
the left and right
each a well-fitting
may
be manipulated.
the microscope freely accessible, the
side walls are capable of being completely
removed along
is divided down
The whole stands on a thick metal plate with
The heating is effected by warming the
three metal feet.
plate from below by means of a micro-burner, the gas
supply of which is controlled by a regulator. Experiments
made in the Carlsberg laboratory have shown that the
with the halves of the back wall, which
the middle.
Keichert regulator described below

apparatus.
With the
side
flaps
is
very suitable for
this
opened or closed the
greatest variation of temperature in the apparatus at 25 C.

or 32 C.
was only
1.
If the
apparatus
is
then the flaps suddenly opened, the temperature
closed
falls
and
about
46
2
It is therefore desirable to
and then keeps constant.
when working with temperatures which
leave the flaps open
allow of
this.
With high temperatures
this is impossible.
","'"-'m^
Fig. 17.
Reichert's
L.
Pfeiffer's
Regulator.
thermoregulators which
Microscope-heating Apparatus.
There
may
are
numerous forms of
be used in combination with
the thermostats for higher temperatures described above.
In the course of long use in the Carlsberg laboratory
THERMO-REGULATORS
and other
being due in no small degree to
The
construction.
by heat and
closing
natural size
18
Fig.
in
c is
simple
at
the bulb
about
filled
the
inlet
The apparatus
diminishing the gas supply.
represented
its
principle of this regulator consists in
the mercury expanding
is
proved extremely
places, Reichert's regulator has
efficient, this
tube, thus
47
one-fourth
of
its
with mercury, the ther-
mometer tube widening out above into a cylindrical space

which communicates with the inflow tube. A, by a ground
air-tight joint
thermometer begins, and has a

ing at
down
the tube, A, reaches
where the widening of the
to the point
The gas passes
open-
fine
to
the
burner through the side tube, B.
In
a.
off
order to adjust for various temperatures,

there
is
another side tube
thermometer stem and

cury,
fitted to
filled
with mer-
end being closed by an
its
adjustable iron screw, S.
The
ing takes place in the following
the
easily
regulat-
way The
:
tube, A, is turned until the opening,

is
opposite the tube,
set so that the
mercury just begins to
the wide space
when
ture
is
reached
forced upwards
flame begins to
a,
the screw, S,
is
fill
the proper tempera-
the mercury
is
then
of the closing of the tube. A, the burner
the opening,
a,
Fig. 18. -^Reichert's
by the screw until the

As a result
get smaller.
is
Thermoregulator.
fed only through
the size of which can, in some forms of the
regulator, be varied
by turning the
tube, A.
After being in use for some time, a black powdery

ssubstance
is
deposited on the surface of the mercury in
consequence of impurities in the coal gas, the sensitiveness

of the regulator being diminished.
To remove
this
it
is
48
sufficient to
take out the inflow tube for a
moment and
remove the impurity from the mercury with a brush.

Reichert regulator allows the controlling of
from
to
The
temperatures
all
above the surrounding temperature almost to the
boiling point of mercury.

Fig. 19 represents the
improved form of the Reichert
regulator, the bulb being omitted.
Fig. 19.
Eeichert's
When
the temperature
Improved
Thermoregulator.
Fig. 20.
Muencke's Thermoregulator (after Lothar Meyer).
becomes too high, the mercury
and thus cuts
off part of the
form there is a by-pass at

Muencke's Form of
raises the floating valve, a,
gas supply.
As
c.
Lothar
Meyer's
Regulator.
Regulators are often employed in which there
is,
This principle was
first
above the
mercury, a liquid which boils at a low temperature,

alcohol or ether.
b,
in the simpler
e.g.,
proposed by
THERMO-REGULATORS
49
Lothar Meyer, and has been applied in several ways.
regulator of this kind (by Muencke, Berlin)
in
is
shown
the illustration (Fig. 20).
The inflow gas tube
is
a metal one with a steel end
it
passes through an air-tight stuffing box, and can be fixed
by means
of a screw.
provided with a millimetre
It is also
scale so as to control its position.
This regulator
is
used in
many
laboratories,
and
is
But
haps, under certain circumstances, to be preferred.

for
most cases the Reichert regulator

Roux's Regulator.
quite sufficient.
is
Roux's regulator
per-
is
the one usually
employed with the Schribaux thermostat mentioned on

page
This regulator consists of a steel and a zinc
42.
plate soldered together

is
fixed
by
and bent
in a
shape.
a screw, the other remains free.
One limb
When
the
temperature rises the two liinbs separate, the free limb thus
displacing a cone ventilator fitted into a metal box, so that
If the tempera-
the gas can only enter through a by-pass.
moves back and opens the
ture sinks, the free limb

so that the gas passage
becomes
Soxhiet's Regulator.
free.
different regulator
above must be used for low temperatures.

of Soxhlet,
which
is
shown
valve,
in Fig. 21,
is
from the
That
suitable
for use with the Petersen thermostat at 15 C.
Cold water flows down from a constant level

cistern
through the upper tube on the
right.
If
the temperature of the water in the thermostat

is
normal
all
the cold water runs
overflow vessel on the
left,
off'
which
is
through the
always
full
but, should the temperature of the thermostat Soxhiet'sTher-
water
rise
01 above the normal, the ascending
mercury column
and the
closes the
opening of the syphon,
""forîow"
t^^^P^ratures.
cold water flows through the horizontal tube
on
the right into the water of the thermostat, until the normal
50
temperature
sunk
again reached and the mercury column has
is
opening of the syphon free
so far as to leave the
The bulb on the
few drops of ether

some other liquid of low boiling point.
Koch's Lamp, For heating the above described thermostats, gas lamps are sometimes used which are provided
again.
right contains a
or
with an automatically acting apparatus that cuts off the

gas supply as soon as the flame
lamp, belonging to
this
type,
is
manner a metal tongue consisting

and bent downwards projects into
:
end
is
following
of iron and brass sheet
the flame.
The lower
bent round and acts as fulcrum for a lever loaded
with a weight.
If the flame
is
extinguished by any cause,
the metal tongue cools and approaches the burner
motion the lever

position,
and by
loses its
It
is
about 10 em. long with a
rests in a
this
off"
the gas.
convenient to use thermometers
maximum
reading of 25 C. for
They can be
fixed
which has small grooves down the
sides
measuring temperatures in thermostats.

in a bored cork
by
support and takes up a vertical
so doing turns
Thermometers.
and
Koch's
extinguished.
works in the
wide-mouthed
The
glass vessel.
air in this
communicates with that in the thermostat through the

grooves on the cork.
such a
glass, it
without fear of
If the
thermometer
is fitted
up
in
can be taken out and the temperature read

its
changing, as would be the case
if
the
thermometer alone were removed from the warm thermostat.
It
is
not out of place to remember that ordinary
thermometers cannot always be depended on, but must be

also necessary to
do
from time to time, as the thermometers change
in
checked before they are used.

this
course of time.
thermometer
It
is
is
checked by comparison
with a standard thermometer, the zero point of which has

been exactly determined previously by immersing
in finely
pounded
ice.
possible error
is
its
bulb
thus removed.
STERILISING APPARATUS
51
Sterilising Apparatus.
5.
With Dry Heat. Glass and metal apparatus are sterilised by means of dry heat.
This is done by using an oven
made of iron, coated with lead and of the shape shown
in Fig. 22.
It consists of a double-walled chamber of
fomi
cylindrical
on the
inside
the outer wall
and
is
is
coated with asbestos
conical at its lower end;
a large
gas burner projects into the conical
com-
part, so that the products of
bustion rise between the walls and

leave the apparatus
by the chimney
on the top. The door is also double

walled.
The burner can be raised
or lowered,
its
and the
position
proper size of flame being deter-
mined by experiment.
There
is
quite a large
number
of diflferent designs of these hot

air
is
The
chambers.
principal point
that they must give a tempera-
ture of 150 C, and

able.
regulator
must be dur-
may
along with them, but
be used
it
is
not
necessary in most cases since

is
it
immaterial whether the glass
articles are heated to a little
more
than 150 C.
On
or a
little less
the other hand,

the
gypsum
if
fig! 22. -Apparatus for'steriiis

ation with Dry Heat,
the hot air chamber
is
used for
blocks to be referred to later,
it
sterilising
is
of great
importance that these should not be submitted to a higher

temperature than 120 C, because they lose water of crystallisation
The
and afterwards crumble on being placed in wate*is to use an iron box, the walls
simplest way, however,
52
of
which are coated with asbestos paper and which
An
vided with a regulator.
pro-
is
ordinary Bunsen burner
is
temperatures not exceeding 120 C.
sufficient to obtain
Culture media are sterilised by boiling on a sand bath
or by means of steam.
sand bath
shallow rectangular iron box on four
is
made out
best
of a
Below the box
feet.
are placed two horizontal and parallel iron tubes closed at
one end, connected with the gas supply and pierced on the
At the end
top with holes which serve as burners.
of the
tube where the gas enters there are holes like those on
The
a Bunsen burner.
best dimensions for such a sand
On
bath are, length about 42 cm, breadth about 22 cm.
room for 10 to 12 i-litre Pasteur flasks.

simpler however to sterilise flasks containing
the above there

It
is
is
an autoclave to
media in
culture
by steam
Sterilisation
be
described
often necessary and
is
is
later.
the most
frequent means employed.
Steam
Sterilisation.
indispensable
An
autoclave or digester
purpose,
for this
and with
it
can be effected with or without pressure
steamer cannot be used with pressure.
an ordinary
The accompanying
sketch (Fig. 23) shows a Chamberland autoclave
Wisnegg
of Paris.
escape
if
is
a tap,
c.
by means
b,
no pressure
manometer,
of
required, a safety valve, a,
is
Below the
is
clave, the height of which
is
60- cm.,
is
and a
surrounded by a
In an auto-
the inner space used
for containing the objects to be sterilised
When
the
may
two concentric
vessel there are
jacket of sheet iron provided with a door.
is
On
which the steam
rings of gas burners, and the whole
deep, and
made by
It consists of a steel vessel with a cover
which can be closed tightly and screwed down.

cover there
quite
is
sterilisation
is
about 32 cm.
divided up by several horizontal partitions.
the autoclave
is
about to be used, distilled water
put into the vessel, the amount depending on the size of
STERILISING APPARATUS
the latter.
The
objects to be sterilised are thereupon placed
on the shelves and the cover screwed down.

flames are then lit, and after the water in the
Fir,. 23.
is
is
is
extinguished.
If
no
required, the tap on the cover remains open
during the boiling, otherwise

sure
Ail the gas

vessel begins
Cliamberland's Autoclave.
to boil, the outer ring of flames
pressure
53
indicated
it
remains
by the manometer and
The presregulated by the
closed.
is
54
source of heat.
If the liquids to be sterilised froth strongly
during the boiling,
one ring of burners
is
heating then proceeds more slowlj^ and frothing
is
As mentioned
onlj'
lit
the
avoided.
above, an ordinary steamer can bo used
for sterilisation without pressure, tliat
for simply heating
is,
by means of steam.
It is fitted up in practically the
same manner as an autoclave, only it need not be so strong,
and the valve and manometer are omitted.
A
is
steamer of this kind used in the Carlsberg laboratory
made
is 1 metre high and has an inner

The inner space for holding the
is 65 cm. deep and is divided into
of tinned copper,
diameter of 42 cm.
objects to be sterilised
three stages by perforated platforms.
It is
charged with
5 litres of distilled water.
6.
We
After
shall
all
now
Culture Vessels.
describe various flasks
and culture
vessels.
the openings have been closed with cotton wool
such vessels are sterilised in a dry heat for two hours at

150'
of treatment of glass ware.
mode
They
must be tolerably dry before
being-
C,
this being the usual
put into the
steriliser.
The Pasteur Flask.
We
are
indebted to Hansen for the im-
proved modification of this flask
now
in
use (see
Fig.
flask consists of a glass
The
bulb drawn
24).
out into a l^ng tube wide at the

beginning, bent twice and with a
The Hansen ModifiFig. 24.

cation of the Pasteur Flaslc.
bulb between the two bends.
An
inoculation tube, a short straight

side
tube, proceeds
from the bulb, and
rubber tube and glass rod.
The Pasteur
is
closed with a
flask is
commonly
CULTURE VESSELS
55
used in three
sizes,
respective! J^
As
rule, a ring of
cork or pasteboard (see Fig. 24)
having capacities of
J-,
the bottom of the flask
and
is
not
litre
flat as
is
used as
a support.
It is
specially important that all the Pasteur
flasks,
small and large, in the laboratory should have side tubes

of the
same diameter as those on other
to later;
vessels to be referred
otherwise the tubes do not
fit
into the
same
rubber connections when the flasks have to be joined with
Fig.
one another.
25. The Pasteur
Vessel.
Standard flasks ought therefore to be kept
as patterns for
new
stock,
and in giving an
order, the
external diameter of the side tube should be given exactly

6
mm. is a suitable diameter.

When Pasteur flasks containing
culture solution either
alone or with cultures are put aside, the bent tube should
be closed with a small asbestos plug which

air
that
may
penetrate into the flask
temperature changes.
cold situation,
When
filters
any
as the result of
flasks are placed in a
very
and therefore usually a very damp one.
56
wool should be used instead of asbestos, as
salicylic cotton
moulds cannot grow through the former.
These flasks
have the great advantage of only allowing a very slight

evaporation of the liquid;
they
may remain
for
many
Thus
years without any notable evaporation occurring.
Pasteur flasks with cultures have been standing in the

Carlsberg laboratory for twenty years during which no
appreciable diminution of their contents has taken place.
Hansen introduced the above-mentioned expansion

the bent tube, in order to avoid infecting the culture
by germ-laden
air
bubbles being carried into the flask
itself
which takes place
after
as a consequence of the sucking back

boiling.
These bubbles are caught in the
bulb,
and
should,
always heated to redness before beginning to
therefore, be
the flask, in order to kill the germs which
have settled
may
there.
The Carlsberg
of metal,
little
The tube and bulb
deposit their germs on the glass.
work with
of
medium
e.g.,
Vessel.
Large culture vessels are made
tinned copper, as
it is
tical grounds, to use glass flasks of
and because,
not convenient, on prac-
more than
1 litre capacity,
in general, vessels containing 7 to 8 litres of
nutrient solution are used in preparing pure yeast for brew-
ing purposes.
Pasteur was the
shown
first to
use a vessel of this
on fermentation
by a two-holed rubber bung
fitted with a short straight tube closed by a rubber tube
with glass stopper for introducing the yeast, and a long
kind, as
experiments.
It
in Fig. 25, for carrying
was
closed
bent tube for allowing the carbonic acid generated during

fermentation to escape.
On the top there were two windows,
and near the bottom an ordinary metal stopcock for drawing

ofi" the liquid and yeast.
But this form had somewhat great disadvantages, as it
was found that the contents
of the vessel were often infected
through th^ windows and stopcock,
it
being very
diflBcult
CULTURE VESSELS
to keep the latter
On
sterile.
this account
57
Hansen and
his
assistants gradually evolved the Carlsberg vessel described
below.
for its
There are
indicate
some who prefer the Pasteur
still
window and tap
it is,
vessel
therefore, not out of place to
its failings.
There are two modifications of the Carlsberg

older, cheaper form, and a
vessel,
an
newer.more expensive one they are

Figs. 26 and 27. There are two straight side tubes
;
shown in
on this vessel, one, a, on the
Fig.
26. The Carlsberg
the bottom
Figs. 27
the bent tube
Fig.
is fitted
other,
a little above
b,
27. Tlie Carlsberg
Vessel,
new modoL
either to screw off (see
and 28), so that both vessel and tube can be easily
part connected
fixed to the vessel
it is
by means
in the middle,
filled
e.
is
the
more expensive, as
The bent
to be preferred.
it is
tube, at the end of which a
a glass tube
and the other
of a piece of rubber tubing, c (see
The first-named form
has been remarked, but
panded
and the
Vessel, old model.
cleaned, or a part of
Fig. 26).
top,
filter,
This
d,
filter
with cotton wool
can be
was
;
fitted, is
in the older
in the
ex-
model
new model
it
58
consists of a metal cylinder filled
with asbestos, provided
with a loose-fitting top, and screwed on to the tube. Rubber

tubes are fitted to both of the side tubes, and are closed
glass stoppers
the rubber on the lower side tube
The yeast is introduced
provided with a pinchcock.
is
by
also
into the
liquid in the vessel through the upper side tube, the liquid
and yeast being removed through the lower

purpose of this vessel
is
one.
The
chief
the growing of yeast in large
quantities, as, for example,
when such
is
required for the
CriD
>k
\J
^v\A
Fig. 28.
Mode of connecting the
Fio.
29. Prior's
Vessel.
Bent Tube with the Carlsberg Vessel.
pure culture apparatus mentioned later

for
it
is
also used
fermentation experiments with larger quantities of
liquid.
The manner
in the
fits
new model may be
into
the
connection
joint
of connecting the bent tube with the vessel
is
vessel
effected
seen from Fig. 28.
by means
by means
of
The tube
conical joint
of a female screw.
must be made very accurately, otherwise the
useless.
the
This
flask is
CULTURE VESSELS
59
The most convenient capacity for this vessel is about 10

litres.
The largest amount of wort with which such a vessel
can be charged
from
is
7 to 8 litres if it is to
fermentation experiments, for
it
be used for
must not be completely
In general 125 to 150 grams of thick yeasty sediment
filled.
can be grown in the above quantity of wort.
Sometimes
happens that the bent tube becomes
it
blocked during the sterilisation of the wort, so that
draw
impossible to
diiEculty
surmounted by passing
easily
is
As a
a thin, doubled copper wire.
heating the tube, since
tube
is
stopped.
it
It
is
into the tube
rule little
is
gained by
known at what point

advisable, now and then, to boil
it is
is
This
off the contents of the vessel.
not
the
out
the Carlsberg vessels with solution of soda, to remove the
hop
resin, etc., clinging to the sides.
Prior's Vessel.
E. Prior has constructed a modification
of the Carlsberg vessel,

side tube,
e,
is
which
is
and glass stopper; the
by means of
tube carrying a pinchcock, and the tube,
two
tubes,
r,
are connected
a piece of rubber
a, is
with a short rubber tube and pinchcock.

fitted
on at /.
also provided
The
filter
further information see page 77.
The Chamberland Flask and

to be seen
is
This modification of the original model has
For
been designed chiefly to allow aeration of the wort.
is
represented in Fig. 29.
closed with rubber
from
Fig.
its
iWodifications.
As
30, this flask consists of a flat-
bottomed bulb with a short neck provided with a ground

cap which
is
drawn out
into a
somewhat long tube
filled
with cotton wool.^
A more
known
1
frequently used modification of this flask
as the Freudenreich flask.
is
that
It (see Fig. 31) differs
Ordinary cotton wool (not fat free) should always be used for plugging
Fat-free cotton wool attracts moisture and can thus set up in-
flasks.
fection.
60
from the Chamberland
jrards the size of the flask a total height of 10 cm.
may
of tube 2 cm.
of the base
is
neck about
As
flask in being cylindrical.
re-
and a length
The external diameter
be recommended.
about 2 5 cm., the internal diameter of the
The inner diameter of the tube ought

02 cm., and the tube itself should not
cm.
not to be more than
be shorter than the length mentioned above, otherwise too
medium
great an evaporation of the nutrient
flask like this holds
about 20
c.c.
not contain more than 10 to 15
Fig.
30. The
This flask
Cliamberlaiid Fla,sk.
is
c.c.
takes place.
but, as a rule,
it
should
of liquid.
Fk;. .31. The Freuileiireich Flask.
very frequently used, as
it
does not take
up much room, and on account of its small size also, workBut the flask has this failing
ing with it is not expen.sive.
It is therefore advisable
that it is somewhat easily upset.
to place the.se flasks in small tin boxes made of various
The height
sizes, e.g., for 6, 10, 15, 25, 50 and 100 flasks.
of the box
may
be
made
3 5 cm.
the other dimensions are,
coiTCsponding to the above numbers

cm., 9
X 14
When
cm., 14
X 14
cm.,
9 cm., 6
X 14
14 X 28 cm. and 19 X 38
using the Freudenreich
flask, care
must be taken
CULTURE VESSELS
to
make
the cap fast
the cap, for
may
it
61
the flask ought never to be lifted by
easily
happen that the cap comes
the hand, and the growth in the flask
off"
in
is thus infected.
The Hansen flask forms another modification of the

Chamberland model (see Fig. 32). It is distinguished by
having a
.side
tube, the lower part of the flask being either
The
globular or cylindrical.
it
flask has this advantage, that
can be connected with the side tube of the Pasteur
flask.
li
Fig.
32. Tlie Cylindrical

Hansen Flask.
FreudenFig. 3-3.
reich Flask with Rubber
'I'ulje
Fig.
.34.
TheJorgenseu
Fla.sk.
and an S
Tube.
The
side tube
is
either closed with
an asbestos plug and
sealing wax, or with a rubber tube and glass stopper.

first
method
is
The
used specially when cultures are to be kept
a long time in such flasks.
Freudenreich and Hansen flasks are used not only for

cultures in or
upon culture
nutrient gelatine, which
is
liquids,
order to obtain a larger surface.

is
but also for cultures on
allowed to solidify obliquely in
Although the evaporation
but small in Freudenreich and Hansen flasks,
it is
advis-
62
able,
on
when
the latter have to remain a long time with cultures
gelatine, or in or
upon
glass tube to the cap
During
33).
an
removed from the
by means of a short rubber tube
may
(Fig.
and rubber are
experiment the tube
If the flasks only contain nutrient
flask.
mouth
of the cap
wax
to prevent
fitted a
small hook-
gelatine or liquid without cultures, the
tube
S-shaped
liquids, to attach a small
be closed with a
sealing
little
drying up.
For
this purpose Alfr.
Jorgensen has
shaped tube to the cap of the Hansen flask
The Freudenreich and Hansen
(see Fig. 34).
flasks are well
adapted
for sending yeast specimens, the latter especially, as the
culture liquid can be directly transferred to

flask provided with a side tube.
on the bottom
flask
with yeast
c,
is
of
placed,
which a layer of
and the
has also a cotton-wool plug,

in the ordinary
way with
b,
from another
fat-free cotton woo]
side tube of
with asbestos and wax, d and
it
Fig. 32 represents such a
which
The neck
c.
is
closed
of the flask
the tube of the cap being
cotton wool,
filled
a.
In order to prevent interchanging of caps in cleaning,
both flask and cap should be etched with the same number.
Pipettes
in
must be used
in
working with culture liquids
Chamberland and Freudenreich

Flasks.
meyer
For
many
flasks.
experiments, the ordinary Erlen-
flasks (with a capacitj^ of
200 to 250
c.c.)
are very
suitable because they can be used for solid as well as liquid
nutrient media.
The layer
of
medium has
a large surface,
Fig. 35 re-
so that air has free access to the cultures.
presents one of these flasks which
ably wide neck which
down with
'
When
is
closed
is
seen to have a toler-
a cotton-wool plug tied
a double layer of sterile filter paper.
flasks covered in this
way
are to be put
away
in a
damp
place,
paper and the string which ties it

This can be avoided by using a filter paper impregnated with a
mould growth soon forms on the
down.
by
filter
CULTURE VESSELS
63
The cotton-wool plug must always be passed through a

kill any germs on it.
As soon as the plug is quite dry after sterilisation, but
flame before and after inoculation to
not before,
filter
it
is
advisable to replace the double layer of
paper by a tightly
fitting
rubber cap after the upper
portion of the cotton -wool plug has been sterilised in the

flame, in order that the culture
dry up when
left for a
medium may
not completely
long time.
Globular flasks, not having such wide necks, are better
adapted than the Erlenmeyer flasks for preserving small

quantities of culture gelatine which are to be used for plate
cultures.
Fig.
The
is
35. The Erlenmeyer
Flask.
size in general use
charged with about 15
Fig.
36. Globular
Flask.
has a capacity of 80 to 90
c.c.
of gelatine.
c.c.
and
It is not advis-
able to use smaller flasks than these, as the melted gelatine
cannot be shaken up vigorously enough.
The form
of the globular flask
may
It is closed with a cotton- wool plug

filter
paper.
rubber cap
If the gelatine
may
is
be seen in Fig. 36.
and a double layer of
to stand for a long time, a
then be used instead of the
filter
paper, the
same precautions being observed as were described above.

Of the flasks already described the Pasteur flask is the
10 per cent, alcoholic solution
can be similarly treated.
of salicylic acid
and then
dried.
The
string
64
most
reliable
one to work with.
and takes up much room
This flask
expensive
is
though not standing so well,
the small cylindrical flasks, being cheaper, are coming into

use wherever suitable.
laboratories
many
Test tubes are also used in
but they are
less
adapted for cultures in nutrient
reliable
and are not well
liquids.
In wine-manufacturing establishments large glass
flasks,
with double-bored stoppers are used instead of the Carlsberg

flask.
Two
tubes are fitted to the stopper as in the Pasteur
flask.
Dishes.
Petri
Petri dishes consist of

A
glass dishes (see Fig. 37).
lower dish
is
two
a set of
suitable diameter for
flat
the
about 9 cm. and for the upper 10 cm., the
Fig. 37.
height being about
1 '5
cultures.
Each
paper and
sterilised.
set is
Petri
Dishes.
These dishes are used for plate
cm.
wrapped
in a double layer of filter
Apparatus for Spore Cultivation.
7.
of
Gypsum Blocks and their Containing Vessels. Blocks

gypsum are generally used for the cultivation of the
Fig. 38 represents a block of
spores of saccharomycetes.
gypsum
placed in a covered glass dish with sterile water.
The block
is in
of the vessel
Carlsberg
the form of a truncated cone, and the cover

quite loosely.
fits
laboratory
(Vogelnapfe).
measurements,
A
is
The dishes used
the so-called
are
"
in the
"
bird troughs
suitable size for these, taking outside
as follows
of the bottom, about 7 cm.
Height, 4-5 to 5 cm.
The gypsum block
diameter
is
3 cm.
SPORE CULTIVATION
high
the diameter of the lower surface
The manner
the upper surface 3 8 cm.

is
made on
When
the
" bird
gypsum
troughs
"
is
in
65
5 '8 cm., that of
which the culture
block will be described further on.

are used which have a
somewhat
convex bottom, the gypsum block must be concave under-
To make
a gypsum block, 2 parts of powdered

mixed with f part of water and the mixture
poured into a tin mould. The block should be hard, and
the mould must not be rubbed with fat, oil or such material.
neath.
gypsum
are
Sometimes glass
latter are to
plates are used instead of glass lids, but the
be preferred.
gypsum
culture on a
block in such a vessel cannot, as
a rule, be kept free from bacterial infection, for the cover
Fig. 38.
must not be
access of
air.
Gypsum Block In a glass
rlisli
with water.
tightly closed down, but should allow free
When
therefore a spore culture
is
to be pre-
served in a pure state, one uses not gypsum, but a shallow

layer of water in a culture flask
these and other methods
But as a richer spore formation is got

gypsum blocks than on other substrata, it
are described later.

in the cultures on
sometimes of importance to be able to produce bacteriumSchionning has

free spore cultures on gypsum blocks.
is
method
therefore described a
in Fig. 39, there
into
is
for doing this.
taken for
this
As may be seen
purpose a Hansen flask
The promade with a
which the gypsum block has been moulded.
cedure
is
as follows
diameter a
little
First a paper cylinder
is
smaller than that of the neck of the flask.

5
66
This paper cylinder

parts of
used as a mould, and a mixture of 2
is
gypsum and
f part of water
glass rod
is
is
poured into
is
gypsum has
taken
gypsum
off,
solidified the
paper
a suitable length of the
cylinder cut
depression
After
ture to drive out air bubbles.
the
it.
used for stirring the mix-
made
off,
and a shallow
at both ends.
The
upper depression serves later for holding the yeast, the lower one enables
the cylinder to stand better on the
curved bottom of the

39. Gypsum Block
Hansen Flask (after
Pig.
a
as the cylinder
As soon
flask.
has been placed in
in
side the flask it is fixed in position
SchiSnning).
by some
of the
gypsum
paste being
cautiously poiu-ed on the bottom of the flask through a small
paper
The
filler
side
without disturbing the position of the cylinder.
and top tubes are closed with cotton wool.
These flasks and the ordinary glass dishes with gypsum

blocks are sterilised for 1 to 1| hour at 110 to 115 C, the
wrapped in a double layer of filter

As mentioned above, the temperature should not rise
above 120 C. The ordinary gypsum blocks and cylinders
glass dishes being first
paper.
are sterilised in a moist condition, but are difficult to get

perfectly sterile.
Spores of bacteria clinging to them will
often survive the heating.
The rubber tube

which
is
fitted
on the side tube in Fig. 39,
closed with a glass tube filled with cotton wool, is
only fixed on after the culture

tube
is
at the
same time used
is laid
on the block.
for connecting
The
with another
flask holding sterile water.
The
large
above (Fig.
and
is
gypsum block standing
38), is
in a vessel, as described
used for spore cultures in nearly
extremely convenient.
The blocks
of
all cases,
gypsum can
SPORE CULTIVATION
be used several times.
water;
if
They
are cleaned
67
by immersion
in
very much contaminated they are boiled, being
afterwards brushed with a
stiff
brush, and finally shaved
with a piece of glass or something of the kind, after which

they are washed with water.
Sterile
is
Water
Holder,
brought into use in
FiG. 40.
many
holder with sterilised water
kinds of experiments, for ex-
Holder for supplying Sterile Water.
gypsum block cultures. Fig. 40 represents a

holder made in such a way that some of the water can be
drawn off without the remainder being infected.
The apparatus consists of a globular or conical flask
ample, in
with a capacity of about 2

rubber stopper.
litres.
Through the one hole a
to the bottom of the flask;
it is
rubber tube with a pinchcock

filter,
which
It has a double-bored
is
consists of a glass
attached to the end.
tube packed with cotton
wool and covered with a loose glass cap,

other hole of the stopper.
glass tube passes
twice bent outside and a
is
placed in the
68
The whole apparatus is sterihsed in the following way

The flask is filled with the necessary amount of distilled
water, the stopper put in place and the long glass tube
adjusted so that
end
its
hole in which the
is
above the water surface.

be placed
filter is to
of a glass stopper and the pinchcock
The water
now
The
shut by means
is
removed from the
on a sand bath for an hour,

the steam passing freely through the glass and rubber
tube.
tubing.
boiled
now removed from

pushed down to the bottom,
The
glass tube
is
flask is
the sand bath, the

the glass stopper
The apremoved and replaced by the sterilised filter.

paratus is again placed on the sand bath and the water
again made to boil
the water
is
now
on account of the increasing pressure

driven through the glass and rubber
tubing; the pinchcock

flask
now
is
now
and the
fitted to the latter
removed from the sand bath.
All that
to get a continuous stream of water
is
is
required
to open the
To prevent
pinchcock, the tube acting as a syphon.
the
apparatus from becoming infected when not in use, the

glass jet at the
end of the rubber tubing
is
passed through
the cork of a test tube or small flask containing alcohol.
Before use, a
alcohol.
little
water
is
allowed to run
The whole apparatus
should not be too low.
is
off"
to
remove
placed on a stand which
In more delicate experiments
it
is
preferable to use sterile water taken from such flasks as
are only opened once.
8.Moist
Chambers.
Moist chambers are employed in investigations on de-
velopment, for producing pure cultures,
Hollow Glass Slips.

paratus
is
The
the hollow glass
etc.
simplest form of this ap-
slip,
by which
is
understood
a slip having an oval or circular depression in the middle.
drop of culture medium or liquefied nutrient gelatine
MOIST CHAMBERS
69
containing the micro-organism to be examined
on a cover
drop
and
glass
The cover
below.
is
placed
is
laid over the depression so that the
glass
then fastened to the
is
with vaseline.
glass slip
Ranvier's
Moist Chamber.
We
may
use Ranvier's
moist chamber for the same purpose (see Fig. 41).
It con-
which there is an annular groove.

The medium containing the organism is placed on the part
sists of a glass strip in
of the glass slip inside the groove, which
the outer part of the strip
is
thinner than
may
a drop of water
be put
into the groove, this
however not being always necessary.
carefully laid on so that the drop does
cover glass
is
now
To keep
not flow into the groove.

it is
Fig. 41.
Ranrier's Moist Chamber.
liquid condition
thus
the cover glass in position,
smeared with vaseline, which may be applied in the
made
to
by means
fit
42. Bottcher's Moist Chamber.
Fig.
of a brush.
The cover
glass
is
In addition the edge can be
air-tight.
painted with a liquid mixture of
part of vaseline and 2
parts of beeswax.
Bottcher's
Bottcher's moist
to a glass slip.^
of the chamber,
Moist
Chamber.
chamber
in
Fig.
which a
drop of water
42
glass ring
is
represents
is
and the cover glass carrying the culture
medium and the micro-organism next laid on the

culture
is,
cemented
placed on the bottom
ring.
The
of course, on the under side of the cover glass,
by means of melted
be stuck on to the ring
the latter being fixed on the glass ring

vaseline.
'
The cover
glass
Good strong chambers
Altmaun, Berlin.
may
of this
also
kind
may
be obtained from Messrs.
70
by means
glass slip
of fish glue
by means
on the glass
and then the ring fastened to the
The ring must be adjusted
of vaseline.
slip so
that the adherent
medium
is
not dis-
solved by the water. In most cases only one drop of

water is placed in the middle of the chamber, so that even
when
be
the adherent substance
little
chance for the ring to
is
An
not employed.
a loose adhesion
medium can
turpentine
little
Syndetikon,
painted on after melting.
It is certainly
loose.
adhesive
wax and
be made from a mixture of

is
soluble in water, there will
become
medium when
best to use an insoluble
with vaseline
is
fish glue,
it
water
glass, etc., are all soluble in water.
Two
use,
18
sizes of rings are in
general
having diametei's of 30 mm. and
mm.
The former
respectively.
are used chiefly for preparing pure
where the presence of a
cultures,
large
in
number
of colonies
is
desired
Squared cover
one chamber.
glasses are often used for this.
Fig. 4.3. Stand for Moist
Chambers.
Stand for Moist Chambers.
When
manjr moist chambers with cultures are in use at
the same time
it
is
convenient to have a stand for them
(see Fig. 43).
9.
Additional Apparatus.
Pipettes are used in
many sizes and it is necessary to

Some of these must be gradu-
have a large stock of them.
ated and have a capacity of j

necessary to
pipettes, a
have ready
number
of
c.c. to
various
100
sizes
c.c.
them being drawn out
into long capillary tubes.
Some
It is also
ungraduated
of
of each kind
end
must have
at one
the lower part long and thin enough to reach to the bottom
of the Pasteur flasks through the side tubes.
Such pipettes
NUTRIENT LIQUIDS
as a rule are not
on
sale, so
from a glass blower.

top end
is
is
and
Before the pipettes are sterilised, the
They
and kept
in suitable metal cases provided
and on the bottom of which some cotton wool
lids
placed
that they have to he ordered
closed with a small plug of cotton wool.
are sterilised
with
71
may
or each one
be wrapped in
paper
filter
sterilised.
Red
Rubber Tubing for Flasks.
rubber tubing
used for culture flasks having side tubes.
is
Pieces 8 to 9 cm.
long will be found the most suitable for the Pasteur flasks.
These are
first
washed
and then both ends are
in spirit
closed with glass stoppers, the latter having a length of
about 6 cm. and being drawn out at both ends.

pare a number of such tubes in
sterile condition
placed in a beaker or similar vessel, covered with

tied on,
and kept
in a current of
To
pre-
they are
filter
steam for one hour.
paper
After
the rubber tubes have been once used they must be boiled
in water before sterilisation.
Platinum
Brush.
be mentioned here,
They
Lastly,
platinum brushes ought to
these having
consist of pieces of
fine
often
collected together like the hairs of a brush
the end of a glass rod.

Geissler's successors,
shall
now
Nutrient Media.
describe the nutrient
1.
Beer Wort
is
and fused into
They may be obtained from
employed, their preparation and
solutions.
useful.
Bonn.
II.
We
proved
platinum wire which are
media commonly
sterilisation.
Liquid Media.
one of the most frequently used nutrient
It is not usually prepared in laboratories but
obtained from breweries, and
may
wort or as ordinary hopped wort
be used either as malt

the former, however,
is
72
only exceptionally used.

wort,
i.e.,
wort
If a clear
desired, filtered
is
wort which has been passed through the
in the brewery,
An
can be used.
obtained by setting this aside and allowing
oxygen from the

If the
air
method
is
take up
to
it
a deposit forms at the same time.

is employed
must be diluted with
water on account of the great evaporation which
takes place, so that the concentration

the same after sterilising as before
7 parts of
The
bags
of boiling on the sand bath
for sterilisation of the wort, the latter

sterile
filter
absolutely clear wort
wort and
dilution
is
1 of
may
be approximately
usually a mixture of
water will be found most
unnecessary
when steam heating
suitable.
used for
is
sterilising.
Wort contained
Pasteur flasks
in
sand bath in the following manner

for
an hour
steam
is
during the
is
The wort
on the
sterilised
is
boiled
three-quarters of an hour the
first
allowed to pass out of the side tube through the
open rubber tubing

the flame
is
the glass plug after being sterilised in
then put into the tube so that during the
remaining time the steam only passes through the bent

_
tube.
As soon
the tube
is
as the flask
removed from the sand bath,

The air which
is
closed with the asbestos plug.
passes in as the flask
cools
through the hot tube.
Later on, after the
is
sterilised
by
its
ttibe
passage
has cooled,
the current of air passes very slowly through the tube and
is
thus filtered by the asbestos.
The wort
in the different flasks
is
sterilised
by means
of
steam at ordinary pressure for a half to three-quarters of
an hour, large flasks with 70
c.c.
of wort requiring three-
quarters of an hour, the smaller flasks,

reich, half
e.g.,
the Freuden-
an hour.
After sterilisation
it is
advisable to let the flasks remain
at least fourteen days before use in order to be able to judge

to
what extent the wort
is
sterile.
During
this time the
NUTRIENT LIQUIDS
73
wort takes up oxygen, and the germs which may possibly

be present will develop.
wort
Sterilisation of
in the following
is
in Carlsberg vessels
way Each
:
of the
two
is
performed
straight side tubes
provided with a 10 cm. length of thick grey rubber
tubing which must be very stout and

prevent displacement
asbestos
filter,
for
by
two hours
itself in filter
After
at 150 C.
in the lower rubber tube which
pinchcock.
closed with
is
upper one has a loosely fitting metal
sterilised
is
chamber
To
The
exactly.
fastened with copper wire.
it is
the lower opening of which
cotton wool (the

cover),
fit
this,
about 5
is
The water
is
inserted
is
also provided with a
litres
are poured into the vessel, which
powerful gas flame.
paper in the hot air

glass plug
is
of distilled water
then placed over a
kept boiling for three-
quarters of an hour, during which time the upper side tube
The hot steam thus passes
remains open.
end
of three-quarters of an hour the tube
glass plug
steam
out, until at the

is
closed with a
then allowed to pass for a quarter of
is
an hour through the bent tube, the gas flame being meanwhile turned
down
little
afterwards the hot water
lowed to run out through the lower side tube and

in case the vessel should be used the
of beer wort.
drawn off",
same day for
sterilise
it,
sterilisation
If this is not the case, only about
the rest remaining in until the vessel
is al-
100
c.c.
is filled
are
with
wort, the bent tube being closed with an asbestos plug.

"While the water
is
running
with a gas flame to

replace the water.
out, the bent tube is
sterilise
When
heated
the air which passes in to
the water has been removed
in this manner, the vessel can be filled with nutrient solution.
We
The
and
^
will
assume that
this, as is
usually the case,
wort.
is
vessel is then charged with 7 litres of ordinary

1 litre of distilled water,^
This amount
heating the vessel.
is sufficient
wort
the asbestos plug being taken
when a three-burner Bunsen
is
used for
74
out of the bent tube and the mixture heated to boiling

glass stopper
wort
is
is
also
removed from the upper side
which has been
is
the-
The
tube.
The rubber
boiled for three-quarters of an hour.
of the upper side tube
then closed with a glass plug
and the steam
the flame,
sterilised in
allowed to escape for another quarter of an hour through

the bent tube, under which a gas flame
About 100
now
is
placed.
of boiling hot wort are then drawn
c.c.
off
through the lower side tube, and the rubber tube thus
The part
sterilised again.
cock
or
is
of the rubber outside the pinch-
cleaned either with the aid of sterile
by means
of a hot iron rod,
previously sterilised in the flame
during this the flame
is
whereupon the
is
paper
filter
glass plug-
quickly put into place
kept under the bent
soon as the wort has become lukewarm the
tube.
filter is
As
placed
To do this it is taken out of the filter

paper in which it was wrapped for sterilisation, the cottonwool plug is removed in a flame and the filter screwed on
on the bent
tube.
to the tube, the
mouth
the
Bunsen flame meanwhile being held over

Finally the flame used for heating
of the latter.
the bent tube
is
removed.
Before the wort
is
used
it
must be
This can
aerated.
be done by allowing the wort to stand for several months.

If,
however, an air-pump can be used the aerating and
cooling of the wort can be performed on the same day.
Assuming that the
air
from the pump
pumped through
is
a tube which ends in a stopcock, the procedure
Before the air

free
filter
from stray germs and

;
is
wool, and
is
as follows
it
must be
therefore passed through a
the latter consists of a metal tube
diameter.
is
comes into contact with the wort
filled
with cotton
conveniently about 25 cm. long and 3 cm. in
About 40 grams of cotton wool are necessar}^

and should be sterilised at 150 C. for
for filling the tube,
two hours
previously.
The whole
filter is
then packed in
NUTRIENT LIQUIDS
filter
paper, sterilised in the same
75
way and then
connected
with the tube from the air pump.
While the wort

is
in the vessel
is still
boiling, a gas flame
placed under the bent tube through which the steam
The rubber tubing
escaping.
of the lower side tube
is
then
is
connected with a bent sterilised glass tube, and the latter

placed in connection with the air
by means
filter
of a sterile
rubber tube.
The air stopcock above the filter is now opened a little,

and at the same time the pinchcock, which is afterwards
pushed forward over the rubber tubing on the glass tube
the air
now
the vessel
also
is
goes through the wort.
under the bent tube.
until the
hours.
The gas flame under
then removed, and some minutes later the flame
wort
cools to
The aerating
30 to 35 C,
i.e.,
is
continued
for about five to six
If the temperature during aeration falls below 30 C.
the wort usually froths out through the bent tube, and
About 60
of course, ought to be avoided.
this,
litres of air are
required for 7 to '8 litres of wort.
Before the aeration

heated.
is
stopped the bent tube
The communication
is
now
interrupted
again
is
by
closing
the lower rubber tube with the pinchcock, and at the same
time the stopcock above the air
removed from the rubber
The
filter.
glass tube i&
tube, the latter cleaned out
speedily closed with the flamed glass plug.
and
Simultaneously
with the removal of the gas burner from under the bent
tube the asbestos
is
filter is
completely cooled
able here to let
sure that
it
As soon
screwed on.
ready for use
yet
as the
wort
it is also
advis-
stand for fourteen days in order to
make
it is
it is really sterile.
Besides
its
use in fermentation experiments with large
quantities of liquid, the Carlsberg vessel can also be employed
with advantage in storing large quantities of

solution: the latter can
then be drawn
off"
nutrient
into smaller
76
which are connected with the large
flasks,
This
vessel.
is
when one requires an absolutely

then only drawn off" from the Carlsberg
of importance, for example,

clear
wort
it is
vessel after
is
it
has stood long enough.
to be placed in a larger
cedure
If the
same wort
same pro-
of flasks the
While tapping, the
followed.
is
number
filter is
removed
and the bent tube heated.
When
it is
expedient to avoid sterilising brewery wort
again in the laboratory, the Carlsberg vessel
used by putting
may
also be
in communication with the wort cylinder
it
of the Hansen-Ktihle pure culture apparatus,
with the cooled, aerated,
and
brewery wort.
sterile
filling it
For many
experiments this has, in addition, the important advantage

that exactly the
same wort can be treated
treated in the brewery
as
is
is,
of course, very
When,
in the laboratory
the composition of the wort
much changed by
repeated sterilisation.
from the wort cylinder
therefore, the wort
to be filled into a Carlsberg vessel the following process
adopted
The upper
side tube of the vessel
is
is
is
connected with
a cock on the wort cylinder by sterilised glass and rubber

tubing.
This manipulation must, of course, be performed
with due regard to

cylinder
all
precautions,
all
the more as the wort
generally set up in the neighbourhood of the
is
fermenting room, and the conditions of working are thus
more
difficult
tube
is first
than in the laboratory.
right-angled glass
inserted in the rubber of the lower side tube
of the vessel (the pinchcock remaining in position)
this
tube should be about the height of the vessel, and should
have been
sterilised beforehand.
The advantage
this glass tube is that the quantity of
be noted and the supply cut

full.
When
ofl'
wort
when
in using
in the vessel
the vessel
is
may
exactly
the vessel has been connected with the wort
cylinder in this fashion the asbestos plug
the bent tube and the wort
is
is
removed from
allowed to flow, the pinch-
NUTRIENT LIQUIDS
17
cock on the lower side tube being at the same time opened.
When
the
quantity
been
has
vessel
of
wort
the
charged
pinchcock
diately afterwards the tap
of the
with the required
is
shut,
and imme-
wort cylinder.
Before
communication between the vessel and the cylinder

interrupted the sterile
filter is
of the vessel in the usual
is
screwed on to the bent tube
manner.
Then the
glass tubes are
taken out of the rubber of the two side tubes, this being
Before the flamed glass plug
done in the flame.
is
inserted
in the lower rubber tube the latter ought to be sterilised
by means of a hot iron rod. When the right-angled glass

tube is removed the wort remaining in it runs out, and the
rubber tube of the Carlsberg vessel
may thus
be wetted.
If
must be well washed with spirit and afterwards flamed, dried up wort being a splendid culture
medium for moulds and other micro-organisms.
this
happens
it
If a Prior vessel is used, the aerating of the
wort pro-
ceeds as a consequence of the temperature difierence between
the air in the vessel and that outside, the air being sucked
through the wort.
The construction
of the vessel
may
be
seen from Fig. 29.
As soon as the wort in the vessel is sterilised in the usual

way and some hot wort drawn off" through a, thus sterilising
this tube, the flame is extinguished, the air filter, /,
put in
and the rubber tubing connecting both parts of the

The air which
tube, r, completely closed by a pinchcock.
place
is
sucked in at
fied
a,
in consequence of the formation of a rare-
space over the wort, passes through the
the lower part of the tube
and
collects in the flask
r,
filter at
/ into
then through a into the wort,
above the wort, which takes up
more or less of its oxygen. The aerating is continued until

the wort has reached the temperature of the surrounding
air, and is very vigorous in the first stages, as one can hear
from the noise of the bubbling.
As soon
as the temperature
78
has reached a state of equilibrium the pinchcock
is
opened,
thus making communication with the outer air again in

Cultivation of yeast can be proceeded with
the old way.
immediately.
Wort, with the addition of tartaric acid
(to
the
amount
of 0'3 per cent, for mixtures of yeast and bacteria to exclude

the latter),
made by adding a concentrated
is
tartaric acid in sterile
wort
the mixture
difficulty, that a
among
fore,
is
arises this
it
added
is
may
when only
a small propor-
the dilution of the wort
be neglected.
When
it is
is
desired
a specially strong solution of tartaric acid in wort
the acid
now
both solutions can, there-
be mixed after sterilisation,
make
There
sterilised.
fairly strong deposit forms, containing,
tion of tartaric acid
to
water to the necessary amount of
then
other things, albuminoids
then so slight that
solution of
is
Such
dissolved directly in the wort.
bump
acid solutions
violently during boiling
this
tartaric
may
be
avoided by adding to the solution some pumice stone which

has been heated to redness.
Water
is
invariably used distilled, and
cult to obtain completely sterile
always
tolerably
diffi-
ought, therefore, to be
one to two
two or three times
sterilised several times at intervals of
days in a current of steam.

is sufficient
in
most cases
quarters of an hour the
second and third times.
manner
it
is
lies in
Sterilising
the water
first
is
then boiled for three-
and for half an hour the
time,
The reason
for proceeding in this
the fact that certain bacteria spores present
in water can survive a temperature of 100
C, while, on the
other hand, the growing cells of these bacteria are destroyed
The germinating power

can even be increased by heating the water
at this temperature.
left at rest for
some time after the
first
of the spores
is,
therefore,
heating, the spores
germinate and are then easily killed by the subsequent

heatings.
NUTRIENT LIQUIDS
When
for
water
is
79
to be sterilised under pressure
an hour in the autoclave
it is
a pressure of
at
heated
to 1|
atmosphere.
Yeast Water
an extract of yeast and
is
favourable culture
medium
for bacteria
an extremely
is
and yeast
cells.
prepared by boiling J kilogram of pressed

yeast free from starch with 2 litres of distilled water for about
Yeast water
half an hour
is
the liquid
is filtered
warm and
while yet
the solution boiled for another half -hour and
which
it is
then
filtered, after
distributed in flasks and sterilised in a current
The yeast
of steam for about three-quarters of an hour.
water thus obtained is however too concentrated for ordinary
When, therefore, it is about to be used it is

mixed with an equal quantity of sterile water, or as much
as to make the mixture sherry-coloured, and then sterilised
in flasks for three-quarters of an hour without pressure.
Meat Extract is prepared according to R. Koch in the
following manner 500 grams of meat free from fat, and
1,000 grams of distilled water, after being thoroughly
experiments.
stirred together, are left for twenty-four hours in an ice safe,
The liquid is then

expressed, boiled and strained, by which means the precipitated albuminous bodies are removed 5 grams of sodium
or,
during the winter, in a cold situation.
chloride and 10 grams of peptone are dissolved in every 1,000
grams
of the
meat
sodium carbonate.
extract,
The
which
liquid
is
is
then neutralised with
now
liltered at boiling
temperature, sterilised for two hours and preserved in
Pasteur flasks.
Fruit Syrups are most easily prepared from
after sufiicient dilution
with water
fre.sh fruit
the_y are sterilised for
an
hour in a current of steam.

If
no fresh
fruit is obtainable, dried fruit
as a substitute.
For instance, a syrup
from dried apples
in the following
may
manner
may
be used
be prepared
kilogram of
80
dried apples, 5 litres of water and 20 grams of tartaric acid

are allowed to stand for twenty-four hours
then pressed, filtered and
way
In the same
raisins.
But
It is
is
a grape juice can be prepared from
easier to use the concentrated grape juice
it is
obtainable as a commercial product
mann.^
the mixture
sterilised.
recommended by Wort-
prepared in Sicily by evaporating the freshly
prepared juice to about one quarter of
its
volume.
It is
viscous like syrup and contains about 65 per cent, of grape
and
fruit sugar, in addition to live yeast cells which,
however,
do not develop so long as the syrup remains concentrated.
When it is about to be used it is diluted with 3 parts of water,,

clarified, if necessary, filtered
Solutions of Saccharose
tions in
common
and then
sterilised in steam.
and Dextrose.
Other
solu-
use which might be mentioned are a 10
per cent, solution of saccharose in water, and a 10 per

cent, dextrose solution in
yeast water; both are sterilised
for half an hour in steam.
Beer should be sterilised in the autoclave for a quarter

of an hour under a pressure of 1 to 1| atmosphere.
advisable to
because,
as
fill
beer into flasks with rubber connections
mentioned before, the rubber tubing cannot
stand the pressure.

of
It is not
On
boiling on the sand bath the whole
the alcohol would disappear
on the other hand, after
sterilisation in the autoclave the beer contains half of
original quantity of alcohol.
It has
the
been shown that lager
beer containing 5'65 per cent, of alcohol by volume retains

2 8 per cent, after sterilisation for a quarter of an
hour
under IJ atmosphere pressure in Freudenreich flasks. If,

therefore, sterilised beer is required with the whole quantity
of alcohol the proper
amount must be added
either before or
after sterilisation.
1
Concentrated grape juice
is
Sons, Mazzara del Vallo, Sicily.
obtainable,
e.g.,
from Messrs. Favara and
SOLID CULTURE MEDIA

Discontinuous Sterilisation,
from any
substances which,
cause, cannot withstand boiling
by a discontinuous
sterilised
Those
81
process, the
may
often be
media being sub-
jected to a temperature of 56-58 C. for two to four hours
every day for a week.

Sterilisation
by
Filtration,
We have hitherto described
the sterilisation of nutrient solutions
by heat
alone.
In
those exceptional cases in which this method cannot be
employed,
filtration
The best known

In the
filters.
through special
first
substituted.
filters is
and Berkefeld's
of these are Chamberland's
of these, filtration takes place through
a tube of biscuit ware, the liquid being passed through
The pores
pressure or suction.
by
of the tube, however, are
quickly clogged, rendering a frequent cleaning and
sterilis-
ing necessary, the bacteria otherwise spreading through the

filter.
In the last-named
the filtering
filter
medium
consists
of kieselguhr.
It
advisable to have a stock of the various culture
is
liquids in concentrated form, as they do not then occupy
so
much room.
2.
Gelatine
and
Solid Culture Media.
Agar-Agar,
In
the
preparation of
nutrient gelatine care must be taken not to
make
the
heating too long or too strong, as the gelatine thereb}^

loses its power of setting and is thus rendered useless.
The procedure is as follows: The quantity of gelatine is
weighed and placed in the boiling liquid, which has also
been weighed, and diluted with the necessary quantity
of
water
to
prevent
over-concentration.
Heating
is
carried out in a dish on a sand bath, as gelatine solution

is
very easily burned
it is
therefore advisable to
stirring the mixture as long as
it is
over the flame.
soon as the gelatine has dissolved, the solution
is
keep
As
removed
82
from the source
amount
small
of heat
of fresh
and cooled
albumen
is
to about 50
added
C, when a
the latter
is first
beaten up with a
little water, this being most readily done

by shaking up violently in an ordinary medicine bottle.
The white from one egg is sufficient for two litres of liquid.
The
gelatine solution
is
thereupon mixed well with the
white of egg solution, the whole then put on the sand bath
and
cautiouslj' heated to boiling
without
coagulates the white of egg in a few minutes, and

ates out in large flocks
which aggregate
all
now weighed
same
to ascertain if the
the weight
if
is
it
separ-
the suspended
matter and impurities present in the gelatine.

is
This
stirring.
The whole
weight has remained the
too small, sterile water
is
added
otherwise the liquid must be carefully evaporated at a

gentle heat until the proper weight
gelatine solution
warm, the
still
In
many
it is
is
is
arrived
strained through a flannel while
flannel being stretched
cases a perfectly clear gelatine
then unnecessary to
filter
it.
If
The
it
is
on a wooden frame.
is
not required, and
on the contrary perfectly
clear gelatine is required, it is filtered boiling hot
a paper filter provided with a toughened point.

is
at.
through
This
filter
placed in a glass funnel fitted in a copper funnel (see Fig.
The
44).
latter is double walled
There
side tube.
is
and provided with a closed
an opening on the upper edge of the
funnel which allows of the space between the walls being

filled
is
with
warm
water.
Under
the side tube a gas flame
placed which keeps the water boiling during filtration
the gelatine
is
Smaller quantities of nutrient gelatine
with
thus prevented from setting.
least trouble
may be
prepared
on the water bath.
According to Rich. Meissner the use o dry albumen is not to be

as it has been shown that when it is used for clearing the
gelatine, organisms sown on the latter are hindered in their development. This probably arises from the formation of secondary products
'
eoommended,
(ptomaines
?)
during the drying.
SOLID CULTURE MEDIA
83
Pasteur flasks are best for preserving nutrient gelatine

as they do not allow
gelatine while
any drying up.

hot
still
is
The strained or filtered
poured into a
flask,
previously
and boiled for flve minutes on the sand bath. A

smaller amount may be stored in diflerent small flasks
sterilised,
according to the purpose for which
it
is
intended.
As
regards preservation of nutrient gelatine to be used for
ordinary plate culture,
it is
advisable to use the globular
flask represented in Fig. 36,
Fig.
15 CO. of gelatine.
which
44. Hot Water
The
gelatine
is
is
charged with about
Filter.
here also boiled for five
minutes on the sand bath, after the flask has been plugged
with cotton wool and covered with a double layer of

paper.
In case the flask
is
to remain for a long time,
filter
it is
of advantage to use a rubber cap in place of the fllter paper
These flasks are to be used in preference to

Freudenreich
flasks,
test
tubes or
because the liquefied gelatine seeded
with a culture can be better shaken up in the former than

in the latter, it being
above
6*
all
desirable
when making
84
ordinary plate cultures to distribute the germs as
much
as
If the gelatine is to be applied for surface plate
possible.
cultures, test tubes or Freudenreich flasks
which the culture gelatine
mixed with the germs
is
may
be used in
only liquefied without being
but globular flasks are also prefer-
able in this case.
In Freudenreich
of
flasks, gelatine is sterilised for a
an hour in steam.
After the sterilisation
is
quarter
finished, the
flasks are placed in an oblique position until the gelatine
has
When
set.
these flasks are to contain their gelatine
ought
for a long time, the cap tube
with
to be closed
wax
or with the S-shaped tube mentioned previously (Fig. 33),
prevent the gelatine drying up.
to
all cases to
it
is
ought in
Gelatine
stand for some time before use, partly to see
sterile,
and partly because
it
can then
With regard
temperatures better without melting.
latter property, difl'erent kinds of gelatine
Hueppe
recommends
the
resist
behave
to the
differently.
method
discontinuous
if
higher
sterilising gelatine, this consisting of subjecting it
dail3'^,
of
for
four to five days, to a boiling heat for five minutes.
Wort
etc.,
gelatine, yeast
water gelatine, fruit syrup gelatine,
are prepared with a content of 7 to 10 per cent, of
gelatine, that
is,
as
without melting.
much
as will enable
them
to stand 25 C.
Meat extract peptone gelatine
is,
on the
other hand, always prepared with at least 10 per cent, of

gelatine.
of
Ten grams of gelatine are dissolved
meat extract
in the usual
way
sometimes
in
it
100 grams
will be ne-
cessary after adding the gelatine to neutralise with sodium
known, gives an acid

and many bacteria do not thrive even on a feebly
Meat extract peptone gelatine is always
acid medium.
the
carbonate, for
gelatine, as is
reaction,
sterilised
by the discontinuous
Nutrient agar-agar
gelatine
but
it
is
process.
prepared in a similar
must be cut
way
to the
into very small pieces before
SOLID CULTURE MEDIA
85
being put into the boiling liquid, and has to be boiled for
some time before it

case are 100 grams
Filtering
is,
The proportions in this

grams of agar-agSf.
dissolves.
of liquid to 2
as a rule, avoided, as
with
diflBculty,
keep
it
it is
only accomplished
agar-agar requiring a higher temperature to
Hence, white of egg
liquid.
is
not used for clearing.
remove coarser suspended matter, the
If it is desired to
solution can be strained through linen.

If,
however, the agar-agar has to be
he employs
substance
among
Giesenhagen
To
accelerate the agar
filtration in steam,
and distributes the
recommends the following method.

filtration
filtered,
several filters
working simultaneously.
Small tin funnels with turned down edges are used for
and these are provided with
filtering,
flat
enamelled covers
Three funnels are placed in rings
with projecting edges.
round the stem of a special stand arranged over Erlenmeyer

flasks of appropriate size
is
16 'o cm. high
for each flask
Two, or
filters
(each of the three filter stands
8 cm.
and
wide).
fixed in the
is
The wadding plug
meshes of the wire stand.
in high steam chambers even three, such sets of
(i.e.,
6 to 9
can be arranged for steaming.
filters)
The
done through two folded filters. Nutrient agaremployed for cultures at high temperatures, gelatine
filtering is
agar
is
being unsuitable as
it
becomes
liquid.
Mixtures of agar-agar with gelatine are prepared in the

proportions of 100 gi-ams of culture medium,
agar-agar, and 4 or 3
grams
of gelatine.
or 2
grams
The addition
of
gelatine prevents the separation of water which always
takes place
when pure agar-agar
Litmus gelatine
detecting
sometimes used as a reagent for
It is
prepared according to Hueppe in
way An aqueous solution of

made up, sterilised and allowed
the following
is
used.
the formation of acid during the growth of
a micro-organism.
matter
is
is
the colouring
to cool.
The
86
gelatine
is
liquefied at 30 C. (the agar-agar at 40),
and
is
The
then mixed with the equally hot litmus solution.
latter should only be so strong that its action as a reagent

is
just distinguishable.
Other Solid Media.
is
made
steam
^Bread
into a paste with a

rice
might be mentioned as one
media which are sometimes employed.
of those solid
may
little
water and
is
several
sterilised
times with an interval of one or two days.
employed,
by
Manure
sterilised
be sterilised in the same way.
(with or without addition of water)
It
These substrata
cultivating moulds.
Potatoes are
frequently used for the culture of bacteria.
The potatoes
are
e.g.,
in
are cleaned well with a brush and laid for some minutes
and afterwards
in a 10 per cent, solution of sublimate

O'l
Finally they are well washed with water and then
hours.
sterilised for
two hours
in
steam and cut up.

Method.s.
III.
Microscopical Investigation of Micro-Organisms.
1.
Preparation Making',
ism
is
made
cover glass on
it.
sterile so as to
paration)
little
preparation of a micro-organ-
by putting
as a rule
or in Canada balsam,
and
in a
per cent, sublimate solution for half an hour to fifteen
etc.,
Water
is
in a
it
on a glass
drop of liquid
and lajdng a
slip
when
often used (best
distilled
exclude outside organisms from the preof the
growth
to be investigated is taken
out with a platinum wire or similar instrument and stirred

in the water.
If it is in a culture liquid, a sample can be

taken out with a small glass rod and placed direct on the glass
slip
without addition of water.
The needles and rods used for
taking out samples must of course be sterilised beforehand,
when
the cultures are to be preserved pure
precautions must be observed.
on the drop the enclosing of
When
all
the usual
the cover glass
is
laid
air bubbles in the liquid is to
METHODS OF INVESTIGATION
87
These can, however, when formed, be expelled
be avoided.
by a cautious tapping on the cover glass. Yeast cells and

moulds are usually examined in the unstained condition.
Water-mounted preparations can be kept for some time
if the cover glass is sealed round the edge to the glass slip
no evaporation can take
so that
medium
wax in
wax.
for this purpose

spirit,
If it
is
is
place.
a solution of
very suitable
common
sealing
or a melted mixture of vaseline and bees-
wished to make really durable preparations,
Hantsch's solution (see p. 92)
an ordinary preparation
added drop by drop to
is
water so that after some time
in
the only liquid remaining in the preparation
is
the alcohol and water having evaporated.
The gradual
addition
is
necessary so that the form of the
be altered too
much by
glycerine,
cells
may
Afterwards the edge of the cover glass
glycerine.
sealed either with the above-mentioned sealing
This method
tion or with asphalt lac.
able for preparations of yeast cells
is
wax
the latter are to be

in
is
solu-
especially suit-
and moulds.
For the
preparation of stained bacteria specimens see below.
mounted
not
the water-absorbing property of
made permanent they
If
are mostly
In permanent specimens the
Canada balsam.
form to some extent.

Removal of Grease from Cover Glasses. In preparing
cells
always
lose their natural
a microscopical specimen which
and stained,
glasses.
in
it
is
To obtain
is
to be
fixed,
hardened
necessary to use perfectly clean cover

these
it
is
not sufficient to clean them
the ordinary way, but means must be employed to
remove the thin layer

The cover
the glass.
of grease
which always adheres to
some strong
mineral acid (hydrochloric or sulphuric), then washed with
water and boiled in a soda solution it is again washed
glass
is
laid in
first
with
distilled water, dried,
again dried.
washed
in absolute alcohol
and
88
Fixing and Staining of Yeast

glass
carefully cleaned in
is
culture
spread over
is
and the cover
this
under a glass
glass left
has completely dried up.

solid substratum, a little of
cell
the cover
drop of the
bell until the
drop
If the culture is present in a

it is
distributed in a water drop
and the mixture spread on the cover

the staining of a yeast
After
manner
thin a layer as possible,
in as
it
Cells.
preparation,
As regards
glass.
e.g.,
with an aniline
dye, the preparation, thoroughly dried in air by the above
method,
taken up by means of a pair of forceps with the
is
prepared surface upwards and drawn through a small gas

flame three times with imiform speed describing a vertical
circle
with a diameter of about one-third of a metre, the
The
three motions occupying about three seconds.
men
is
thus fixed and hardened.
solution
is
now
put on the cover
some minutes and then washed
The clean
oft'
side of the cover glass
paper, and the specimen
The distinguishing
is
is
of the staining
little
glass,
speci-
allowed to act for
with
distilled water.
next dried with filter
then ready for examination.
of dead cells from living ones has
been assiduously carried on in most brewery laboratories
came into general use. But the value

by the reagents employed for this
purpose has been very much overestimated. The quessince the microscope
of the indications given
According to
tion seems to deserve proper investigation.
Wehmer, a
the dead
half per cent, methylene blue solution will stain
cells
indigo blue, while the living
cells
remain
colourless.
Staining of Yeast Spores.

solution (see
of yeast
cells.
page 92)
As soon
in the above-described
is
Ziehl's
carbol
fuchsine
used for colouring the spores
as the preparation has been fixed
manner,
it is laid in
a small crucible
or watch glass with carbol fuchsine, heated for a short
time to boiling, and then washed with water, afterwards
with dilute acid (5 per
The
and then again with water.
cent.),
spores are then usually coloured red
Sometimes
less.
other
may
spores,
and
remain
colourless.
it
also
cell
nucleus
is
the rest
is
coloured bodies appear
happen that some
Staining of the Yeast Cell Nucleus.

the
89
no easy matter.
colour-
besides
single spores
The
detection of
Janssens and Leblanc
recommend a modification of Moeller's method, viz., the

following A few drops of a solution of iodine in potassium
:
iodide (1 part potassium iodide, 100 parts of water, iodine

to saturation) are placed
yeast in question
is
on a glass
slip
stirred in.
and a
then spread on a well-cleaned cover
The specimen
now hardened
is
lie
out of the iodine solution, placed
first
cent.,
and, finally, in
stain, the
colour of the cells must be completely removed.

this is not effected
by the 80 per
taken
is
in water, then in
Before proceeding to
cent, alcohol.
put into the
for twenty-four hours.
33 per cent, alcohol, next in 80 per

95 per
is
the cover glass
cent, alcohol,
yellow
In case
an aqueous
solution of potassium iodide (1 to 3 per cent.), or ether
be used.
The specimen ought
to
hours in the 95 per cent, alcohol

harmful, but
is
unnecessary.
staining, the cover glass being
is
Immediately
glass.
after the mixture has dried, the cover glass
iodine solution and allowed to
of the
little
drop of the mixture
lie
may
at least forty-eight
a longer soaking
Carbol fuchsine
warmed
is
not
is
used for
in a little of this
watch glass. The cover glass is then

washed several times with water, and, finally, with very
liquid contained in a
dilute sulphuric acid.
Heidenhain's method
cedure being as follows
hours in a solution of 2
distilled
water;
it
is
may
also be adopted, the
pro-
The fixed specimen is laid for four

5 grams of iron alum in 100 c.c. of
then placed for twelve to eighteen
hours in a solution of 0^5 gram of haematoxylin in 100
c.c.
90
Lastly
of distilled water.
decolorised in the usual
it is
manner.
Fixing and Staining of Bacteria.
bactei'ia is stained
preparation.
is
and fixed
in the
preparation of
same way as a yeast
drop of an alcoholic aniline dye solution
then allowed to act for some minutes, after which washing-
with
water takes
distilled
special
method
place.
of staining
described
is
which has found extensive application,
by Chr. Gram,
chiefly because
it is
used as a method of diagnosing certain species of bacteria.
According to this method the fixed specimen
is
stained from
one to three minutes in a hot saturated solution of gentian

violet in aniline water,
and then immersed for one
minutes or longer in a solution of iodine
precipitate
is
thus formed which
The preparation
bacteria.
is
is
in
to three
potassium iodide.
only deposited on the
then washed with absolute
alcohol until every trace of the colouring matter has been
removed.
Staining
stained
by
of
Bacteria
carbol fuchsine (in
some cases
the evaporated liquid
washed
Spores.
Bacteria
spores
are
boiling the fixed specimen for a long time in
is
for
an hour, during which
constantly renewed);
it
is
then
in alcohol.
Aujeszky has recently communicated a simpler method

for staining spores.
spores
is
drying in the
is
little
air,
warmed over
is
Bunsen fiame
in a porcelain dish until
When
this point is reached the
removed, and the cover glass,
fixed, is laid for three to four
preparation
is
is
a half per cent, hydrochloric acid solution
bubbles begin to appear.
Bunsen
of the culture containing the
spread on a cover glass, and while the smear
now
dried, but not
minutes in the
liquid.
The
afterwards washed with water, dried, fixed
and treated with Ziehl's carbol fuchsine, then held in forceps

over the Bunsen flame and heated until it fumes. As soon
91
as the staining solution begins to fume, the preparation is
drawn out of the flame for some seconds, this heating being
twice repeated. The preparation is then allowed to cool
one to two minutes more, after which decolorising with 4
to 5 per cent, sulphuric acid follows.
The latter process
should not be carried too far in case the spores again
become
colourless.
we shall describe the method given by Alex.

who found that spores easily become stained without
Finally,
Klein,
any previous treatment if the dye is allowed to act on them

in the moist state.
The process is the following Prepara:
tion of an emulsion of the spore-containing material in 0'7
per cent, salt solution (in a watch glass) and addition of an

equal quantity of filtered carbol fuchsine solution, then
gentle heating, steam being given off at the surface for six
minutes, dust being kept off by covering with a second
watch
allowed to
The preparations are tlien spread out and

dry in air and fixed by passing twice through
the flame.
Decolorising
glass.
acid acting for one to

is
is effected by 1 per cent, sulphuric

two seconds, and lastly the preparation
washed with water.

Staining of Flagella according to
assumes a special
of
when
.significance
Loffler.
detecting the motile organs, the flagella,
There are several ways of doing

being that described by Loffler.
been carefully fixed
it
is
this,
.
Staining
the question
of
is
one
bacteria.
one of the most used
After the preparation has
treated with a mordant, which
consists of 2 parts of a 20 per cent, solution of tannin,
some
drops of an aqueous saturated solution of ferrous sulphate
and
part of logwood extract (1 to
Some drops of this mixture

and warmed directly over the
8).
are placed on the cover glass
flame until steam begins to
form.
Afterwards the cover glass is washed with water
and stained with carbol fuchsine or with Loffler's solution.
<)2
of which several drops are filtered

glass.
warm on
This staining solution consists of 100
aniline water,
to the cover
c.c.
of saturated
of a 1 per cent, solution of soda
1 c.c.
and
4 to 5 grams of gentian violet, fuchsine or methylene blue.

Washing with water takes place after staining.
Reagents. The reagents used most frequently in our
microscopical investigations are the following
Absolute alcohol.
Concentrated
spirit.
Ether.
Chloroform.
Dilute soda solution
(1 to
Dilute sulphuric acid

Dilute nitric acid
3 per cent.).
(5 to
(5 to
10 per cent.).
10 per cent.).
Perosmic acid (01 to 1-0 per cent, aqueous solution, kept in a brown
or black bottle in a dark place, e.g., in a tightly-closing cardboard box).
Iodine-potassium iodide solution (2 parts of potassium iodide, 300
parts of water, 1 part of iodine).
Tincture
of iodine (a
saturated solution of iodine in strong alcohol).
Iodine-zinc chloride solution.
Hantsch's solution
(3
parts of 90 per cent, alcohol, 2 parts of water, 1
part of glycerine).
Carbol fuchsine
part of fuchsine, 5 parts of crystallised carbolic
(1
acid, 10 parts of alcohol, 100 parts of distilled water).
Tincture of alcanna (alcoholic extract of the alcanna root).
Development
in
Moist Chambers.
If
it
desired to
is
study under the microscope the development of a microorganism, the moist chambers described on pages 68 to 70
Bottcher's chamber (Fig. 42)
are used.
is
best suited for
the cultivation of an organism which requires plenty of air

in order to
is
grow
The micro-organism
well.
to be
examined
seeded either in a hanging drop of culture solution or in
a thin layer of nutrient gelatine on the under side of a suitable cover glass.
One
or
two drops
of
water are placed on
the bottom of the chamber and the cover glass stuck to the
ring with vaseline.
fastened
if,
The cover
glass can
in addition, the edge
mixture of wax and vaseline.
is
be
still
better
painted with a melted
In examining organisms
METHODS OF INOCULATION
93
may be
on so as to leave a small opening, or the chambers are
requiring a large quantity of air the cover glass

laid
The chamber should then
provided with special air tubes.
Hollowed glass
be placed under a moist bell jar.
slips
can
be used in the same way.
Ranvier chambers
(Fig. 41)
may
be used with the same
Bottcher chamber for
facility for liquid, as the
solid,
media.
In using a Ranvier chamber the quantity of nutrient solution
must not be
cover glass
down
fixed
is
when the
so great as to run into the groove
groove when necessary.
a drop of water
placed in the
is
In this case also the cover glass
can be adjusted so as to leave the groove in communication with the outer
air,
and the chamber
is
then, like the
former, placed in a moist glass enclosure.
2.
Inoculation of Liquid
Experiments with Various Flasks.
and Solid Culture Media.
The Manipulation of Nutrient Liquids requires
We
able practice.
will give in the following a description of
the various devices so far as this
pose that
it is
growth in
it,
table
is
Let us sup-
practicable.
required to transfer a liquid, with or without a
from one Pasteur iiask to another, without
in-
we should proceed in the following manner.

on which we are working is first moistened with
fecting the liquid
The
consider-
the mixture of spirit and water previouslj' described, the
gas burner and tubing being also washed.

tion which
is
This
is
a precau-
to be observed in all such experiments.
sleeves should
fit
tightly to the wrist
rubber bands are used,
or, still better,
and for
this
Coat
purpose
a linen overcoat with
Above all it is desirable that

tightly fitting arms is worn.
no dust should be introduced. The gas flame (the tubing
should be connected to the
and between
it
left) is
placed directly in front,
and the operator the tinned copper
already mentioned which
is sterilised
in the gas flame.
vessel
The
94
flask
the
from which the solution
left,
to be
is
poured
placed on
is
the other on the right, and both flasks as near the
The copper vessel is placed between

The arrangement may be seen from the accom-
gas flame as possible.

the two.
panjang sketch in Fig. 45. K^ is the flask from which we

wish to pour solution into K^. Both flasks as well as their
supports are then carefully sterilised on the surface by
means
of the gas flame
if
they contain cultures this must
be done with great care, so that the organisms are not killed
by
The bent tube
the heat.
is
now
heated to redness,
beginning at the bulb in the middle of the tube and then
Fio.
45. Arraugemoiit
tube
of the
of
G, the burner
work
going downwards
taken
If it
out.
the flask
is
two Pasteur Flasks (viewed from above).

Kj and K^, the flasks B, the copper dish
;
S, the gas
T, the edge
table.
to the end.
is
The asbestos plug
is
then
wished to transfer an average sample
shaken up, the lower bend of the tube being
held in the flame during the shaking up, so that the air
passing in
may
be
point of the tube
sterilised.
is
Care must be taken that the
kept out of the flame, otherwise gas
would be sucked into the flask with danger of an explosion.

The gas flame is put in its place again and the glass plug of
Kg loosened, without being taken out (if this is done
must be in the flame), so that it remains only with its
flask
it
end
is
fitting loosely in the
now
rubber tube.
The Bunsen burner
regulated so as to give a luminous flame because the
non-luminous
latter is not so hot as the
then taken in the

held,
left
the flask, Kj,
is
hand, the body of the flask being
and the rubber of the
The rubber
right hand.
95
is
with the
side tube is loosened
then quickly taken
ofi"
in the
flame and laid in the copper dish, the opening of the side
tube remaining in the flame, while the tubing of the

Kj,
is
squeezed with the right hand so that
hand holds Kj so that the opening

and the right hand is occupinching the rubber tube of K^ the side tube of K,
is
as follows
of
its side
pied in
is
now
The
flask,
glass plug
For the moment the situation
the copper dish.
falls into
its
tube
is
left
in the flame
fitted into the
should
all
rubber
is
rubber of
K.^
in the flame.
This
be done so quickly that neither the tube nor the

over-heated.
flasks are now in communication with each

we leave them in this position without transferring
the liquid so that the heated tube of Kj may cool
The two
other and
any
of
down.
This takes place in quite a short time, whereupon
the burner, the flame of which

is
held in the left
red hot, after which Kj
run from
it
may
be
again made non-luminous,

side tube of
sterilised.
is
The
as
being poured so that the air passing
When
the required quantity of liquid
glass plug of
Kj
is
right hand and sterilised in the
luminous
can
K^ being heated
has been passed over, the Bunsen burner

place.
Kj made
tilted so that the solution
is
into Kj, the bent tube of
long as the solution

in
is
hand and the bent
is
put back in
the glass plug being held between the
first
second fingers of the right hand, the rubber of Kg
between the thumb and
first
its
now taken up with the

flame, which is then made
finger of the
is
and
held
same hand, and
the side tube of Kj disconnected, being placed quickly in
the flame
the glass stopper
is
immediately inserted in the
rubber of Kg and the rubber of K^ carrying
its
glass stopper is
at once lifted out of the copper dish with the right hand
96
and placed on the

still
side tube of
Kj the opening
which
of
is
in the flame.
This procedure
may perhaps seem somewhat complicated
through the above description, but
after reading
been tried in practice

difiiculty
found to
will be
it
offer
The very
but only requires practice.
if
it
has
no special
first
exer-
can be performed with flasks which contain ordinary
cises
water
then flasks
may
be used containing sterile wort,
allowing them to stand between experiments in order to
which contain
flasks should be used
or meat extract in order to see
constant use
this is
After some practice
has been avoided.
see if infection
if
much more
sterilised yeast
water
these remain sterile with

difficult, as
most bacteria
develop readily in these liquids, which does not happen in

wort.
If it
seen that these flasks remain sterile after
is
they have been worked with for several days,

then assumed
that
the
may
it
be
necessary experience has been
acquired.
similar
Hansen
But the
method
flasks
to the above is adopted
in
using
which are also provided with side tubes.
air entering does
not require to be heated after
passing through the cotton wool in the cap, since the latter
acts as a lilter.
Sterilised pipettes
have to be used in experimenting
with Freudenreich, Chamberland and Erlenmeyer
flasks.
These flasks also are of course always sterilised before-
hand with the
flame.
This has to be done also
part of the contents of a Pasteur flask
one of the above-named
flasks.
is
when
to be passed into
If only small quantities of
liquid are being used, sterilised glass rods or metal wires
may
replace the pipettes.
performed in the
it is
necessary to
sterile
These experiments should be
cupboard or in the
work very
quickly.
sterile
room, and
Exercises ought also
to be performed with the pipettes as with the Pasteur flasks,
and the operator ought not
until he can
keep
97
consider himself efficient
to
water or meat
flasks containing yeast
extracts sterile after repeated manipulations.
Experiments with Solid Culture Media

of a
growth
is
medium, then metal
glass rods are used.
solution or on
such as platinum,
wires,
They
with a glass plate likewise flamed.

This
until quite cool.
otherwise easily
ately
kill
many
are left in the
very important, as one might
Immedi-
the growth to be introduced.
drawn quickly
again
once
cases to take one
point, especially
covered
is
When glass rods are to be used it will be
through the flame.

better in
is
they are
before use
or
Before use they are sterilised in the
flame and placed in a flamed tin box, which
box
medium
to new
to be inoculated from a solid culture
into a flask containing nutrient

solid
If a portion
when
drawn out
into a long thin
small specks of growth
(e.g.,
from a
moist chamber) or a fine mycelium, which clings easily to

inoculation needles
when these are used, has to be introduced,
as the point of the rod can be broken

infected culture liquid.
The
infection
oft"
is
and
left in
the
thus performed
more surely and more quickly than when a metal wire has
to be rubbed against the sides of the flask in order to leave
particles of
growth
Sometimes small pieces
in the liquid.
of platinum wire are used for the
same purpose, being
manipulated by a pair of forceps.
medium
Infection of a solid culture
on or below
its surface.
easily as a streak
made with a metal wire
the culture to be introduced

pipette
may
takes place either
surface culture
is
is
laid
on most
or glass rod
if
contained in a liquid, a
we then sow a
culture medium or we may
be used, by means of which
drop on the surface of the

use a metal loop.
On
solid
the other hand,
be sown in the substratum, this
may
if
the culture
is
to
be performed by aid
of a metal wire (an inoculation needle), which with the

7
98
adherent growth
"
stab
culture
"
thrust into the
is
a so-called
Development
thus obtained.
is
medium
organism then takes place on and
below
Cultures in the body of the substratum
may also
by mixing the growth with
the
the
of
surface.
be obtained
liquefied nutrient gelatine.
Plate cultures, which are employed in the production of
pure cultures, are described on page 103.

of Anaerobic
Cultures
to start a culture of
medium,
Organisms.
If
it
an anaerobic organism on a solid
can be done by covering the medium with a
this
plate of mica
(Koch), which
having a perfectly
is
level surface.
medium
glass plate can be used
organism under examination

with nutrient gelatine, and
which there
test tube in
(1
vol. of
is
According to the
is
this
the
latter
placed in a small test tube
put into a second larger
an alkaline solution of
pjrrogallic
an almost saturated solution of pyrogallic
acid mixed with 10 vols, of potash solution [1
outer test tube
on to the
Of other methods that of H.
(Hesse).
Buehner may be mentioned.
acid
medium
pressed on, the
in place of the mica, or melted gelatine is poured
infected
wished
is
is
-|-
1])
kept well closed with a tight plug.
alkaline solution of pyrogallic acid absorbs
all
so that the culture in the inner open test-tube
the
The
the oxygen
grows
in
an
oxygen-free atmosphere.
The
process
may
also be carried out
atmospheric air by an indifferent gas,
The method
described
by Frankel
ordinary wide test tubes
fitted
through which two glass tubes
by
displacing the
e.g.,
by hydrogen.
consists in
the use of
with double-bored bungs
pass,
one reaching almost to
the bottom, the other ending just below the bung.

culture tube after being
inoculated,
through
it.
filled
with the gelatine
This
is sterilised,
and then has a current of hydrogen passed

After
all
the air has been driven out, the glass
tubes are closed by fusing.
PURE CULTURE METHODS

Suppression
is
of Bacteria
in
99
Growths.If
Yeast
it
required to encourage the development of a bacteria-
infected culture of an alcoholic yeast, the
adopted
is
to
cultivate
nutrient medium.
way, or are
method usually
the impure growth in
Nearly
all
an acid
bacteria are killed in this
to a great extent hindered in their development,
so that the alcoholic yeasts in the mixture preponderate.
But
not always possible to use such an acid medium,
it is
as the organism to be cultivated
adversely.
may
also be influenced
In such a case the cultures can be exposed to
the action of light, as bacteria can withstand the action of

light only to a small degree.
This was proved several

Downes and Blunt. Experiments in this
direction have also been made at the Carlsberg laboratory,
and have shown that spore cultures of saccharomycetes on
gypsum blocks can be kept free from bacteria if exposed to
years ago by
light.
3.
The methods
Preparation of Pure Cultures.

for preparing pure cultures described in
the following pages are only those of practical importance
and
application.
Pure Cultures for Investigations in Morphology

AND Development. The preparation of pure cultures for
investigating morphology and development

early, the
the microscope.
(1821)
cell
was begun very
development of the single cell being observed under
later,
in the
examination
This was perhaps
first
done by Ehrenberg
Mitscherlich studied the budding of the yeast
same manner.
as
The process
a means of
studying
of
microscopical
morphology and
development was largely employed in investigating fungi

belonging to the most widely separated divisions of the
system.
The technique was brought
perfection, especially
by Brefeld
7*
to a high degree of
(1875).
100
Brefeld's Glass Slip Cultures.
method
this
is
essential point in
mass of
spores, or a small
spores,
from the mould in question,
a Mucor, with the aid of a fine needle
them
in a
drop of
all
Brefeld takes a fruit carrier with
stages of development.
e.g.,
The
the direct microscopical observation of
sterile
water
and distributes
he continues diluting until
only one or two spores are present in any drop, and one of
these drops
then placed on a glass
is
He
slip.
nutrient solution, the position of the mould spore
then adds
marked,
is
and, to prevent evaporation, the glass slip culture
under a moist
investigation
by
when not under
bell-jar
is
medium
is
replaced
These Brefeld glass
gelatine to prevent evaporation.
which
is
kept
If the
observation.
prolonged, the culture
slip cultures are quite open,
is
some importance in
of
the investigation of the higher fungi as they have then

sufficient
room
way exposed
growing the
for
to infection
culture,
from the
air.
this danger, a paper shield is placed
however,
In order to mitigate
e.g.,
way
of little importance
it is
be made, as the culture

the whole time.
is
is
those of
excellent in
its
not found a place in this book,
all
trary,
is
to lead to
the more so as they have
(See the moist
68.)
If the pure culture, on the con-
when
the former
is
designed only
morphological and developmental purposes.
latter case it is of
used
an absolutely pure mass culture, other
considerations arise than

for
subtilis
The
work and have
been recently replaced by a better design.
chambers mentioned on page
own sphere.
Recklinghausen.
v.
latter are only suitable for morphological
Pure Mass Cultures.
no error can
under microscopical observation
This technique
viz.,
But
a Penicillium spore, find their
Brefeld in his investigations on Bacillus
moist chambers,
in this
on the microscope.
even when foreign germs,

into the culture,
is
no importance
if
In the
a foreign organism
is
present along with those being cultivated, for the whole

investigation
101
carried out on the stage of the microscope
is
under continued observation.

the physiological experiment
taining mass cultures
It is quite otherwise
another technique
when
carried on with flasks con-
is
here necessary.
is
These pure culture methods are divided into two groups,

one comprising those based on the principle of dilution,
whilst in the second the physiological behaviour of the
species forms the basis.
method, and that in
It
undoubted pure culture
is
only by use of the dilution
most developed form, that an
its
is
medium and by
Such a culture
obtained.
prepared by sowing out a single
cell in
I.
way
further cultivation in such a
foreign organisms are able to force their
The Dihiticm Methods.
is
a sterile culture
way
that no
in.
Dilution methods may be again
divided into two groups, according as the dilution takes

place
nutrient liquids or
in
nutrient liquids the method
germs in a certain unit
solids.
is
In the dilution of
to count the
of volume,
and then
a calculated quantity of the liquid until there
per unit of volume.
pure cultures of a
Hansen
perfection
is
of
with
one
cell
This method was used by Lister (1878,

lactic acid bacterium), Nageli, Fitz
The method was brought
(1882).
number
to dilute
and
to its greatest
by Hansen.
Hansen's Dilution
Method.
The
dilution
method as
used by Hansen's predecessors was quite uncertain.
It
known whether those flasks in which a

was
growth was developing contained a pure culture or not,
never really
i.e.,
whether the seeding consisted of one or several
The counting method
in use
was not exact
cells.
but even with
exact counting the seeding might consist, in certain flasks,
more than one cell. Therefore, Hansen added to the

method two elements, by means of which it gained in
of
certainty, viz.
(1)
an indication
to
decide
whether the
infected flask has received one cell or several; and (2) an
102
Hansen applied the

cells.
The yeast (Hansen's method can only
be used for the heavier cells, and thus not for bacteria)
was mixed with sterile water, in which the cells were disA drop was
tributed by continued and violent shaking.
aid to the exact counting of the
following method
taken out, and the number of
cells in
a drop determined
by means
of the squared cover glass (Fig. 4) described
page 30.
The squares
sible
to perform
of the cover glass rendered
on
pos-
it
The mixture was
an exact counting.
thereafter diluted to such an extent (by calculation) that
there was, at the most, one
cell
in every
two drops.
drop of the mixture was then sown in each of a series of

flasks containing wort, after
which the
flasks
were shaken
some time in order to separate the cells,

in case it should happen that more than one had been introduced they were next set away and left undisturbed,
up vigorously
for
so that the cells could sink to the bottom.
Those flasks
which only a single yeast spot (colony) formed had

thus received only one cell, and contained an absolutely
in
pure culture.
This,
which
later
formed the starting point
for Koch's plate cultures in nutrient gelatine, constituted,

in conjunction with the direct counting of the cells present
in the drops, the exactness of the method.
this
method Hansen prepared the
first
By means
of
pure cultures of his
Saccharomyces species.
In order to control the exactness of his method he

instituted
special
species of yeast
experiments in which he mixed
two
which could easily and with certainty be
distinguished from one another under the microscope
mixture consisted of Saccharomyces
cerevisice
Pastorianus with Saccharomyces apioulatus.
monstrated the exactness of the method
the
or Saccharomyces
The results dewhere only one
yeast spot had formed there was but a single species in

the flask.
PUKE CULTURE METHODS

The
direct
sowing out of a single
cell can,
done also with the aid of the squared cover

the easier gelatine
103
of course, be
Since
glass.
method described below has been placed

method is only used in
at our disposal, the above dilution

isolated cases.
This happens,
when
number
the
when it is intended
much emaciated cells,
e.g.,
prepare a pure culture from very

of living cells
is
to
or
to be determined in
a growth of which most of the individuals have died.
Emaciated
in
wort
Dilution
medium
in
mentioned above, do not develop at
as
cells,
gelatine,
and on Solid Substrata,
When
used for dilution a plate culture
is
all
but do so in wort.
Schroter was the
first
to
introduce
them
observed, on slices of potato exposed to
is
a solid
prepared
He
(1872).
air,
gradual
formation of spots of different shape and colour, produced
by the
bacteria
in
the
air.
On
investigation
of
these
spots he found that each, as a rule, contained only one

species.
R.
Koch used (1881)
solution
for
gelatine
mixed with a nutrient
the preparation of pure cultures.
tributed the germs in the solidified gelatine
by
He
dis-
by inoculation
The number of germs introduced into the

becomes less and less for every additional streak,
streaks.
gelatine
so that the colonies in the last streaks are isolated ones.
But
it
does not follow from this that they contain pure
cultures.
Koch's
Plate
Koch in the year 1883 reby his plate cultures and obtained
a more complete separation of the
Culture.
placed his streak method
by means
cells.
of the latter
This method consists in distributing the germs in
liquefied
gelatine,
the mixture being
horizontal glass plate, which
is set
and protected from outside germs.
poured out on
under a moist
The
cells
bell jar
are fixed
by
the solidification of the gelatine, and colonies develop, which,
104
in the course of a
The number
few days, become
naked
visible to the
eye.
of cells in the gelatine should not be too large
room
there must be sufficient
for the
development of the
Instead of pouring out the gelatine on a glass
colonies.
plate, Petri dishes (see Fig. 37, p.
now commonly
64) are
As, however, the dish does not afford
complete security against infection, special precautions must
The procedure
be used to ensure safety.

of the
little
growth
various organisms,
is
as follows
is
mixture of
to be separated, usually a
placed in sterile water, for example in
a Freudenreich flask or better in an ordinary globular flask

(see Fig. 36, p. 63), in
distributed
by shaking
which the
;
it is
cells in
the water can be
here necessary to separate and
Some
distribute the cells in the water as well as possible.
nutrient gelatine contained in a similar flask
water bath heated to about 35

liquefied
small
quantity of the latter
gelatine,
and
this
When
C.
and the water mixture

is
placed
The
melted on a
the culture
is
shaken up, a
sufficiently
the liqueiied
in
mixture now well shaken up, care being
taken, however, that no air bubbles

gelatine.
is
flask containing the
with a plug of wadding
mouth
the
formed
are
gelatine
is
in
the
provided
of this flask
is
put
and simultaneously the cotton wool

plug removed by forceps and again replaced.
After cooling,
(juickly into the flame
the plug
is
again taken out and the liquefied gelatine mix-
ture poured quickly into a Petri dish which

closed with
its
cover.
The dish
undisturbed, until the gelatine

ture
is
is
is
must be quickly
then allowed to remain
quite firm
then the cul-
brought up to the desired temperature.
It is not
advisable to place the dish with the liquid gelatine on ice in
order to cool
it
more quickly,
as the air then passes in too
quickly, and outside germs easily find their
way
in.
The
simultaneous preparation of several plate cultures with
varying additions of the growth of organisms
is
to be

recommended.
105
Beginners usually get far too
many
cells
in each plate.
It is of assistance in the investigation of the
water mixture
if
The preparation
out in the
a counting
is
done under the microscope.
of plate cultures should always be carried
sterile
chamber.
This pure culture method
is
men-
specially adapted, as
tioned before, for the separation of the various elements

of mixed cultures.
There
well.
is,
It has,
however,
for instance, no security that the developed
colonies arise from single
cells.
Hansen tested the Koch method
own
disadvantages as
its
in the
same way
as his
pure culture method, following the same procedure,
and using the same yeast mixtures
were mentioned on
as
page 102, and prepared some plate cultures by
The result was that
1*5 per cent, of the colonies
its
means.
were formed
of both species, while the remaining colonies were pure
Holm found
cultures, either of the one or the other.
the source of error
is
usually larger
that
he carried out a
thorough research, the result of which was, as regards the

yeast
cells, that,
from 108
cells.
on an average, 100 colonies were formed
He
difficult to separate
found, further, that the cells are
more
from one another at the beginning of
the fermentation than at the end.
Therefore the error
is
made with cells in the latter

other
hand,
large number (25 5 per cent.)
on
the
a
But,
stage.
of cells do not then develop on account of their weakened
condition.
This number is reduced to 4'5 per cent, if the
Wort
cells are taken at the beginning of the fermentation.
smaller
gelatine
if
the plate culture
as
compared with wort
favourable to development.
an impure brewery
it
is
If
it
is,
is
on the whole,
less
wished to separate
yeast, so as to isolate the culture yeast,
should be noted that the wild yeast preponderates at the
end
of the fermentation.
Miquel carried out similar investigations with respect to
106
bacteria
he found that 100 colonies were formed from 134
thus giving a
cells,
still
more unfavourable
Surface Plate Cultures,

culture
The
is
result.
modification of the plate
the so-called surface plate culture (W. Kruse).
process consists in pouring the melted gelatine (before
infection) into a Petri dish
and
become quite
allovsring it to
After complete setting, a suitable quantity of the
firm.
water mixture containing the organisms
is
placed on the
and spread out carefully over the surface by means
gelatine
The
of a sterile platinum brush (see page 71).
that
all
is
the developed colonies can be easily taken out.
Second
Hansen's
Pure
Method.
Culture
same year, 1883, that Koch introduced

described above,
ture
result
method
Hansen worked out
for yeast
cells.
In doing
In
the
his plate culture
his second pure culso,
he took advantage
made by Koch, viz.,

but he added a new element to
of the technically important step
the
use of culture gelatine,
the
process in controlling the development of one cell into a
colony under the microscope.
made
absolutely certain
starting point.
On
Only by
this
that the single
the other
hand
this
cell
means
is
it
forms the
method cannot be
used for most types of bacteria, as they are too small to

be observed singly in the gelatine.
The method
is
as follows
suitable mixture
from the yeast growth by means of
number
the
liquefied
of cells after
wort gelatine'
is
made
sterile water, so that
mixing a drop of the mixture into
is
not too great (see below).
however, requires practice.
This,
Some information may be
obtained, as mentioned above, from the microscopical ex;
the approximate
by
aid of a squared
amination of a drop of the water mixture
number
'
of cells in the drop is counted
At ordinary temperatures a 4 per cent, culture gelatine can be used.

in the course of seventy-two hours in such a
that they can be removed.
The colonies then develop
way

cover glass
it
can then be calculated about
107
how many
drops of the water mixture are to be added to the gelatine,
number of
This number
in order to have a convenient
cells in
of the gelatine mixture.
is
if
a Bottcher chamber with a ring of 30
But
used.
after some time
one drop
20 to 30
cells
mm. diameter
is
will be possible to prepare
it
the proper mixture without counting or calculation.
When
a suitable mixture of yeast and sterile water has
been thus prepared, several Bottcher chambers are
sterilised
and placed under a sterile bell jar or sterile

protect them from dust all these experiments
in the flame
beaker to
A very
are carried out in the sterile cupboard.

of sterile water
is
small drop
then placed on the bottom of each
chamber and some vaseline melted in a small saucer over a
The edge
flame.
of the ring of the Bottcher
chamber
is
painted with the melted vaseline, the latter substance being
used because, after solidifying, a completely homogeneous
mass without
air bubbles is
glasses are then flamed

bell jars
gelatine
is
obtained
The
requisite cover
and likewise placed under
or small beakers.
sterile
Finally a flask with wort
placed on a water bath at 30 to 35 C. to liquefy
the gelatine.
little
of the yeast water mixture
is
mixed with the
proper quantity of liquefied wort gelatine in a globular
and after being shaken vigorously, the formation of

air bubbles being avoided, a drop is taken out with a thin
glass rod or a fine pipette and spread out in a thin layer on
flask,
the cover glass.

jar for
The
some minutes
latter is left
under the
until the gelatine has set.
placed with the gelatine layer
sterile bell
It is
downwards on one
then
of the
Bottcher chambers and pressed firmly round the edge so

that the vaseline closes
it
completely.
The edge
is
with a melted mixture of 2 parts of vaseline and

of
wax
to prevent the cover glass slipping.
painted
1 part
The Bottcher
108
chambers are often
way
such a
with
and the ring
fish glue,
by means
slip
mentioned previously, in
set up, as
that the cover glass
is
fixed to the loose ring
then fastened to the glass
is
of vaseline or with a
wax and
mixture of
vaseline.
We
now
proceed
to investigate the
Some
too great a magnification.

finding the cells
and
it
deeply embedded
isolated cell
either
is
found,
by means
scale or fixed
When we
is
desired,
its
its
position
of the object
mark
have
bj^
(see
required in
is
to investigate
whole thickness, so that any
may not escape
by the use of squared cover

a
practice
must not be forgotten
the gelatine layer through

cells
chambers with not
is
notice.
marked
When a
well-
this is
done
marker of Klonne and
glasses, or
by using
pages 28, 30 and
some means marked
Miiller,
a stage with
33).
as
many
cells as
and have convinced ourselves that there are no
other cells in their immediate neighbourhood, the chambers

are put
away
at the temperature of the
If precautions are not taken,
what
To prevent the formation
at 25 C.
water drops are usually formed
on the under side of the cover

of the gelatine and,
room or
is
glass, especially at the
worse, on the gelatine
edge
itself.
of these water drops, the chambers
are placed under a moist bell jar
brought to a temperature a
little
the chambers are to be kept.
which has previously been

higher than that in which
is
advisable to examine the
chambers after twenty-four hours
in order to confirm the
isolated positions of the
When
It
marked
cells.
the colonies have become large enough, they are
cautiously transferred to the nutrient liquid
by means
forceps or by
(c/.
page 97)
platinum wire held by a pair
either
of a piece of
of
a very thin glass rod, the point of
which
can be easily broken off in the liquid to be infected.
In order to be able to use the object marker of Klomie
and
Mtiller,
already mentioned, the objective
is
un.screwed

and the point
10&
the object marker coated with a dye
of
Since the chambers are often placed under a
solution.
moist bell jar as described, a colour must be chosen which
Holm found
can withstand moist air and does not spread.
may
that a suitable colour
be prepared from 0"25 part of
fuchsine dissolved in 2'0 parts of aniline and mixed with

2'0 parts of a xylol solution of
Canada balsam.
drop of
spread in a thin layer on a small glass plate
this colour is
such as a glass
The point
slip.
of the object
marker
is
now
pressed against the medium, so that the edge of the opening

is
Care must be taken that the colour
distinctly coloured.
film does not spread over the opening;

it
this
if
happens
can be easily removed by blowing through the other end
The
of the apparatus.
object
marker
is
now screwed on
to
the tube of the microscope, and so adjusted that the point
The
almost touches the cover glass of the moist chamber.

tube
is
then screwed downwards very cautiously for a small
distance
by means
of the micrometer screw so that the
point of the object marker touches the cover glass, with
which
it
is
allowed to remain in contact for about ten
seconds, after
which
it is
stamped on the cover

cell
under observation.
raised again.
It
is
red ring
which
glass, inside of
is
not advisable to
marker on to a revolving nose-piece,
is
thus
the isolated
fit
the object
for it often happens that
the field of view of the objective and the opening of the

object
lies
marker do not exactly
coincide,
and the
cell
therefore
outside the coloured ring.
Sometimes
it is
little difficult to
mark
distinctly
on the
by
cover glass. This is
marker not being quite plane and partly by the coloured
Under such cirliquid not having the right consistency.
caused partly
cumstances
it is
found that the best
the point of the object
way is
to allow the first
layer of colour placed on the point of the object marker to
dry on and then
to
apply a new
layer.
The
first
dry layer
110
then acts as an
The use
elastic cushion.
of this apparatus
requires practice and, as maj' be inferred, a light hand to
avoid breaking the cover
Squared cover
may
squares,
In the
first
above, the isolated
page
the
numbers
fixed point
find the cell again.
is
set
made
up as described
in the squares (see Fig.
6,
number and the position

number is drawn on a piece
of
its
with respect to this
cell
is
being marked in the following
cells
If there are
30), the square with
paper.
case the squares can be
The chamber
larger than in the latter.
with or without numbers in the
be used, as already mentioned, instead of the
object marker.
manner
glass.
glasses,
thus obtained and
If there are
it
of
will be easy to
no numbers in the squares
page 30), each square can be designated by means

two numbers, the one being the number of the horizontal
row, the other the number of the vertical column which
contains the square.
For example 3, 4 means that square
(Fig. 5,
of
which
lies in
one
cell
row and
the third horizontal
in the fourth
In this case there must, of course, be only
vertical column.
in each square.
Lindner's
Droplet
Culture
The methods described
above can, of course, be varied in several ways

these, for instance,
is
one of
Lindner's droplet culture (1893).
He
diluted a wort culture until only one cell was found in every
streak or dot which he

glass.
The cover
glass
made with
drawing pen on a cover
was then turned over and fixed with
vaseline on a hollow glass slip or on a Bottcher
and the preparation examined microscopically.

lets
showing only one
cell
upper side of the cover
chamber
Those drop-
were marked with ink dots on the
glass.
After a few days the growths
have developed and those droplets which contain the growths

originating in one
cell
are sucked
piece of sterile filter paper
gelatine in a tiask,
up by means
the latter
is
and a drop of wort
of a small
then placed on wort

is
added in order to
Instead of taking up the drop on
accelerate development.
filter
paper, a
little
111
may
gelatine
be added; the whole
is
then taken up on a platinum wire or similar instrument
and introduced
with a
liquid,
Lindner accordingly begins
into the wort.
then uses gelatine, and only after a growth
has formed on the gelatine are the mass cultures prepared

in flasks containing nutrient liquid.
Schonfeld's Method,
Of
the dilution methods that of
Schonfeld remains to be described.
According to him a
is prepared by means of liquefied culture gelatine,

and small spots are placed on a cover glass from this gelatine
mixture by means of a drawing pen.
Each spot ought, as
dilution
far as possible, only to contain one

is
further considered
it
cell.
When
this
method
ought
will be seen that the spots
only to be so large that the whole of each
may
be in the
view when using a medium magnification, and
field of
it
more gelatine so that

the small gelatine spots may not dry up and in order that a
growth may take place at all. The method is, as may be
seen from the foregoing, a combination of those of Hansen
and Lindner.
found necessary to add a
will be
IL Physiological Methods.
organism which
sent
in
is
little
It
often
happens that the
to be cultivated in a pure state
comparatively small numbers, and the
is
mentioned dilution methods cannot then be applied.
must then
resort to a physiological method.
however, are by no means exact, and
otter
pre-
above-
We
These methods,
no certainty of
obtaining a perfectly pure culture.

Fractionated
method
Culture
in general use
bacteriologists
Others.
the older physiologists
and
after
among
was a combination
Klebs
and
of an imperfect dilution
method with a physiological method. Klebs's method, the

so-called fractionated culture, is an example of this, as it
consists in inoculating
new
sterile culture liquid
with the
112
previous culture as soon as that has developed.
way
Klebs expected to obtain
species present in greatest
finally a
numbers
preponderance of one species

the use of the method
therefore a condition
may
not survive best, so that
questionable in the case where a pure culture
whether
is
for
any
culture obtained will, at
rate,
it is
obtained
The pure
consists of the species looked for.
it
The
at the beginning.
but that species which was in the
majority to begin with
is
In this
pure culture of the
belong to that species
which increases most strongly under the prevailing conditions
which
but this will not in every case be the species of

desired to obtain a pure culture.
it is
Method
Pasteur's
Pasteur (1876) also employs this
He
physiological principle.
gives
some
indications as to
the possibilities of obtaining a pure culture, by making use

of the various physiological properties of micro-organisms,
but chiefly of the greater or

different culture media, or,
less
if
capacity of increasing in
the culture liquid
is
an un-
favourable one, of the greater or less resisting power.

struggle between the species
possibly the weaker
species, too,
is killed,
but this
is
not certain
peaceably together, and on this account also
for
the
medium,
is
obtained.
it is
in order to favour the
cases
uncertain
development of one organPasteur's use of tartaric
may
be instanced
affords a distinct proof of the uncertainty to
may
a method
which
live
This holds in general
acid in the preparation of pure yeast
of
those
addition of chemical substances to the culture
ism at the expense of the other.
it
which are equally strong will be able to
whether a pure culture
thus brought about, in which
is
it is
lead.
desired to
make
unknown, the method
reason that
species are
it
which such
Since the properties of that species
is
a pure culture are in
most
also less attractive for the
simply assumes that the properties of the
known.
The above holds
also for the
employment

of certain temperatures for the
method
logical
paratory one
is
The physio-
same purpose.
only important in so far that
single cell culture
113
it is
must be employed
a pre-
for the
preparation of a genuine pure culture.
Pure Cultures of Bacteria.

In the foregoing we
have had chiefly in view the pure culture of yeast cells
with regard to bacteria, the physiological method may be
;
employed
in general for a preparatory cultivation, otherwise
Koch's plate culture
is
the most suitable means,
since
bacteria are too small to be recognised with certainty in an

isolated state in gelatine or liquids.
The
plate culture
is
repeated several times, the starting point each time being
from a colony
The probability
in the previous plate culture.
of obtaining a pure culture increases with the

plate
cultures.
means
affords a
of determining
been reached, as this

species
number
of
Sometimes the appearance of a colony

is
whether a pure culture has
often characteristic of the single
microscopical examination of the
cells also assists,
of course, in the elucidation.
Pure Cultures of IWould Fungi.
In
order to prepare
a pure culture of mould fungi, a single sporangium

of
Mucor) or the conidia of a single conidiophore
of Penicillium) is touched with
sterile
needle.
{e.g.,
{e.g.,
The
adhering sporangium spores or conidia are then inoculated

into a nutrient
medium such
an additional step
may
as
wort or wort
gelatine, or
be taken, the spores being dis-
tributed in water and a plate culture in wort gelatine
prepared.
In the foregoing, reference has been made to
Brefeld's investigations on the development of
mould
fungi.
In his treatises are to be found very valuable directions on

this point.
114
Methods of Preservation.
4.
and Moulds,
Yeasts
Method
Saccharose
Hansen's
of
It
for
Preservation
the
of great
is
importance in
physiological fermentation laboratories to be able to keep
pure cultures of the various micro-organisms in such a
way
new
that
it is
culture
not necessary to
medium
make
frequent additions of
Hansen
preserving yeast fungi and many
in order to
has elaborated a method of
keep them
living.
mould fungi,
viz., by storing in a 10 per cent, cane sugar

As regards yeasts the process is as follows A
strong young growth of the species of yeast to be preserved is
cultivated for twenty-four hours in wort at 25 C.
The top
solution.
liquid
is
all
poured
off
from the settled yeast and a small
quantity of the latter placed in a 10 per cent, aqueous solution

of saccharose,
which
is set
away
in a flask at not too high a
Ordinary room temperature
temperature.
working temperature, and
Hansen
if
is
the flask used
is
the highest
a Freuden-
must be kept in a dry place so

that moulds may not grow through the tube of the cap.
The evaporation of liquid in a Pasteur flask is quite insignificant some cultures in saccharose have been kept in
reich or a
flask, it
Pasteur flasks in the Carlsberg laboratory for more than
twenty years, as previously mentioned, without showing any

noteworthy evaporation of
the other flasks
is
in the cap tube
is
liquid.
The evaporation from
also tolerably small,
not kept too
loose, the
proper length and the cap fitting well.
if
the cotton wool
tube being of the

Flasks of the latter
kind can also be kept for several years without a renewal

of liquid being necessary,
beginning.
So
(Gf.
pp.
61,
if
they are well
filled
at the
62.)
far as the saccharomycetes are concerned the vitality
appears to be almost unlimited

in the above manner.
if
Numerous
the preserving takes place

cultures of these
have been
METHODS OF PRESERVATION
kept alive for more than twenty years.
taken
place,
and then only
few
in a
has thus given excellent results.
115
Death has seldom
species.
It has been
This method
recommended
that the pure cultures should be allowed to stand in the
fermented wort, that
But
it
is
in beer,
and preserved in
has been shown that there
is
great
variations.
saccharomycetes
therefore of
cases
little utility as
^this
holds
for
Beer
years.
is
a preserving medium, since the
must be renewed every two or three months
cultures
all
in beer only for a
several
for
when
life
used being subject to
is
The same species

lives in some cases
few months, in other
way.
then no certainty that
the growths will be kept alive, the duration of
such a method of preservation
this
of course, almost impossible
do
to
in
this
it is,
an ordinary
laboratory.
been advanced against the saccharose preservation
It has
method that the saccharomycetes increase in it and form

yeast rings and films, of which the cells vary in morphological
and
from the original seeding,
phj'^siological characteristics
and give a progeny which' inherit these new properties.
On
investigating the numerous old saccharose cultures of
the Carlsberg laboratory, the author arrived at the result

that yeast ring and film formation
saccharose solutions
when
only take place in
the seeding, and therefore also
According to Hansen's
the increase, has been too great.
researches the increase from a scanty seeding in a saccharose

solution
is
limited, but
if
a larger
amount
of yeast
is
seeded,
a vigorous increase, as mentioned, takes place, accompanied
by the formation of films and yeast rings even when the

yeast is washed beforehand this can occur, the stronger
Under these
cells living at the expense of the weaker.
;
circumstances
it is
possible that
numerous generations are
and that thereby a

The author obtained proof that the
cultivated under abnormal conditions

variation
may
ensue.
8*
116
above views are correct by instituting a comparative series
German bottom yeast
of experiments with culture yeast (a
and Sacch.
I.)
cerevisice
and wild yeast
{Sacch. Pastorianus
In no single case was the smallest trace of a yeast ring
I.).
or film observed,
when
to a trace
becoming turbid; but the contrary
only, the liquid just
took place in nearly
amounted
the seeding
where 5
all cases
to 7 drops of
bottom
washed or with adherent culture medium, were

Freudenreich flasks. Experiments with two of
yeast, either
placed in
Hansen non-sporulating varieties of Sacch. cerevisim

Sacch. Pastorianus I., which had been seeded in the
same way, demonstrated that these forms which had lost
the power of film formation simultaneously with the power
the
I.
and
of spore formation, naturally
rings even
the
when
formed neither
But when
the seeding was considerable.
surface of the
saccharose solution
with the scanty
seeding of the original forms as well as of
was examined macroscopically, single
Now
about.
for, first,
do these
cells
cells
nor yeast
films
varieties
its
were seen floating
belong to the surface film
No,
the macroscopic film and the yeast ring are wanting,
and, secondly, these single surface cells
a,re
found in the non-
sporulating varieties named, which form absolutely no film,

as well as in the original forms.
Thus there
no question
is
here of a film formation, but only of difi'erences in the specific
gravity of the single

to sink to the
There
is
cells
therefore
some
of the cells are too light
no danger of a yeast ring or
in the saccharose cultures,

in the
bottom of the denser saccharose solution.

forming
the seeding has been performed
if
manner described above.

large proportion of the moulds are capable of pre-
servation in a 10 per cent, saccharose solution.

e.g.,
film
for Mucor, Aspergillus
and
This holds,
Penicilliuvi, besides for
other
fungi, such as Monilia, Oidium, Torula, Mycoderma, Dematium,
Cladosporium,
etc.
The
life
of these fungi also
may, ac-
METHODS OF PRESERVATION
117
many
cording to Hansen's researches, be prolonged for

years.
Preservation
on Cotton Wool or Filter Paper (after
When
saccharomycetes are to be preserved for
Hansen).
a shorter time, cotton wool or filter paper can, according
when
to Hansen, be used, especially

to be sent away.
yeast sediment
placed on
is
paper
wrapped up
is
used, a small
sterile
little
cotton wool in a Freudenreich or

filter
a pure culture has
small quantity of freshly cultivated
piece
Hansen
hygroscopic
folded
is
When
flask.
once,
then
and
in four or five thicknesses of filter paper,
the whole sterilised.
After this a few drops of the thick
yeast liquid are cautiously poured on the inner sides of the

folded paper and one of the coverings
when
wrapped round
the latter has absorbed the moisture
and replaced by a second covering, and
it
is
finally placed in the
This process must of course be per-
remaining coverings.
formed carefully so as to prevent infection taking

pure culture
envelope
months.
may be
transmitted in this
the duration of
way
life is limited,
pure culture
may
in
place.
also live longer
than in the
may
an ordinary
however, to a few
be preserved safely by the
above-described preparation on cotton wool, and the
kinds of moulds
it
removed
filter
paper covers.
cells
Also most
be kept alive for several years by
these dry preservation methods.
Preservation
of Bacteria.
tures of bacteria there
is
For
the Hansen saccharose method.

tories
preserving pure cul-
no method which corresponds
to
In bacteriological labora-
pure cultures are preserved on or in the particular
nutrient
medium in such a way that they are always renewed
after a certain time, a very troublesome process.
Bacteria
in the spore condition can, however, be preserved in the dry
condition in
many
cases.
tions acetic acid bacteria
According to Hansen's investiga-
remain alive in beer for several years.
118
Preservation of Ordinary Brewery Yeast.
In
tion with the preservation of pure cultures, a
may
be
also
brewery
said
about
preservation
the
connec-
few words
of
ordinary
Experiments were made and methods
yeast.
described more than 100 years ago concerning these more
Thus, the yeast was mixed with
or less impure mixtures.
ashes and the moisture removed from the mixture

of a cloth, or the yeast
verised
wood
by means
was mixed with sugar or with pul-
or animal charcoal and the mixture then dried.
Beer in a cold
cellar
was
also
employed as a preserving-
medium.
The
O. Reinke described a method some years ago.
well-washed and quickly pressed yeast

enclosed in
is
two
then pressed
is
very rapidly
The yeast
sheets of sterile filter paper.

flat,
rolled
up again
in a sheet of ordinary
white blotting paper, sprinkled with traces of sterilised

boric
acid,
plates to
and then pressed between
The
remove the water.
sterilised asbestos
latter are subjected in a
hermetically closing metal box to a strongly cooled air

current, sterilised
and dried by concentrated sulphuric
acid.
After thorough drying the packets are arranged in a metal
way
is
surrounded with a
layer of cold sterilised burnt gypsum.
Finally the metal
receptacle in such a
that each
boxes are soldered up.

Will
has also
made experiments
of
washed and pressed the yeast and mixed

the
following substances
scraps of
filter
paper,
kieselguhr,
wood
this
it
kind.
He
with one of
asbestos,
gypsum,
wood shavings
especially the wood
charcoal and
the two latter gave the best result,
The drying was done as quickly as possible on

an oven at a temperature between 25 and 48, being begun
shavings.
at the lower
When
temperature and continued at the higher.
the yeast
was dry
it
was
filled
into tin boxes
were hermetically soldered and stored
at a
which
temperature of
TRANSMISSION OF YEAST
2 to 7 C.
Some
contained living
119
of the specimens preserved in this way-
cells after
the course of eleven years, this
Heron
happening both with culture yeasts and wild yeasts.

has also described a method quite recently.
In
all
yeast the
these methods for preserving
common brewery
washed and
Different experi-
latte)- is
pressed.
menters perform the drying in somewhat different ways.
Whichever
it
may
be,
the entrance of bacteria and other
foreign organisms during these manipulations cannot be
Fig. 47.
Section through the
side tube of the Hansen
Transmission Flask showing the method of closing.
Fig. 46.
Hansen's
of
Flask for the transmission

pure yeast cultures.
Even when one begins with
avoided.
the pure culture apparatus, a yeast
which
is
always more or
less
Transmission of Yeast.
is
a pure yeast from
obtained in the end
contaminated.
In
sending small samples of
yeast the methods described on page 117 using cotton wool
and
filter
paper are taken advantage
of.
tity of pure yeast culture is to be sent,
If a larger
which
in the liquid condition in the pure culture apparatus,
recommends the employment
quan-
will be used
Hansen
of a flask the appearance of
120
may
which
The Hask is made

bottom.
The yeast
be seen from Fig. 46.
strong thick glass and has a
flat
of
is
passed into the flask through the side tube, after which the
latter is closed.
when
closed
with a small
Fig. 47 gives a section of such a side tube
c is
the tube, the
collar, b
mouth
which
of
provided
is
a tightly closing rubber stopper, a a
strong rubber cap tightly stretched over the stopper, being
The binding may be seen

fit exactly and
fastened at d by copper wire.

at a (Fig. 46).
The rubber stopper must
be easy to take out after the
cap
To add
removed.
is
to
the security the bent tube
is
divided into two parts, which

are
connected
tube
the glass tubes
pletely close
with
filled
is
up the
poured
b is
and
glass
tube, c
filter
ofi'
when
is
and
the
through the
Both the rubber
side tube.
by wire
cotton wool
used as an air
yeast
Jorgensen's Metal Flask for

the transmission of pure yeast
rubber
fastened to
is
pinchcock which can com-
Fig. 48.
by a
the latter
tubing should be
sterilised separately.
cultures.
glass flask naturally has
advantages over a metal flask as the contents are
which
in
is
working with
of importance both in
it
visible,
and
also
the transmission of yeast through the Customs, as
now
so frequently
done
but
breakage during transmission
For
this
is
it
if
is
exposed to the risk of

the
packing
is
faulty.
reason metal flasks have been frequently used
of late as substitutes.
Such
Alfr. Jijrgensen represented
for instance
in Fig. 48.
exactly the same as that of the
Hansen
is
the flask of
The
flask.
principle
is
SPORE CULTURE METHODS

The above-mentioned transmission
chiefly
121
flasks are constructed
with regard to brewery requirements.
Only small
glasses or bottles are necessary for wine fermentation, as

is
it
then generally a question of sending small quantities of
yeast.
5.
Spore
Preparaticm of Spore Cultures.
Cultures
of
Saccharomyces.
Even
at
the
present day the statement sometimes occurs that a spore
formation will be produced merely by sowing a
on a moist gypsum
little
yeast
block, a potato, or slice of carrot.
In
so far as the conditions of spore formation are treated, quite
now
incorrect statements are even

e.g.,
not infrequently made,
that the yeast should be well washed beforehand, that
the whole process depends upon a starving condition,
The
etc.
old error of the formation of spores at a low tempera-
ture
is
now
l&ss
frequently met with.
stances just mentioned
it will,
Under the circum-
however, depend on chance,
as regards most species of Saccharomyces, whether they form

spores or not.
The essential part of the method does not consist in the use
of any particular substratum, such as gjj^psum blocks, potato
The substratum on which the cultivaor carrot slices.
tion proceeds is in the main unimportant. A shallow layer
of water in a culture
flask, gelatine, etc.,
equal advantage, as will be shown

lies
later.
simply in the use of a moist surface.
io the method that
we
should
know
favouring the function in question.
may
be used with
The
But
chief point
it is
essential
the best conditions for
These conditions were
ascertained by Hansen and the essentials published in

(A
1883, additions being made in later communications.
more
is
detailed explanation of the physiology of this function
given in the next section.)
According to these investiga-
tions a copious formation of spores takes place,
if
(1)
the
122
growth
consists of strong
ture
employed
is
young
cells
the supply of moist air
as follows
C.)
and
wort
flask containing
of spore formation
infected with a
is
small quantity of the yeast species in question, shaken
and
placed in a thermostat at 25 C.^
is
hours sufficient
gypsum
the seeding out on the
supernatant fermenting wort
up
In general a quantity
of yeast sediment forms in twenty-four

to perform
(3>
is plentiful.
The technique of the Hansen method

is
a high tempera-
(2)
most species about 25
(for
poured
off,
block.
The
and a small
quantity of yeast taken out by means of a pipette and
gypsum block
spread in a thin layer on a dry, sterilised

in a glass dish (see
make
Fig. 38, p.
the yeast layer thin
It
65).
of the air has no access to the lower
the layer of. yeast
water
is
is
is
important to
with thick layers the oxygen
spread on the
cells.
As soon as
gypsum
block, sterile
poured into the dish until the gypsum block
immersed
is
to about two-thirds of its height.
The addition
made by means of the water

holder described on page 67 (Fig. 40).
During this manipulation the cover of the glass dish must not be raised any
higher than
is
of
water
is
necessary and the whole operation must be
done as quicklj' as
possible, because
gypsum
are very easily infected from outside.
operation
As soon
is
as the
recognised
is set
skilfully
by
away
The use
done the infection
gypsum block
is
block cultures
However,
is
if
soaked with water, which
at the desired temperature.
of
gypsum
'The culture instead
blocks
was proposed by Engel.
it is
e.g.,
of standing for twenty-four hours at 25 C. may

hours at the room temperature. If the growth is old
advisable to freshen it once or twice at the ordinary temperature before
used.
In determining spore curves this has always to be done.
left for forty-eight
it is
is
the glistening of the yeast layer, the culture
Similar substrata were recommended later by others,
be
the
inconsiderable.
SPORE CULTURE METHODS

earthenware cubes by Elion and
"
chamotte
123
"
blocks
by
According to experiments made by the author
Wichmann.
the latter are very inferior to the
gypsum
blocks, the for-
mation of spores beginning later and the number of sporecells being fewer than when gypsum is employed.
The porcelain cubes mentioned were found to be almost as
bearing
good as the gypsum blocks.

If
it is
desired to obtain a bacterium-free spore culture
of a Saccharomyces according to Hansen, a thin layer of
water
may
be worked with in a Freudenreich or Hansen
flask or in a moist
access of air
chamber,
e.g.,
Ranvier's chamber, with
or a seeding out on gelatine without addition
of nutrient substances
may
be also used.
Good
results
have
been obtained from shallow water layers in flasks and moist

chambers.
In
spores
many cases somewhat more copious formation of

may be obtained on the gypsum blocks than in thin
water layers
if it is
gypsum block
desired at the same time to protect the
culture from infection,
it
may
be placed,
according to Schionning, in a Hansen flask (see Fig. 39,
page
Sterile
66).
water
is
then added from another Han-
sen flask, the two side tubes of the flasks being connected.
The yeast, on the other hand, is put on the gypsum block

by means of a pipette through the neck of the flask.
It has been shown on page 99 that the influence of light
may
also be
employed in the preparation of bacterium-free
spore cultures of saccharomycetes.
The
spores of saccharomycetes
may
sometimes be con-
fused with other formations, especially with fat or
oil
drops,
(li the
which are frequently found inside yeast cells.
is treated with perosmic acid it may be easily
preparation
ascertained
if
the bodies in question are of a fatty nature, as
they then become brown or black.

also in alcohol
and
ether,
Fatty particles dissolve
and are again precipitated on the
124
The
addition of water.)
practised microscopist will,
ever, soon learn to distinguish spores
There
is
no
from other
decisive colour reaction for spores
usually stained
by means
how-
objects.
they are
of Ziehl's carbol fuchsine (see page
88) and retain their colour after the preparation has been
Experiments made by the
decolorised with dilute acid.
author have shown
by
stained
further that spores are sometimes not
this process,
uncertain.
and that on the other hand
may
other than spores
The mode
particles
Thus the method
be stained.
and germination furnish
reliable characteristics for distin-
guishing whether a particle
is
a spore or not.
Spore Cultures of Bacteria.
There
method for inducing spore formation
is
no perfected
in bacteria similar to
that which Hansen has described for saccharomycetes.
nearly
is
of formation, the anatomical structure
In
spore formation occurs with-
all species of bacteria,
out using any special method of cultivation, being brought
about merely by allowing the cultures to remain after the
substratum has become poor in nutriment or has become

unsuited for the growth from any other reason
(as, e.g.,
by
the accumulation of fermentation products).
Spore Cultures
of Moulds
Zygospore formation
a phenomenon frequently observed in the

section III.).
there
is
The conditions
of this are
therefore as yet no definite
Mitcorinece (see
still
method
is
unknown, and
of producing
it.
Bainier states that Mucor racemosus forms zygospores on
gypsum
blocks which are placed in dextrose solution.
The
author has tested this statement, but has obtained no

positive result.
In the MucorinecB and Aspergillece sporangia and conidia

respectively are always formed
when
the mycelium grows on
the surface of the culture medium, and
other respects in a
fit
when
the latter
condition to act as a food.
immersed in liquid forms neither sporangia nor
is
in
A mycelium
conidia.
FILM CULTURES
In
Aspergillus, ascospores are
]2.>
only known in those species
which are classed under Aspergillus glaucus and A. repens

(see Section III.).
Although the particular conditions of
known for these species, yet it is

easy to produce them, as such spores always form when the
ascospore culture are not
culture
is
allowed to stand.
In Penicillium glaucum, which likewise comprises several

species, a formation of sclerotia precedes the formation of
ascospores (see Section
by Brefeld by
conidia,
and placing this between two glass plates which were
pressed tightly together.
Sclerotia developed in the course
They were then washed and spread on moist
of three weeks.
filter
These sclerotia were obtained
III.).
infecting coarse bread, free from sourness, with
paper, after
which
asci
developed in their interior.
6.
In
unknown.
this species also the exact conditions are
Preparation of Film Gidtures of Saccharomyces.
Since film formation in the saccharomycetes plays a

considerable role in the characterisation of the species,
be necessary in
many
it will
cases to prepare film cultures.
The
conditions for a vigorous film formation are, according to
Hansen, the seeding out of a strong young growth on a

favourable culture
medium
to
which
air has free access,
and
the placing of the culture in complete quiet at a moderate
temperature.
Film cultures are best formed by seeding
out the Saccharomyces in an Erlenmeyer or Pasteur flask

half filled with wort, this being set
position at the
room temperature.
away
Hansen, Will and others
have determined the cardinal points
When
the optimum temperature
employed.
is
in an undisturbed
for
known
some
species.
this is of course
126
Gounting of Yeast Cells and Seeding with a Definite
7.
Number
of Cells.
sometimes necessary to determine the multiplying
It is
power of a yeast species under certain conditions, or

be intended to seed a certain quantity of yeast
a culture liquid.
the
cells,
and
For
this purpose it is necessary to
in doing so the following procedure
is
may
it
cells
in
count
adopted,
the details of which have been gradually evolved in the
Carlsberg laboratory.
For
counting in a liquid
cell
an exactly average sample.
it
is
required to obtain
If this is not obtained, then,
The average sample

is obtained by vigorously and repeatedly shaking up the
flask with the culture, and taking from it, by means of
of course, the counting
is
of no value.
a graduated pipette, a small measured sample, which
put into a test tube.
This operation
is
is
then
repeated.
withdrawal by means of the pipette must be done
The
(juickly,
so that the cells do not begin to settle before the sample

is
removed.
If the culture is in a flask
provided with a
side tube, the specimen can, of course, be poured out into
a glass from which small samples can then be easily taken
by a
it is
pipette.
desired to
As soon as the sample is withdrawn, and

retain unchanged for some time the number
of cells present at the
moment, the culture must be
away on
very low temperature
ice,
an increase
or at a
in the
number
may
of cells
set
otherwise
take place during
the counting, which requires an appreciable
amount
of time.
two samples are withdrawn, the one to check the other, and, for the same reason,
several drops are examined from each specimen.
Each
In order to obtain a reliable
sample
In
is
result,
treated as described in
many
what
follows.
cases the cells to be counted are in wort.
since cells present in
wort are hardly separated at
all
But
by
METHODS OF COUNTING
127
mere shaking, and whereas

to
this liquid is very inclined

form froth when shaken, and an increase of the cells
in the sample withdrawn must be prevented during the

counting, the samples are, according to Hansen, treated with
dilute sulphuric acid (1
part concentrated sulphuric acid
and 10 parts water). This furnishes, in addition, a liquid

in which cells do not sink to the bottom too quickly, an
important point when single drops are taken out for counting purposes.
Supposing, for instance, that a test tube
contains 3
of a sample of wort with yeast
c.c.
will be necessary in
most cases
The
1 c.c. of dilute sulphuric acid.
proceeded with further than

the observed
by the
plied
number
is
few
(or
cells
of
it
dilution should not be
absolutely necessary, since
of cells must, of course, be multi-
dilution coefficient,
consequently increased.
a,i"e
cells,
to treat this with exactly
Besides, the presence of too
many)
too
and experimental errors
increases
the
difficulty
of
counting.
In counting, the counting chamber described on page 32

is
employed
(see Figs. 7
and
8).
After the test tube with
the average sample and the sulphuric acid has been sub-
jected to a prolonged and vigorous shaking (this being
done most
easily
by placing the thumb over the mouth of

is taken out by means of a fine
the test tube), a sample
pipette as quickly as possible (before the yeast cells sink

to the bottom)
and a drop of the contents rapidly placed
the central part of the counting chamber.

often repeated that
rapidity
otherwise
it is
it
absolutely essential to
may happen
in
It cannot be too
work with
that the cells in the
pipette sink to the bottom and the drops then contain too
many
cells.
The cover
glass
is
put in place immediately the
drop has been deposited in the counting chamber.
ought
The drop
to be so large as to touch the cover glass, but not so
large as to be pressed out
by the cover
glass over the edge
128
into the surrounding space
this
if
happens the chamber
should be carefully cleaned, dried and provided with a fresh
As soon as the cover glass has been put in position^

the chamber is laid under the microscope, and if a hsematidrop.
meter
piece
"
being used as counting chamber, the
is
It is not advisable to use
required.
is
magnification than
the counting
is
is
net
a
eye-
"
greater
After waiting a short time,
necessary.
proceeded with when
the cells in the
all
The "net" eye-
preparation have sunk to the bottom.
piece consists, as described previously, of a large square
divided into sixteen or twenty-five smaller squares, the
The
latter being used as aids in counting.
the
square are counted
large
inside
cells
how
does not matter
it
the cells lying on the side lines of the square are counted,
if
the same
applies
of
rule
the
to
is
Many
The same also
followed.
counting of (apparently) dead
buds which are
cell.
always
still
in
may
squares in each preparation
by displacing the hsematimeter.
It is to be
always to count a certain number of squares,

in the middle
and eight along the edge
and
mother
cells
connection with the
be counted
recommended
e.g., ten
two
As
of the drop.
soon as these ten countings are performed, the hsematimeter

is
well cleaned and dried, the second test-tube well shaken
and then a drop taken from

manner. This alternation
is
is
it
and counted
same
repeated until a constant average
obtained.
In the following example, which
made by
apparent.
number
is
in the
the
author,
When
is
the
exactness
is
particular
viz.,
that of a
of
the
method
is
the
same unit of volume
column of liquid of which
the large square of the
magnification
from a counting-
not necessary to determine
of cells in a given volume, the
always employed,
the base
it
is
"
net " eye-piece for the
employed, the height being the
thickness of the perforated cover glass.
METHODS OF COUNTING
The mixture, 3
c.c.
of wort with yeast cells and 1
sulphuric acid, gave the following results
129
c.c.
of
130
and
of tube, magnitication,
make
each time in order to
When
"
net
eye-piece
"
must be used
a proper comparison.
number of cells is very large, dilution with

the sulphuric acid must be carried further, often to four or
five times the original amount of wort.
If the actual number of cells in a certain volume is to
be calculated, the size of the space unit must be determined.
It
is
the
then necessary to
liquid,
i.e.,
know
the height of the column of
the thickness of the perforated cover glass.
Hayem and Nachet
hsematimeter designed by
The
has one with
a thickness of 0'2 mm., but that in the Zeiss hsematimeter

is
mm.
usually O'l
The value
of the square in the
"net"
must further be known,

or squared cover glasses are used of which the size of the
squares is known.
In Thoma's chamber the column of
liquid is 01 mm. high and the large square etched on
The
the bottom of the chamber contains 1 sq. mm.
volume of the liquid prism, of which the base is the large
eye-piece for the magnification used
square,
is
When
water
is
thus O'l cubic

it is
mm.
intended to sow a definite
number
of cells,
usually added to the yeast to be used as sowing
material, the cells being thus
more
easily separated
from
one another on shaking; also no appreciable increase of

the
cells
takes place, especially
if
the flask
is
subjected
to a low temperature after the sample has been with-
The yeast
drawn.
is
therefore shaken
up vigorously and
continuously with sterile water, and an average sample
removed
in the
manner described above.
There are three
now considered, viz., (1) when we only

know how many cells are present in a certain
of the water-yeast mixture (2) when it is intended
different cases to be
wish to
p irtion
number of cells into

when it is desired to sow
seeding the definite number of
to inoculate a previously determined
the liquid to be dealt with

so
many
cells,
and
that after the
(3)
METHODS OF COUNTING
cells
desired
may
be present in an arbitrary space unit,
when making comparisons of

In the
species.
attention
is
of
it
of cells
is
it is
two
required to determine
which are
to be seeded,
know
only required to
and no
;
in
the relative
Finally the following must be remembered
to be a definite
is
cases
but regard must be had to the quantity of
cells,
liquid seeded.
If there
two
e.g.,
the multiplying powers of
paid to the quantity of liquid inoculated
the last case
number
first
number
the actual
131
volume
in the flask after seeding,
then, in the case where the seeding
not to be made in
is
water or where the concentration of the liquid is of some

account, no water must be used in shaking up the yeast.
In this case the same culture liquid must be employed.
The same quantity

from the
of
culture liquid
flask before seeding as will be
then
is
removed
added when seeding
takes place.
The procedure
in the
Thoma
haematimeter or in the
cells
above three cases
is
as follows
is
After shaking, a drop of the water
1.
is
placed in the
chamber, and the number of
On
determined in the usual manner.
seeding a
measured portion of the water mixture we thus know how

many cells have been sown.
2.
As above.
After the counting
it
is
determined by
how much of the mixture must be taken out in

number of cells may be sown.
As above. In counting we learn, for example, that
calculation
order that a definite

3.
a
to
cells
are present in a certain volume.
know
It is here necessary
the quantity of culture liquid in the flask to be
assume
inoculated
to seed so
many
this
cells
amount
to be
If it is desired
c.c.
that there will be aj cells per unit of
volume, the number of cubic centimetres x of the wateryeast mixture, which must be added in order to arrive at
this, is
found from the following equation
9*
^^,
or
132
number
the
of
the water mixture (the seeding
in
cells
same proportion to the number of cells after

seeding as the whole amount of liquid after seeding has to
the amount of the seeding liquid. The quantity of liquid
liquid) has the
in the flask after seeding has taken place
From
the given equation x
thus
is
Example
found that the seeding liquid contains 75

of
volume and the
of wort, and
x.
It is
per unit
cells
70
flask to be infected contains
c.c.
further desired to have 5 cells per unit of
it is
y^K
5 X 70
volume after inoculation, accordingly x =

to be
p +
withdrawn from the seeding
The
liquid.
result
^'^'
may
be checked by another counting after seeding. If the result

is
more
incorrect either
But
in exact
Suppose
and
work
it is
of
&i cells
liquid or
more
cells
must be added.
this contingency does not arise.
wished to sow
a species
ctj
cells of
a yeast species
in a flask containing
A
of
p c.c.
two seeding liquids containing a and b
volume respectively, the number of cubic
culture liquid, from
per unit of
cells
centimetres, x
is
and
y, to
be sown from
found from the following two equations
a_^
p+x+y
B respectively
and
:
^^^^^ p + x + y^
the quantity of liquid after infection being p + x

this
we
X =
ab
-~
a,bp
a^b - aÔj
and
y =
from
ab,p
ab- a^bf- - a^b^
Combinations of the above three cases

occur, but
may
from the explanations given here
difficult to solve
more
+y
find
detail.
them.
It
would lead us too
it
of course
will not
far to
be
go into
BIOLOGICAL ANALYSIS OF YEAST
133
The Biological Analysis of Yeast.
8.
Preliminary Investigation. The biological analysis of

a yeast specimen
by its origin
and the eventual use to which the yeast is to be put. In
general it is no small labour to determine all the elements
is
guided, to a large extent,
of a specimen of yeast.
nary experiments
analysis.
With
This
is
begun by several prelimi-
in order to obtain a basis for the real
view a microscopical examination
this in
of an average specimen
of the yeast is
and
observed
size of the cells are
dead
cells
practice.
The shape
made.
further, whether
many
are present, as that aflects the use of the yeast in

It can also be
and bacteria
found whether there are any mould
The detection of the latter

by adding dilute soda solution to the preparaby means of which dead particles of organic-chemical
spores
is
present.
simplified
tion,
origin are in general dissolved
with resinous and albuminous

to determine
this is the case especially

It is often difficult
bodies.
by microscopical examination whether the
bacteria found are living or dead, especially
if
they belong
to the non-motile species.
Some information
as to the constituents of
specimen can also be obtained by putting a
wort at 25 C.
the
phenomena
the yeast
little
of
it
in
of fermentation are then
observed on one hand (top or bottom fermentation), and
on the other the time taken to form a

of the liquid.
Lastly, a small sample
film
is
on the surface
placed directly on
a gypsum block at 25 C, and information

in the course of a
few days as
is
obtained
to the conditions of spore
formation.
Separation of the Various
The
Forms
in
the Sample.
actual separation of the different species in a par-
ticular
sample
is
effected
by means
of a plate culture of
a small average sample in wort gelatine.
Yeast and mould
134
few
fungi, but only a
ment.
If
it is
bacteria, are thus
brought to develop-
then desired to recognise the bacteria also
present, a gelatine or agar-agar preparation of either yeast
water or peptone meat extract should be used in making the

at 25 C.
size
away
Plate cultures on wort gelatine are set
plate cultures.
When
grown
the colonies have
to a sufficient
they are examined microscopically and macroscopically.
Those which exhibit differences are inoculated in wort and
The yeast sediment formed
studied more closely.

process
is
examined under the microscope
eventual film formations are set apart
made
in
which
it is
determined, etc.
cultures for the
experiments are
observed whether the fermentation
bottom or top fermentation

is
in the
amount
the
spore analysis
is
is
of alcohol formed
also carried out (see
below).
The comprehension
of these analyses, of course, rests
which has been acquired into
substantially on the insight
the biology of the organisms concerned, and on the use of
by
the scientific results brought out
research.
The analysis
has been perfected chiefly for brewery purposes.
In nearly
all
cases of analysis of a
brewery yeast
it
will
be a question of determining to what extent (1) wild yeasts,

(2) bacteria,
and
(3) various species of culture yeasts, are
The problem
present in the sample in hand.
will
seldom
be that of determining the species.
Hansen's Spore
Method
the
for
ery Bottom Yeast for Wild Yeast.

yeast, Hansen's spore
method
is
average sample of the yeast
cultivated
is
;
of the yeast sediment produced in
way
at 25
testing for wild
generally employed.
25 C. for about twenty-four hours
pared in the usual
Analysis of Brew-
In
in
wort
is
at
gypsum block cultures

this manner are pre-
and 15
C.
after forty
seventy-two hours respectively the blocks are examined

spores are found, wild yeast
An
present.
Holm and
and
;
if
Poulsen
ANALYSIS OF YEAST
have
shown
that
by
this
method yws
135
P^'i't
of
wild
yeast can be discovered in a mixture with culture yeast.

Later, G. Syree proved, with the aid of the
same method,
the presence of the wild yeast species Saccharomyces Pastorianus HI. in a mixture with culture yeast in which it
amounted to only ^^ part of the latter; the two species
had been cultivated for four days at 25 C. In another case
the original mixture was -ji^ Sacch. Pastorianus III. and ^^
Frohberg yeast after they had been cultivated together for
;
was
by Hansen's spore method.
The wild yeast can be most easily obtained by infecting
a wort flask with the yeast sample and placing this away
at 25 C. or at room temperature.
At the close of the first
eight
days,
the presence of Sacch. Pastorianus III.
demonstrated in
this case also
fermentation a specimen of the surface beer
is
taken out
Hansen's investigations have shown that at this period the

greatest quantity of wild yeast
mentioned.
surface beer,
new
new wort flask

and gypsum block
is
is
found in the position
then infected with the
cultures are prepared
from
manner already described.

Spore Method to Top Yeasts.
produced
his method specially for investiWhile Hansen
gating brewery bottom yeast, and the experiments described
above on the sensitiveness of the method were performed
with such yeasts, Alfred Jorgensen has shown that this
the
culture in the
Application
of the
method also gives good

showed further that
results
it is
with brewery top yeast.
He
necessary to perform the analysis
on account of certain top yeast species. The

method has also been used later in the other branches of
at 12 C.
the alcoholic fermentation industries.
The Tartaric Acid Method.

mixture of wild yeast
fails to find
Hansen,
is
it.
The
is
Often,
however, the ad-
so small that the above
method
tartaric acid method, also described
then applied.
An average
by
sample of the yeast
is
136
placed in a 10 per cent, aqueous solution of cane sugar to
which 4 per
culture
tion
is
is
cent, of tartaric acid
set
away
has been added, and this
room temperature.
at the
This cultiva-
repeated four times after every twenty-four hours, or
the culture
is
at twenty-four
from the
away
put
hour
last culture
at 25 C.
intervals.
;
and recultivated twice

wort flask
is
inoculated
then ordinary gypsum block cultures
are prepared at 25 and 15 C. with the yeast thus produced,
In this manner quite small
and these cultures investigated.
In
traces of wild yeast can be detected.

scopical investigation
is,
cases a micro-
all
of course, also carried out.
In the examination of cultures on gypsum blocks regard

is
paid to the appearance of the spores, the spores of culture
yeasts generally containing a less refractive plasma with

vacuoles, thus having
an empty appearance, whilst the wild
yeasts exhibit a strongly refractive plasma.
The above mentioned analysis

cultures on
gypsum
of yeast
by means
blocks can be simplified
if
of spore
the culture
yeast in hand forms spores with extreme difficulty or not

at
all,
as then the simple detection of the spores on the
gypsum
blocks at 25 C.
sufficient confirmation that the
is
sample contains wild yeast or a foreign culture yeast.

Sporeless forms of the saccharomycetes can be prepared
by the Hansen method, described

therefore simplified
analysis
is
kind
employed
is
in
if
in
Section III.
the
a culture yeast of this
Since the asporogenous
practice.
varieties of the saccharomycetes also
form no
films,
this
provides an additional means of detecting the presence of

sporogenous, that
is
to say, foreign species
being set aside to determine

Analysis for Bacteria.
is
a wort culture
film formation takes place.
In
bacteria an average sample
preserved at 25 to 30 C.
if
testing yeast
for
living
placed in yeast water and
When
acetic acid bacteria are
being sought, the beer can be kept at 32 to 33 C.
if
the
ANALYSIS OF YEAST
yeast sample to be examined
not the
case,
little
then subjected to
is still
137
in beer, or
if
If
it is
is
and
Should living acetic
this temperature.
acid bacteria be present these are quickly developed

process.
that
of the yeast can be placed in beer
by
this
required to determine in a yeast sample
the bacteria which are capable of developing in wort, then

of course wort
is
used instead of the liquids named above.
Testing the Contents of the Pure Culture Apparatus.
The
above methods for the biological analysis of yeasts
are applied in breweries to test the contents both of the
pure culture apparatus and of the fermenting

In the
the
case a sample of the top beer
first
pure culture apparatus at the
fermentation
by
close of
vessels.
is
taken from
the primary
Wild
the side tube j (see Figs. 50 and 51).
yeasts are then detected
by means
of the
acid
tartaric
method, and bacteria in the manner already described.

Testing the Contents of
far as the testing of the fermentation vessels

this
may
is
concerned,
be confined chiefly to a microscopical examination
when
of the fermenting wort, especially

is
So
Fermenting Vessels.
not to be used as pitching yeast.
the yeast produced
The appearance
of
the yeast species used will in general be readily recognised
by
daily practice, so that a
foreign admixture will be
detectable by mere microscopical examination.
knowledge gained in
this
way
tage; but on the other hand
is
it
cannot be too strongly
emphasised that a microscopical examination alone
a perfectly
when
reliable criterion,
the culture yeast used
wild yeast
{e.g.,
cultiu-e yeasts
and
is
not
is
this is especially the case
similar in appearance to a
has more or less elongated
which
special
therefore of great advan-
may have found
cells).
Foreign
entrance in certain
cases will, as a rule, not be recognisable
it is
then even
more necessary to apply physiological methods.

The sample is taken from the surface of the beer
in a
138
sterile glass
four to five days before the end of the primary-
In the microscopical examination, the form
fermentation.
of the cells
observed, and whether living bacteria, especi-
is
and Sarcina, are
ally rod bacteria
present.
to test for wild yeast, the sample
deposit has formed
the latter
is
If it
set aside
a yeast
gypsum
placed on
is
wished'
is
till
blocks
and analysed according to the spore methods already de-
The yeast
scribed.
placed directly on the
is
gypsum block
because the wild yeast presumably present has just formed
young
strong
wort flask
yeast generated here

test.
wild yeast
If
is
is
be used for pitching.
At the same time a
juncture.
cells at this
inoculated with a
is
little
of the yeast,
employed next day

found by
in a
This holds also
in the wort,
new
this test, the yeast
when
spore
cannot
Sarcina or other
bacteria are present to an appreciable extent.
always observed
and the
Bacteria are
but are usually dead.
Wild
yeast
is
likewise always found in practice in small amounts ^
when
it
cannot be detected by means of the ordinary spore
method there
The
is
no reason for apprehension.
table on page 139
journal of a fermenting
Lindner's
is
cellar.
Drop Culture.
for wild yeast, uses the
given as an example of the
"
P.
drop
Lindner, in examining
" culture.
This consists in
taking out a certain amount of beer by means of a pipette
and distributing the contents of the
pipette,
drop by drop,
(generally 50 drops) on the bottom and the cover of a Petri

dish.
It
is
thus
known what
quantity there
is
in these
The Petri dish is placed in a thermostat at

C, or left at the temperature of the room. A development is visible in the drops after the lapse of several daySv
If the number of germs is too large, the liquid is diluted
100 drops.
25
with wort to a suitable extent, before the dropping

performed.
If
it
is
is
wished to determine the principal
kinds that are present, the upper dish
is
used, in the drops.
ANALYSIS OF YEAST
<
izi
P3
l=>
o
l-s
<
h;
H
O
iz;
!zi
03
139
140
of which the colonies are crowded at the lowest point, or
are intimately mixed.
with
the
All the drops
which
finger,
been
has
now touched
are
and
cleaned
flamed
which
previously, in order to obtain an average sample,

is
To
then examined microscopically.

yeast from
bottom
advantage
wild
distinguish normal
the property
yeast,
former aggregates in
of that the
taken
is
flakes, whilst
the greater part of the wild yeast cells distribute them-
But
selves like dust in the drop.
Lindner men-
since, as
wild yeast also forms flocks, and the
tions, a part of the
growths of culture yeasts on the other hand can assume
must be
a dusty appearance, other distinguishing features
sought.
From
Lindner's description of his method for analysing
yeast in the brewery,
from
clusions
the
it
seen that he draws his con-
is
cultures in hollow glass slips
as
it
remembered that
is
in
drops
of
which are so
liquid,
cells
often
diflêrent
Under
species.
develop
when they
even
species,
this
cultivated
yield
same
the
by
side
growths,
seem
as to
droplet
be of use so long
one and
of
cells
are
may
side
the cells
to belong
these circumstances culture yeasts
of
several
to
may
also
with an appearance similar to one of the
wild yeasts and
vice
known, subject to
versa.
with
Each characteristic
variation, but this
regard to the form of the
combined
appearance of
microscopical
the
cells.
ordinary
advantage to the practised
is
is,
especially so
This analysis,
microscopical
specialist,
one,
as
is
with
when
is
of
whose eye for the
form and general appearance of the yeast
cells
has been
specially trained.
9.
Hansen's Test of the Stability of Beer in Cask.
Samples from lager casks are taken by boring holes in the

cask.
The place
is first
cleaned with
spirit,
and a try-cock
TESTING THE STABILITY OF BEER

similarly cleaned
beer are drawn
is
oflF
placed in the hole.
141
Portions of the
from the upper, middle and lower layers,
samples being taken from these three different parts of the

liquid in order to get, as nearly as possible,
The weak
an average sample.
point in this and similar analyses consists in the
taking of the samples, which must give a correct average

the analysis
any
to be of
is
described in the following
is
value.
if
The beer analysis
given chiefly for the purpose
of testing the stability, a point of great importance to the
brewer
what concerns him
is
to
have some idea how
the beer will behave after the lapse of a certain time.
Hansen has published the following description of the test

The beer is drawn off in sterile white glass bottles, which
are then closed with sterile corks and placed away in a
dark cupboard at the temperature of the room. As soon
as the samples are taken, their smell, taste, clarification
an appreciable
whether
it
and
how long it takes to form

deposit, and further, how the latter behaves
colour are noted.
It is also
noted
distributes itself easily through the liquid on
shaking, so that this becomes turbid and opaque, or whether

it
forms flocks which quickly sink to the bottom without
substantially affecting the transparency.
kind are caused by

the
liquid
Changes of this
the presence of micro-organisms.
becomes
gradually
turbid
If
and decolorised
without having been shaken, disease bacteria are present.
However,
this takes place but
system
introduced.
is
On
seldom after the pure culture
the other
hand a yeast sediment
always forms after a certain time even in the best beer,
and may
arise
from culture yeast or from wild yeast, but
most frequently due
shown that wild

this stage.
to a
mixture of the two.
is
Hansen has
species of yeast can produce diseases at
For the
made only
rest,
when speaking
of stability, re-
to the formation of yeast sediment
ference
is
and not
to bacterial diseases.
yeast species which gives
142
a stable beer
in this sense, one wiiich not only increases
is,
to a small extent in the finished beer, but
same time
latter obtains either

its
because
competitors to suit
and especially
during
its
is
able at the
to suppress its rivals during fermentation.
to take
it is
itself to
The
in a better condition
than
the conditions of nutrition
up the oxygen,
or because
it
gives
off,
multiplication, products v^hich act as poisons.
Hansen's work has shown that samples from the upper
more quickly
remarked
layers of lager casks produce a yeast sediment
than those taken from other parts.
It is to be
here that beer under brewery conditions
is
when drawn
under pressure of
off
carbonic acid)
(when not drawn
off
but this does not happen
strongly aerated
when
these test
samples are taken, and this materially affects the increase

of the yeast.
Hansen's work showed further that
it
is
necessary to keep the samples at the temperature of the
room and not
at 25 to 27
C,
since the yeast sediment
produced sooner in the former case than in the
is
latter.
Moreover, the varying conditions in practice will be naturally of great importance in such tests,
and therefore do not
admit of the establishment of a general
rule.
necessary as regards separate breweries to

ard which
is
is
obtained
by experiment and
not changed so long as the same yeast
It
fix
is
analysis,
is
therefore
upon a standand which
and so long
used,
as there are no great changes introduced during the
manu-
facture of the beer.
In practice the following procedure

production of a well-stored lager beer
is
drawn about two months
doing
this,
ployed.
white
They
away
adopted when the
view
after the beer
sterile bottles
are set
is
is in
with
is
sterile
in darkness at the
sample
casked.
In
corks are em-
room temper-
ature and observed several times in the course of a fortnight.

It is
is
noted
how
soon a sediment forms
considerable, its constituents
if
the sediment
are investigated.
If
the
BIOLOGICAL ANALYSIS OF WATER
143
may
stability does not appear to be satisfactory, a sample
be taken before the beer

in the
is
When
same manner.
drawn
off,
and
this is treated
the drawing off begins, samples
are again taken from the same casks, but in ordinary bottles,
They
not sterilised ones.
are treated as by the retailers,
being, for example, shaken up.

is
Otherwise the examination
the same as that given above.
The
table given on page 144 will serve as a specimen of
the data entered in a lager
cellar journal.
The Biological Analysis of Water, Air and
10.
In biologically examining water,
air
and
soil,
Soil.
it
of
is
fundamental importance to deal with an average sample;

otherwise the analysis has
very
air
difficult,
and
.species,
soil
little
and in addition to
This
value.
is,
however,
this the organisms in water,
vary considerably, with respect to number and
with the time of year
it is
therefore necessary to
perform a large number of analyses at
different times in
order to obtain a knowledge of the actual
flora of the micro-
organisms and their relative proportions.

vestigations mentioned in the next section
an example
of such a series of analyses,
-object of investigation
Hansen's
may
which had as the
the circulation in nature of saccharo-
mycetes, and especially of Sacch. apiculatus, and, above

discover
in-
be cited as
what organisms
all,
to
are present in the air at different
times of the year.

Principles
Air and
Soil.
the
of
The
of water, air afid
Technical
manner
soil
in
which a
of
Water,
biological analysis
should be carried out depends upon
the object of the analysis.
chief principle is the separa-
tion of the germs in sterile water
mixture.
Analysis
If the question
is
and seeding from the
to find all the species of micro-
organisms present in a sample, the undertaking will be a

very difficult one, especially as regards bacteria.
For
144
1
1
1^
<
'A
1-5
<

isolating
the
145
micro-organisms, several
different
culture
media are employed so that all kinds may develop. We

will not, however, treat of such analyses, as they do not
usually
The questions here
occur in practice.
usually tend only in one direction.
water
brewery
for
information
purposes,
arising
In the analysis of
generally
is
sought for as to the micro-organisms of the water which

are detrimental to the working of the brewery
the non-
injurious forms are of no importance in practice, and are
The
therefore left out of consideration.
employed
in the brewery,
such investigations.
i.e.,
This
culture
wort and beer,
simple
on at one time by Hansen, as

by many workers.
The Hygienic Analysis of Water
it
medium
used for
had
principle
insisted
is
to
be
was neglected
Koch)
(after
is
performed with meat-extract peptone gelatine as culture
medium
a certain quantity of the water
the latter and the
number
In analogy with
determined.
is
distributed in
of colonies developed
this
from
an air analysis
is
it is
some-
times carried out| as well by drawing the air over culture

gelatine.
The object of these methods is

They are, therefore,
as possible.
to develop as
many germs
also used to test the effici-
by subjecting a certain quantity of water to

plate culture before and after filtering, and afterwards comparing the number of germs developed in the two cases.
Water Analysis for Brewery Purposes (after Hansen).
ency of a
filter
technical biological analysis of water for industrial
purposes
is
made by sowing a
in sterile wort, must, etc.

of water for
may
example of such an analysis
The questions
How does the

How rich is it in
the following
water
brewery purposes as carried out by Hansen
be described here.
and beer
An
certain quantity of the
10
to be answered are
water behave towards wort

such micro-organisms as can
14G
develop in these liquids, and are there

species as can
operations
From
which
dangerous disturbances in practical
cause
the analyses given below will be seen the differences
results
may exhibit
of the above-mentioned
ments with wort

gelatine
is
numbers
used.
:
Hansen found by
While cultures
growths and a simultaneous
always gave
water,
according as the one or the other
methods
his analyses the following
of
among them such
0,
when Koch's
he found
series of experi-
3 and 9 growths per 1
6'6,
0,
in beer
c.c.
meat-extract peptone
was used under the same conditions and with
samples of the same water, 100, 222, 1,000, 750 and 1,500
growths per
c.c.
of
water.
This shows that the Koch
method is inapplicable to such brewery analyses.

The procedure is therefore as follows: When,
tap water in a brewery
carefully cleaning the taps
is
the
e.g.,
to be analysed one begins
and tubing
by
of the water supply,
The tap is then opened

and the water allowed to run for some time, e.g., one hour,
This ensures the washing out of
before samples are taken.
using
all
precautionary measures.
the piping.
The
difficulty here, as in all biological analyses,
consists in getting
is
an average sample.
not for transmission, but
sterile
Chamberland
flasks
however, the water sample
is
If the
to be analysed
water sample
on the
spot,
can be used for this purpose

is
if,
to be despatched, sterile bottles
with glass stoppers are employed, the sample being packed

in ice.
After the sample of water has been well shaken up,

a small quantity
sterile pipette.
and
is
carefully
The water
sterilised beer.
is
withdrawn by means of a
inoculated into sterilised wort
In order to observe with greater ease
the development of organisms that
may
be present
it
is
best to employ a perfectly clear wort without sediment.
Freudenreich flasks are the most suitable ones to use, as

the
number
obvious
is
growth
of
is
large,
and larger
that
power
the
would
preventing
of
many organisms which wort and
in virtue of the constituents derived
the
beer possess
from hops, and
acid,
too great a quantity of the water to be analysed
etc., fails if
is
flasks
much room.
take up too
It
used
to be
147
Holm
added.
has determined exactly the quantity of
water which can be added to lager beer wort (about 14

per cent. Balling) before this happens.
author 15
and 15
c.c.
resisting
In
lation
of
power
many
water, since
fails.
cases
it
may
would give
sample should on
it
to take so
much
many germs that the

many growths. The
inocu-
would be an error
contain so
rise to too
this account be diluted
quantity of sterile water (or wort) or

smaller quantity to each flask.
a definite rule for
make
According to this
wort can be treated with ^ c.c. of water,

of lager beer with | c.c. of water, before the
c.c.
this.
it
water
with a certain
may
be added in
It is impossible to give
In each case
it
is
advisable to
a preliminary test in order to determine approxi-
mately the number of germs.

obtained
when one drop
reliable result is usually
of the water
is
placed in each
of 100 flasks with wort; these flasks are then set
The
at 25 C.
away
infecting of beer flasks can be dispensed
with since Holm's investigations have shown that in his

analyses organisms never appeared which were only capable
of development in beer.
Any growth which
developed in
the beer flasks was derived from such species as might

easily
grow
in wort.
In sowing a drop in each of 100
Freudenreich flasks containing wort, 5
c.c.
of water in all
are used, and in most cases this gives rise to not
than one growth in one
flask.
If flasks inoculated in this
at 25 C.
more
way have
stood for one
week
and during this time no development has taken
10*
148
place,
of
then there was, in the water sowed, no germ capable
development under the conditions
of working.
growths are present in some of the
number
per
1 c.c. of
It is of
flasks, the
contents
macroscopically and micro-
of the latter are investigated

scopically, the
If such
of growths noted
and the quantity
the water calculated.
some moment
sowing at which development,

For
especially of bacteria, takes place in the wort.
when they develop only
obvious that
water
in practical evaluation of the
to take note of the time after
it is
after four or five
days they must have been so feeble as to be capable of
development with great
for
development than
under
in practice, since the
rivalry with the yeast (and the low temperature)
The
all
In the laboratory, conditions are far
practical conditions.
more favourable
or not at
difficulty
is
wanting.
water analysis will therefore always be
result of a
such that rather more germs are found than would have
reached development under practical conditions in spite of
the attempt to copy these conditions as far as possible.
Wichmann
lays special
stress
on the importance, in
evaluating the water, of noting the time

"
truction
when
of the liquid under test takes place.
by adding
to
"
the
He
des-
proceeds
each of four flasks charged with 10
c.c.
of
wort, 1 c.c, f c.c, ^ c.c, and ^ c.c. respectively of the water

to be analysed.
These four flasks are numbered 1, 2, 3 and
4.
The destructive power
water
of a
is
taken as equal to
100,
if all
four flasks exhibit development after the lapse
of a
day
this
of the flasks
ducts.
day
If
number
by
is
got by multiplying the numbers
certain factors
and adding the four pro-
development takes place
this factor
is
after four days 4,
10, after
and
two days
in the flasks after

8,
after five days
one
after three days

2.
Thus
if
all
6,^
four
flasks are turbid after twenty-four hours, the destructive
power
is
10 X 1
+ 10
X 2
-t-
10 X 3 + 10 X 4 = 100.
If

flask
No.
shows development
two days, No. 2
after
No. 3 after four, and No. 4 after five days, the
three,
power
destructive
From
after
149
this it
may
is
1x8
2x6
3x4+4x2 = 40.
Wichmann adds more water
be seen that
Hansen and Holm
to the wort than
in their analyses
found
to be advisable.
Schwackhbfer's Standard of Fitness of

Water.
Schwackhofer
a Brewery
has proposed the following scale
as a standard of the fitness of a
water for brewing purposes,
twenty-five flasks being each infected with one drop of
In those cases in which certain micro-organisms
water.
develop neither in wort nor in beer, he describes the water

If development takes place in 10 per
as specially good.
cent, of the
flasks, the
wort flasks at the most, but not
water
is
good
if
in the beer
development takes place in 50
per cent, of the wort flasks and in none of the beer flasks
the water
is fit
for use
more than 50 per
if
micro-organisms are present in
cent, of the
19 per cent, of the beer
wort
flasks,
flasks, the
employed in cases of necessity, and
and in at most
water
is
only to be
finally if the percentage
of flasks of both categories exhibiting growths
than that above mentioned, the water

purposes.
It
is
higher
is
unfit for
brewery
must be remembered here that chemical
analysis has been entirely left out of account.
Holm's Results.
In Holm's analyses of water from the
Carlsberg Breweries, bacteria, moulds and yeast-like
cells
(Tomla, Mycoderma) appear in the wort and beer, but no

species of Saccharomyces.
The presence of the
latter is never-
theless not precluded, but their appearance in the water
at
any
rate rare.
The
species of
is
moulds were especially
numerous, not only in wort but also in beer the same remark
;
applies also as regards the
number of growths.
Bacteria were
found along with these in the wort, whereas they appeared but
seldom in
beer. Yeast-like cells
were rarely observed.
Among
150
the organisms observed
by Holm
in
water the following
may
be named Rhizopus nigricans, Penicillium glaucum, Mycoderma

:
Bacterium
cerevisicB,
addition, Jorgensen
latter investigator
and Bacterium Pasteurianum.
aceti,
and Lindner have found Sarcina.
found
it
In
The
frequently, and supposes that a
prolonged existence of the Sarcina germs in water renders
them more capable of germinating

With regard to the importance
in
hopped wort.
of the micro-organism
content of the water for brewery working, the germs present in the steeping water will be of
the manufacture of malt.
There
consequence in
little
are, in fact,
many germs
on the surface of the barley corns, and the few which are
added with the water are of

boiled, these
little
of
some
When
account.
organisms are killed
is
significance during mashing.
The danger
greatest in the fermentation and lager cellars

factors appear here, viz.,
the wort
they may, perhaps, be

is
naturally
but other
low temperature and the large
quantity of healthy yeast with
its
suppressing power, which
restrain the development of the water germs.
In carrying out a water analysis
we must
direct our
attention to the cistern or the well from which the supply

proceeds.
large
Surface water, as
number
extent
of germs,
well
is
and these
known, contains a
will increase to a large
the piping and the cistern are not kept properly
if
clean.
Hansen's
Analyses of
Air,
It
is
more
diflBcult
to
obtain an average sample of air than of water.
The same
Hansen in his
principles hold here as in water analysis.
time used flasks with wort for investigating the circulation

of yeasts
and the organisms
of a brewery.
open at
Large numbers of these
diflferent
also
flasks
were
left
times of the year at those places where
he wished to analyse the
were
of the air in the various parts
air.
The
so-called
employed for the same purpose.
vacuum flasks
The latter are
BIOLOGICAL ANALYSIS OF AIR

flasks charged
with culture liquid which are sealed during
the boiling of the liquid;
after cooling there
exhausted space over the culture

is
now broken
the
air,
151
off at the place
where
a certain quantity of air
The germs contained
an
is
sucked into the
in this air are
end
wished to examine
it is
is
air-
If the sealed
liquid.
flask.
brought by shaking
into contact with the culture liquid in
which they then
develop.
In his
last treatise
on this subject Hansen recommends
-.^^Tf^
Fig.
49. Miquel's Flask, B,
in
combination with an aspirator, A.
the passing of a certain quantity of air through
sterile
water, which retains the germs and can be subsequently

analysed.
An
same manner
air analysis
is,
in short, performed in the
For
as a water analysis.
ated pipettes are used or,
where
this purpose gradu-
Miquel's flask,
possible,
which is represented in the accompanying drawing

in union
with an
an outflow tube,
with a pinchcock
aspirator.
a,
The
latter,
A,
is
(Fig. 49)
a bottle with
near the bottom, to which rubber tubing
is fitted.
by a bung through which a
The neck
of the bottle
glass tube passes
is
which
closed
is
bent
152
once and
is
is
shaped somewhat like a Chamberland flask and
with two side tubes,

glass tube,
During
tube
filled
b,
and
but the neck
d,
is
latter
provided
is fltted
with a
which reaches almost to the bottom.
sterilisation,
with a
carries a glass cap
little
with cotton wool (as in the Chamberland and
Freudenreich flasks)
at
The
connected with the Miquel flask, B.
by a wool
which can be
is
likewise closed
At c a very loose wool plug is fitted

blown into the water in B at the end of
At d there is a rubber tube which is closed
plug.
easily
the experiment.
by means of
sealed.
The
the one side tube
a glass tube
drawn out
flask further contains a
to a fine point
and
measured quantity of
water in such amount as to immerse the opening of the

tube,
In this condition the
b.
the dry state,
is
again
flask,
sterilised.
then set up at the place where

air,
the tube,
c,
The experiment now

retained by the latter
a,
The whole apparatus
is
desired to examine the
being connected with
by a rubber
tube.
consists in sucking the air to be
examined through the water

cock,
it is
previously sterilised in
in B, so that the
this is
so that the water in
germs are
done by opening the pinch-
The
runs out.
be drawn too quickly through the water
air
must not
the water in
A is
As the
experiment proceeds the pinchcock at a may be opened more
and more as the flow of water gradually lessens. The wool
plug, e, retains those germs which may be carried off" by the
air without being taken up by the water in B. It is obvious
that the volume of air sucked through the water in B is the
same as the volume of water which runs out at a. The
quantity of water in A must therefore be known. This
therefore allowed to pass out at a drop
bottle,
by
drop.
however, cannot be completely emptied through a
while in an upright position, and this should be kept in
mind when the quantity

is
advisable to
first
is
determined.
For
pour as much water into
this reason it
as will
fill
BIOLOGICAL ANALYSIS OF AIR

it
over the inner opening of the tube
and then to mark

a volume of water
a,
the level of the liquid in the flask;
qual to that of the air to be analysed
As soon
the tube,
There are
is
then added.
as the desired quantity of air
the water in B, the pinchcock,
on B
still
is
c,
a, is
closed
passed through
is
and the cap placed
then disconnected from the rubber.
some germs in the
reached the water
153
the tube,
until the water rises in
until b has been
washed
blown or pushed
into the water.
out.
which have not
b,
blown through
therefore
is
c,
This
b.
tube,
is
repeated several times
Finally the wool plug,
is
now
e,
is
well shaken up,
partly in order to distribute in the water the germs which
are on
e,
and partly to
a thorough and uniform
effect
The
germs throughout the water.
distribution
of the
procedure
then exactly as in water analysis, the water
is
being dropped through d into the flasks containing culture

This
liquid.
is
done by cautiously breaking
of the thin glass tube which
is fitted
The dropping can be regulated by holding

the opening of the tube,
If there
inoculating,
water.
If,
is
the finger over
c.
no more water in
it is
not necessary to
than
know
however, only a part of this
is
the
know how much
used.
of the total
amount
Suppose for example that 6
with 10
C.C.
of water,
follows that
each, 5
c.c.
if
have been
B was
charged
and assuming that one drop = 0'05
of water in
it
will
necessary
it is
of water has been
all,
or, in other
c.c,
words, the half of
the water has been used, and therefore the
Sometimes
of this
which
100 flasks are inoculated with one drop
in half the quantity of
it is
amount
litres of air
passed through the water in B, and that
it
used for the
used,
is
probably be the most frequent occurrence,

to
off the point
into the rubber tubing.
air,
i.e.,
happens that the
number
in 3 litres,
is
air is so rich in
of
germs
determined.
germs that
necessary, as in water analysis, to dilute the water.
154
If
it is
required to quickly obtain some idea as to the
purity of the air with regard to germs, Petri dishes with
wort gelatine
may
be
open for
left
The
fifteen minutes.
covers are then replaced and the plates put into a thermostat
at 25 C.
The germs then develop.
Hansen's Results,
organisms of the
retical considerations,
nature,
especially
In
on the micro-
his researches
Hansen had partly
air,
in
view theo-
such as the circulation of species in
that of
and he
the saccharomycetes,
partly followed out purely practical problems
concerned
As regards the solution of the latter the

was examined in different parts of the brewery (ferment-
with brewing.
air
In connection with
this,
experiments were carried out to discover whether
the
ing
cellar,
cooling vessels,
etc.).
vapours from the grains carried infection by means of the
numerous bacteria they
Hansen arrived
contain.
result that this does not take place
at the
on the other hand,
dried grains become dangerous in a high degree as soon as
they are carried by the wind as dust in the

it is
grains in the neighbourhood of the brewery

to
Therefore
air.
not advisable to employ any apparatus for drying the
Hansen, the bacteria are not killed by
therefore, drying apparatus is set
up
for,
this
according
drying
the dry particles with numerous bacteria can find their
on to the cooling vessels and into the fermenting
much harm may
if,,
way
cellars,
be caused by this means.
Hansen further found that the purest

Carlsberg Brewery was in the fermenting
due to the fact that the
cellars are
which has been previously

in the
in such a position that
purified.
air in the
cellars, this
Old
being
provided with cold air
In analyses of the air
fermenting cellars of other breweries which were
without purified air bacteria were observed, among which
were Sarcina and various species of wild

forms also being found.
The cooling
yeasts, disease
vessels are
exposed
SOIL ANALYSES
from the
to infection
air,
juicy fruits are ripe,
155
especially at the time
when
when
sweet,
the dust from the ground
very rich not only in yeasts but also
is
in bacteria.
Analyses of air should be performed in breweries from
time to time
a clear idea
of the different
mishap.
It
is
Analyses.
Soil
thus obtained of the progress,
and
many
will in
cases avert
the air of fermenting cellars and coolers in
which
particular to
is
processes,
special attention
In
soil
must be
paid.
analyses a small sample
is
placed in an Erlenmeyer dask containing a culture liquid
chosen with regard to the organisms to be sought.

flora of the soil is a
and mould
bacteria
The
very rich one, especially as regards

If saccharomycetes are to be
fungi.
sample in wort to
looked
for, it is advisable to
which
tartaric acid has been added, since this prevents to
seed the
soil
a great extent the development of most bacteria.

case also
In this
desirable to allow the cultures to remain for a
it is
considerable time (about fourteen days) at 25 C, and then

to prepare a second culture in wort, as saccharomycetes are
generally tardy in their development.

It has also
sample of
soil
been proposed to mix a small quantity of the
with liquefied nutrient gelatine and then to
prepare plate cultures.
However, the
result
is
in
most
number of germs in the soil being too large.

Others prefer making a paste with the soil in sterile
cases bad, the
water
plate cultures are then prepared from an average
sample.
These analyses, in common with
all
the foregoing,,
have the object of discovering the source of the infection
which
may
for this
gations.
take place in a brewery.
was furnished by
The groundwork
Pasteur's and Hansen's investi-
156
Hansen's Pure Culture System in Fermentation Industries.
11.
The
Culture System
Pure
Breweries.
The
in
Bottom Fermentation
following information on the introduc-
the systematically selected yeast race
tion of
bottom fermentation brewery
The
Practical Studies in Fermentation.
be the yeast which has proved

in practice,
into the
extracted from Hansen's
is
its
starting point
superiority in
and has yielded that product which
must
working
wished
it is
should be the future normal production of the brewery.
Since the species which lends
character in great measure
its
to the product will be present in superior numbers,
it
will
also be the most easily isolated.

If there is wild yeast present in the stock yeast
ployed,
it will,
amount
according to Hansen, be present only in small
in the surface beer at the beginning of the
fermentation, or
the reverse
is
it
the case.
sample of the surface beer
formed in the fermenting vessel
the race or species to be isolated
sample
is
is
now made with

employed
in preponderance,
and
used for the preparation of absolutely pure
single cell (see page 106).
laboratory.
is
when a frothy head has

we are then certain that
the starting point being
cultures,
primary
will be totally absent, while at the finish
therefore taken just at the time
this
em-
these
made
as usual from a
Preliminary fermentations are
pure
cultures
It is advisable to use the
in
flasks
same wort
in
the
as that
and this should not be re-sterilised

The method of procuring this is described
While fermentation proceeds in the flasks,
in the brewery,
in the laboratory.
on page
76.
certain preliminary observations are
use later on.
It is observed
to
made which will be of

what extent the wort
whether the yeast lies compactly on the

whether
bottom, and
the beer has any peculiar smell and
A microscopic examination and also a spore
taste, etc.
remains
clear,
HANSEN'S PURE CULTURE SYSTEM

gypsum block
culture on a
characteristics
the
of
yeast are
happen that the growths
made
are of course
in fact, the
investigated.
in the flasks
It
same
somewhat
we must now make
We
species.
here confronted by individual differences, which
be met with, and
may
which contain pure
cultures of the required species are nevertheless

varied, although they belong to the
157
a choice
are
may always
from among
these growths also.
After preserving some of the pure culture (on the preservation of yeasts, see page 114)
solution
and partly
the procedure
Four or
is
partly in saccharose
in the dried condition on cotton wool,
as follows
Pasteur flasks, containing ordinary
five 1 litre
aerated but sterilised wort from the brewery, are inoculated
from the
are set
flask containing the perfectly pure culture
away
at the temperature of the room, and
they will contain a considerable yeast sediment
in a
these
week
four such
flasks will generally be sufficient, the fifth being really
After pouring
reserve culture.
each of these flasks
week
as
much
the beer, the yeast of'
introduced into a Carlsberg vessel
is
charged with about 7
oft'
litres of
brewery wort.
After one
yeast sediment will have formed in these
vessels as is necessary to prepare stock yeast for 1 hectolitre
of
is
wort in the brewery.
vessel of li hectolitre
then set up in the fermenting cellar
means
of a gas flame
aerated brewery wort.
this
is sterilised
and charged with a
The contents
volume
by
hectolitre of
of the four Carlsberg
vessels are then poured into this vessel.
If the partially
not to be added
fermented wort in the vessels
is
previously drawn
latter case it is advisable to
let
off".
the flasks stand a
the yeast
brewery
may
is
In the
little
also, it is
longer, about ten days, so that
sink more completely to the bottom.
some distance from the laboratory
always necessary to draw the beer
it
If the
will be
off beforehand.
The
15,s
mentioned above
flasks
page 119) are then used for
(see
transporting the thin yeast liquid.
mentioned
brewery.
is
The
If the result is satisfactory^
duced into
hectolitre of
wort
fermented under the normal conditions of the

the yeast
is
intro-
practical working.
It is conceivable that a
brewery may have operated con-
tinuously with a mixture of different species of culture yeast,
and that the combination
of the latter
has formed the
It is
on no account advisable to
isolate these species separately
and then to employ a mix-
character of the product.
them
ture of
as stock yeast, for the relative proportions
of the species cannot be controlled during fermentation.

Besides,
We
it
would be
far too
much
trouble in practice.
have mentioned before that a pure culture yeast of
this kind introduced into practice can only keep sufficiently
pure in the fermenting vessels for a certain length of time.
The
resisting
powers of the different races against wild
yeasts and bacteria
is
extremely varied.
It
therefore,
is,
necessary from time to time to introduce fresh quantities of
There
pure culture yeast into the brewery.

cases
where a race has remained pure
menting
vessels of practice for
are,
however,
in the ordinary fer-
more than a year without
having been renewed.
The Hansen-KUhle Pure Culture Apparatus. In order

hand the requisite amount of pure culture
to have ready at
yeast, Hansen, in conjunction with Kiihle, has constructed
a pure culture apparatus for the continuous production in
mass
of absolutely pure yeast.
paratus
is
given below
description of the ap-
The apparatus (Fig. 50) consists of three

an air pump. A, with an air reservoir,
viz.,
principal parts,
B, a fermenta-
The air pump

receives the air through a filter and pumps it into the
reservoir which is provided with a manometer and safety
tion cyiinder, C, and a wort cylinder, D.

valve.
The
can pass from the reservoir through the
air
communicating
159
pipes,
which are provided with outlet taps
and
for condensed water, through the cotton wool filters, g
m respectively,
into the fermentation or wort cylinder.
The following parts

cylinder,
Fig. 50.
water in a
79),
c,
which opens under
Hansen-Kiihle Pure Yeast Culture Apparatus.
vessel, d; (2) a glass tube,
/,
with marks (10, 20,
which indicate the amount of liquid
taps are fitted at
and
are connected with the fermentation
(1) a doubly bent side tube,
distribute
an outlet cock,
and h;
in the cylinder
(S)a, stirring apparatus,
b,
to stir
up
any bottom yeast formed in the cylinder (4)

I, for beer and yeast
(5) a short side tube,;,
;
provided with rubber tubing and glass stopper
the pure
160
culture of yeast previously prepared in the laboratory is
introduced through this tube, the side tube of the llask
being connected with
it
a pair of windows,
set at
are only added
way
requested, being best omitted as they are
if
in a spot
is
sometimes a water jacket (Fig.
the stopcocks,
r,and s;
q,
brewery into the cylinder
doubly
vessel, o
(2)
in communication with the
s is
is
passed from the
a sprinkling ring and a cold
(3)
The sprinkling ring
cooling the wort.
p, for
(1) a
opening under water in the
n,
pipe through which the boiling hot wort
water pipe,
be
to
where the temperature must be regulated.
Connected with the wort cylinder, D, are

bent side tube,
(6)
an angle to one another, which
round the cylinder when the apparatus has
51) fitted
up
in a flame in the ordinary
Lastly there
superfluous.
set
a,
is
perforated with small holes on the side next to the cylinder.
The cylinder
t,
and which receives the
through the sprinkling
employed instead
more
box which
fitted into a
is
an outlet tube,
expensive.
In Fig. 51 a water jacket
ring.
The
outlet cock,
I,
is
in
k.
from outside
The construction may be seen
fitted so that infection
obviated during tapping.
from Fig. 52
which the cone valve
is
shown
shut.
The
arrows give the direction of flow when the stopcock

screwed open.
The construction
stirring apparatus,
When
whether
b, is
the apparatus
it is air-tight.
As soon
as
it is
in turn
some
and
time.
is fitted
For
up,
this
it is first
all
is
led into
other cocks being shut.
found that the apparatus
closed after the
tested to see
purpose steam
k, all
by means of steam,
When
is
of the lower part of the
given in Fig. 53.
the cylinder through the pipe,
is sterilised
ia
it is more efiicient but also

The wort and fermentation cylinders are
of this ring
connected by means of the pipe,
is
provided with
is
cold water flowing
is
steam-tight, it
the cocks being opened
steam has passed through for
the apparatus
is
sterilised,
the wort-
W.MMHSHIUJ'XA..
Fig. 51.
Hansen-Kiihle Pure Yeast Culture Apparatus.

11
161
162
with boiling-hot
cylinder
is filled
brewery
this has
taken place, a part of
it
is
sterilised
wort from the
then cooled, aerated through

it
is
and, after
passed over into the
fermentation cylinder by means of air pressure.

cultivated yeast from the laboratory
stirring apparatus set in motion,
and
is
later
The pure
then added, the
on more wort
is
As soon as the proper amount of yeast is formed,

drawn off, and the sedimentary yeast stirred up
and mixed with the small quantity of beer remaining this
added.
the beer
is
mixture
Fig.
is
then taken out, to be used as pitching yeast for
.'>2.
Tlte Construction oT the
Outlet Tap of the Hansen-Kuhle
Pure Culture Apparatus.
Fia.
53.
The Coustructiou of tlie Lower
Part of the Stirring Apparatus of the
Han.sen-Kuhle Pure Culture Appara-
tus.
fermenting vessel
a small
wort).
Wort
is
(with about 8 hectolitres of
then added for the next culture.
detailed description and guide for the use of the pure
yeast culture apparatus described here as well as of the pure

culture system in general
was given by E. Chr. Hansen
himself in his Practical Studies in Fermentation, London,

1896.
of
Exact directions are given which are the outcome
very comprehensive experiments, which he made in
the Old and
New
Carlsberg Breweries, Copenhagen, as well
as of extensive observations in these and other breweries at
home and
abroad.
Some
modifications have been
made
in

the apparatus just described, which
is
the most
163
exten-
sively used form, and changes have been introduced
by
among others Bendixen, Bergh & Jorgensen,

Brown & Morris, Elion, Lindner, Marx, Thausing and
Wichmann.
However carefully one may work there is always the
the following,
possibility of infection taking place in the apparatus
some unfortunate means.
It
therefore
is
by
necessary to
subject the yeast produced in the apparatus to a controlling

analysis from time to time (see page 137).
The yeast in the
apparatus should not be changed without good reason.

the apparatus
is
manipulated carefully
it will
If
remain free
from infection for years.
As already stated, Hansen began

on pure culture yeast
races in 1881,
sive experiments in practice
Carlsberg Brewery.
his laboratory researches
When
and carried out conclu-
two years
later in the
Old
the new reform had gained a
firm foothold there and in several other bottom fermentation breweries,
it
naturally resulted in
its
extension to the
other branches of the fermentation industry.
The Pure Culture System in Top Fermentation BrewThe first to experiment in practice with purely
cultivated top yeast was Alfred Jorgensen, who, in 1885,
eries.
introduced a yeast of this kind into a Danish top fermenta-
The method
tion brewery with most satisfactory results.
was the same
by Hansen in
it was
as that described above, used
bottom fermentation breweries, but in some cases

found advisable to aerate the wort a
proceeded somewhat slowly.
little,
as clarification
Subsequently a large number
of good species of beer top yeast were isolated
by Jorgensen,
Schonfeld and others, and the pure culture system introduced
numerous top fermentation breweries on the continent,

although not to such an extent as in bottom fermentation
into
breweries.
11 *
164
Modifications of the Hansen-Kiihle pure culture apparatus
have heen designed by Jensen, Jorgensen, Kokosinski and

Wilson for use
in top fermentation breweries.
Pure Culture System in Wine Manufacture.

Hansen pure culture system has attained great
importance in the preparation of wine.
The systematic
selection from the numerous wine yeast races is here probably
The
The
'of
even greater significance than in the other branches of

In
the fermentation industry.
demands
are
made on the
yeast,
this case the
most varied
which are probably directed
for the most part to obtaining a better product from inferior material.
completely
all
In this domain also pure yeast has realised
reasonable expectations, although
it
cannot be
denied that many expectations went beyond reasonable
limits.
The first to use Hansen's system in wine manufacture

was one of his pupils, L. Marx (1888) later on, Hotter,
;
Mach, Miiller-Thurgau,
Portele,
Wortmann have extended
Seifert
and
especially J.
the application of the system in
this sphere.
The technique of pure culture in wine manufacture is

somewhat different from that in the other branches of the
In a brewery, a
fermentation industry.
dealt with, a wort
it
but
would take up the
it is
sterilised liquid is
not possible to
sterilise
would thereby depreciate
in
quality.
must, as
and the wine
so-called boiled flavour
This circumstance
must in the preparation of wine.

The germs present in must are, however,
as a rule so weakened that they only develop after some
time a large quantity of a vigorous young growth of the
entirely prevents the use of sterilised
selected pure yeast
is
therefore added at once to the must,
and foreign germs are by

is
this
means suppressed.
This action
by the chemical composition of

also which is responsible for the
assisted very considerably
the must; and
it is
this
fact that spontaneous fermentation has been advantageously
165
employed for so long a time, a good product being obtained

without sterilisation or centrifugalising.
According to Wortmann's researches the end products

of fermentation, as well as the quantity of these, are the
same, whether
It is best
tion.
or
little
more yeast added
much
yeast be
added
but the
to the must, the quicker is the fermenta-
when fermentation
proceeds quickly,
as,
in
consequence, the foreign germs present in the must are more
completely suppressed
too violent
a part of
an excessive quantity of yeast
if
result of this
the fermentation may, however, be
is,
that the
thus
it is
lost,
must not only

but,
is
added
froths over easily
the
and
by the vigorous production
of
carbonic acid, bouquet substances are also carried off at the
same time and the wine
loses in quality.
With regard to the quantity of yeast to be employed, the

following numbers given by Wortmann may serve as a
guide With light musts, i.e., musts containing about 18
:
to 20 per cent, of sugar, one can rely with certainty on the
fermentation being controlled by the pure culture yeast and
consequently on a good result

yeast
is
litres of
added.
By
must
fresh
if
from
;|
to ^ per cent, of
this is understood that to every
added
is
into vigorous fermentation
:^
to ^ litre of a
by means
100
must brought
of a pure yeast.
Good
with the larger addition of j to 1

But Wôrtmann found the effect of still larger
results are also obtained
per cent.
additions
of
yeast
be too
to
great
with light musts.
With heavy musts the addition of yeasts may be much

larger, i.e., up to 1 per cent, or even more, without fear
of bad results.
If
it
is
desired to re-ferment wines
not
thoroughly fermented, or sugared wines, the addition of

pure yeast should be
over.
still
greater, in fact 2 per cent, or
The same holds good
for the
employment
yeast in the preparation of sparkling wine.
of pure
If wines
which
have stopped fermenting are to be forced with pure yeast,
166
Wortmann recommends
a yet gi-eater addition of yeast,
up
to 10 per cent., according to circumstances.
The temperature of the fermenting room requires

ful
watching
usual to keep
should be kept lower than
it
it,
it is
care-
otherwise
so that in fermentations to be carried out on
may
the large scale with pure yeast, the fermenting
not be
too violent.
It
is,
condition that,
continue
employ the yeast at the

The yeast must be in such a
in addition, important to
proper stage of development.
its
when
placed in the must,
as short a time as possible.
which he then increases

Since the
is large,
can immediately
Therefore the practitioner
from the laboratory small
obtains
it
development, so as to obtain the mastery in
number
of
quantities
of
yeast,
in definite quantities of must.
wine yeast races brought into use
and the fermentation of grape must
is
limited to
a few weeks in each year, a pure culture apparatus such as

that which has been described for use in brewing for the
continual production of yeast in mass

this branch of
a large
number
is
not applicable in
Laboratories would require to have
work.
of such pure culture apparatus,
they possessed them
it
would be impossible
and even
if
in the short
time at disposal to produce the quantity of yeast necessary

for the
work.
Consequently stations and laboratories
dis-
pose of the pure culture yeast in small quantities which

the practitioner then increases for himself.
In cultivating the yeast, laboratories employ sometimes

the concentrated must mentioned on page 80 and some-
times a must pressed from home-grown grapes

is
diluted with water.
the former
In cases where a specially vigorous
and fermentations
employment of well-nourished,
non-aerated yeast is to be recommended.
Pure yeast is supplied to wine producers in a thin
yeast
is
required,
e.g.,
in re-fermentations
of unfinished wines, the
167
by the stations, e.g., by that in Geisenheim

and by the fermentation laboratory at Klosterneuburg the
liquid condition
flasks contain as
much yeast
as
produced
is
in
05
to 2 litres
of culture liquid.
The Geisenheim
station has given the following direc-
tions for the use of this pure culture yeast
The
flask
with
the pure culture ought not to be more than two weeks old
at the very most.
Some days
actual vintage, about
12
before the beginning of the
litres
fermenting must from good,
of freshly prepared, not
ripe,
sound grapes are allowed
skimmed carefully
down to the tempera-
to boil for about five minutes, being
meanwhile, and then allowed to cool
ture of the room, the pot being covered.
After cooling, the
contents of the yeast flask should be poured into the must,
the flask washed out several times with must, and the pot
securely covered
again,
and placed away in a dust-free
situation at the temperature of the room, until the must,
two to three days, exhibits violent fermentation. The

must thus brought to fermentation is then put into the
fresh must to be fermented, the quantity of which depends
on the conditions at the time.
The selection of the pure yeast is of the highest imafter
portance in the preparation of sparkling wines.
It affords
the certainty that the after-fermentation under the ex-
tremely
difficult
conditions obtaining proceeds unaided,
which was formerly more or

the loss of large sums of
further renders
produce
little
it
less accidental and often caused

money; the use of pure yeast
possible to choose such yeast races as will
turbidity of the wine in bottle in spite of
vigorous fermentation, as they separate easily and remain

clinging to the
The use
coi'k.
of selected yeast races has also proved of value
in the preparation of sweet wines.
in the fact that,
by the
Its value lies chiefly
aid of a yeast which
is
equally
168
vigorous to resist and to ferment in a high concentration
and
of sugar
alcohol,
wines
may be produced
with certainty
which contain always the same amount of alcohol
that these wines are ready sooner,
and
further advantage
is
and preserve better than those spontaneously

W. Seifert has the merit of having first made
are finer toned
fermented.
researches in practice on this subject,
and
of having intro-
duced the pure culture method into this branch of wine

production in the large businesses in Austro-Hungary.
The
Cider.
Pure
The
Culture
System
employment
manufacture of cider
is
the
in
of selected
no
less
important than in wine
and Nathan have worked specially at

is
this subject.
the same as in the preparation of wine.
results are also
and
The
The
very satisfactory here, and ciders prepared
by means of pure wine yeasts assume a more or

taste
of
Besides the above named, Jorgensen, Kayser
manufacture.
procedure
Manufacture
pure yeast in the
less
vinous
smell.
The Pure Culture System in the Manufacture of

and Pressed Yeast. The credit of bringing the
Spirit
pure culture system
manufacture
is
prepared
P.
bj'^
general
recognition
due to the station in Berlin.
Lindner (Races
I.
and IL,
in
spirit
The yeasts
especially the
emploj'ed in numerous distilleries and have given
latter) are
good
into
results.
During the last few years the principles of race selection

and pure culture have been applied in the preparation of a
lactic
bacterium
acid
industry
for
use
in
the
above-mentioned
a mass culture from this bacterium
is
used for
souring in order to prevent the injurious butyric acid fer-
The
mentation.
practice
was
first
by
all
The use of lactic acid

mash has been considered
Fr. Lafar.
souring the yeast

evil
to introduce this pure culture into
the leading technologists.
bacteria for
a,
Recently,
necessary
Wehmer
169
has attempted to use lactic acid directly instead of the

bacteria.
now
A commercial
lactic acid,
not absolutely pure, can
be prepared somewhat inexpensively and he has ob-
tained good results with
it
in practice as a souring material.
Finally, the pure culture system has been applied in
recent times in the manufacture of pressed yeast.
The
Berlin station has been especially active on this subject
and the use
of Race V., there isolated, has spread to a great
extent.
Other yeast species and races applicable to the manufactures
mentioned have been isolated in the technical
fermentation laboratories to be found
now
in every country.
SECTION
III.
THE MICEO-OEGANISMS OF MOST IMPOETANCE IN

THE FBEMENTATION INDUSTEY.i
The
micro-organisms to be described
now
are partly useful,
partly disadvantageous to the alcohol fermentation industry

their importance is therefore of widely different character.
They
all
belong to that branch of the plant kingdom called
The fungi are divided into two large groups true

The
fungi (Eumycetes) and fission fungi (Schizomycetes).
first of these two groups is divided into that of the algae
fungi (Phycomycetes) and that of the higher fungi {MycomyOf the numerous fungi belonging to the phycomycetes)}
fungi.
only a single group comes to be considered here,
cetes
namely, that of the Zygomycetes, and in
this
only the
family of Mucoracece.
Among
the
mycomycetes we
sac fungi (Ascomycetes)
shall refer partly to the
taking representatives of the four
orders, the gjrmnoascese with the family of saccharomycetes,
the
perisporacesB
sphseriacese
with
the
family
with the family of
of
aspergilleae,
the
and the
dis-
sphaerieae,
comycetes with the family of pezizaceae
partly to a large
group of fungi, the imperfect fungi (Fungi
no
less
'
bibliography
We
imperfecti),
of
importance, but which cannot yet be classified

is
given at the end of the boolf.
wishing a more detailed description of the fungus

system and the general morphology and physiology of fungi to Zopf's
2
refer those
Handbiich der
Pilze.
170
MUCORACE^
they are in
171
probability only stages of development of
all
other forms of fungi.
review of the systematic connection of the micro-
organisms to be described here

classification
name given
the
organ
is
made by
aid of the
which the vegetative
to those fungi of
a mycelium.
This consists of long threads which
growing points and exhibit true branching.
possess
first
be
True Fungi (Eumycetes)
I.
is
may
given on page 173.
division
distinguished
is
by
this
The
means .from the
second, which contains the fission fungi.

AlgcB fungi (Phycomycetes)
1.
The whole mycelium

single,
consists as a
very much branched
special conditions,
tion begins.
and
first
rule only of
one
Septa only appear under
appear normally
when
fructifica-
Endogenous spores are formed in sporangia.
The only group belonging

consider here
cell.
is
to this division
which we
will
that of the
Zygomycetes.
fungi partly by
by means of the socalled zygospores, and in some forms by budding, by
gemmae (chlamydospores) and by conidia.
Multiplication takes place in these
means of spores
in sporangia, partly
MucoracecB.
The
spores develop from the mass of plasma in the
interior of the sporangium, a part of the
plasma being
left
which swells by taking up water. This happens as soon

as the spores are ripe, and the wall of the sporangium
bursts, setting free the spores.
These fungi can propagate
themselves not only by means of endospores, but also by
172
The
zygospores (Fig. 55, V. and VI.).
duced in the following manner:
two
on
develop
ings
Two
are
latter
pro-
club-shaped swell-
neighbouring
threads;
mycelial
grow towards one another until their ends touch,

The two flattened end memflat.
these
which then become
branes then coalesce.
septum
then formed in these
is
club-shaped growths, so that an end

cell (Fig. 55, V., c)
and a suspensor
Finally the two end
appear.
zygospore
is
and others on
species with
third
;
and
with others
a).
it
Some
this subject
do not hold as regards those
means
of
propagation
When
a).
possessed
is
which their
may grow
cell
the mycelium
numerous dividing walls
members
are thus formed,
walls thicken.
refractive,
The separate members
into mycelium, or develop sporangium carriers
at once (Fig. 58,
b),
or they
and increase by budding

" spherical
by some
gemmae or chlamydois immersed in a
this consists of the so-called
their appearance; short
also
species
seems to be ac-
which swell to a barrel shape and become highly
may
b)
The communications made by Bainier
culture liquid containing sugar,
after
VI.,
melt into one and a
which the author has experimented.
spores (Fig. 58,
make
copulation
in short, the conditions of their formation are
not yet known.
species
(Fig. 55, V.
cells
thus formed (Fig. 55, VI.,
form zygospores easily

cidental;
cell
yeast
behave
" is
may
separate from one another
like yeast
cells;
thus formed (Fig.
in this
59).
the so-called
The spores
manner.
Hansen has established the following general law for

fungi, that the temperature
maximum
for the
development
of the organs of propagation lies lower than the
for the development
of.
the vegetative organs.
maximum
This, of
Not long ago he

described two new species, Mucor alpinus and Mucor
He showed
neglectus, which can both develop zygospores.
course, applies also to the Mucoracece..
CLASSIFICATION OF MICRO-ORGANISMS
-"^
s
5
K e c ^
Q
BO
a
1^
a,
00
aa
S'S'r I-^-s
<a
. B-
t-
a.
173
174
maximum for development of sporangia
that the temperature
and zygospores
yeast
celium,
is
lower than that for development of my-
and gemma formation, and that the
cells
temperature limits for sporangium and zygospore forma-
with the species
change
tion
thus,
Mucor
in
alpinus,
maximum
sporangium formation has a higher temperature
than zygospore formation, whilst for Mucor neglectus the

reverse
is
the case.
Genus
(1)
Pm
Moulds, Mucor, Micheli.
Characteristic of this order

(Fig.
54, III.
sporangium carrier which

"
columella
"
vesicular
at the point of the
undivided in most kinds, a
is
formed by the more or
less
crust of calcium oxalate (Fig. 54, III.,
often present on the outer wall of the sporangium.
The
phytes,
i.e.,
is
end of the sporangium carrier projecting into
the sporangium.
c) is
the ball-shaped sporangium
separating sporangium and carrier (Fig. 54,
This columella
III., b).
is
and IV.) which occurs
species belonging to this order live either as saproi.e.,
on dead animal or plant matter, or as parasites,
They may be frequently seen
on living organisms.
a white, gray or
malt,
etc.,
brown
and they
felt
on dung, bread,
fruit,
Some
also thrive in beer wort.
as
com,
species
can cause dextrin to ferment, others again contain diastase.
Hansen investigated
to
several of these species with regard
their action on the four
lactose
and dextrose.
by no
species,
It
sugars
saccharose, maltose,
is
fermented
and saccharose only by one single species
after previous inversion, while
species
appeared that lactose
on the other hand
investigated ferment dextrose
all
and maltose.
the
The
fermentation of maltose proceeds very slowly and only forms
higher percentages of alcohol after a relatively long time.
Thus, for example, Miwor Mucedo in wort gave after fifteen
days at 23
C only 0"4 vol.
per cent, alcohol
after
two and
MUCOR
175
three quarter months at the room temperature

1 vol., after six
months 3
vol.,
it
had formed
and after one year only
3'1
per cent of alcohol.
vol.
The fermentability
very varied.
of the various species proved to be
While, for instance, Mitcor Mucedo did not
III.
IV.
Fig. 54.
a,
e,
Mucor Mucedo,
sporangium carrier
I.
b,
.Spores.
reach 4
vol.
The
carriers
s,
II.
columella
plasma between the spores.
sporangium
cent.
L.
c,
Germinating spores.
III.,
Sporangium:
calcium oxalate crystals
(After Brefeld.)
a bursting sporangium.
IV.,
a,
d, spores
mycelium with
(After Kerner.)
per cent, of alcohol, M. erectus gave 8 vol. per

facts
communicated about alcohol formation in
the following are from Hansen.
176
The powerful yeast fungi

fermentation phenomena,
with
taneously
the
of
order
this
EmmerHng found
formation
of
alcohol
show
top
that, simul-
during
the
fermentation, glycerine and succinic acid are formed also

in
about the same proportion as in Saccharomyces
fer-
mentation.
V.
Fig. 55.
VI.
Mucin- Mucedu, L. V.
tion cells.
VI.,
Zygospore formation b, suspensors c, copulacomplete zygospore, a b, suspensor-s. (After Brefeld.)

,
</,
The above-mentioned formation of spherical yeast and

gemmae has no connection with the formation of alcohol.
Thus M. Mucedo, e.g., yields alcohol without possessing these
organs, just as the latter are found in species which have
no fermenting power.
veloped in
all
They
are,
however, strongly de-
species of considerable fermenting power.
MUCOR
177
In a 10 per cent, aqueous cane-sugar
Mucor
of
species, like the saccharomycetes, are
solution
the
very tenacious
All the species investigated
by Hansen remained
alive for seven years he proved, with regard to some
species, that they were alive after more than eleven years.
life.
Fio.
56. jV/kco)- racemosus, Fres.
Fig.
branched carrier with larger sporangium at the top and smaller ones on
short side branches.
J"^.
(After Frese-
spores
racemosus,
Fre,s.
are
seen.
^4^.
(After
Fischer.)
nius.)
They
Mucnr
57.
Three sporangia with transparent

membrane, through which the
lived, dried
on
filter
Mucor IWucedo, L.
is first
more than four years.

55).
The mycelium
The sporangium carriers
paper, for
(Figs.
54 and
white, later light brown.
are often about 10 cm. long
the sporangium (Fig. 54, III.

12
178
and IV.)
it is first
is
spherical, large,
mella (Fig. 54, III.

end.
with a diameter of 100 to 150
The
yellow, later grey and nearly black.
The spores
long and 4 to 7
6) is
yu,
a short cylinder with a dome-shaped
(Fig. 54, I.) are ellipsoidal, 7 to 12
The zygospore
/*
membrane and
thick, with a colourless
yellowish contents.
;u.
colu-
which
(Fig. 55, VI.),
was found on dung, is large (90 to 200 ju), spherical, brownish-
Fio.
58. Mnc'j/ rucemusus,

have contracted
to
Fres.
a,
Part of a mycelium, the contents of which
numerous gemmie.
i^.
h,
Five
which have germinated into small undivided sporangium
gemmae
carriers.
together,
*}i.
(After
Brefeld.)
black and with wart-like excrescences.
Under
special con-
ditions (lowering of temperature, impaired nourishment or

parasitical attacks) the
of
branching
sporangium carriers have the power
the branches
without columellse, so-called

does not exhibit
gemma
then often bear sporangia

sporangioles.
formation.
This species
MUCOR
179
Mticor Mucedo liquefies wort gelatine

this
medium.
It
when
have then reached
maximum.
its
of maltose in yeast water
water
it
it
appears to
In a 5 per cent, solution
exhibited a feeble but distinct
it
cent, solution of dex-
In a 10 per
alcoholic fermentation.
after one
grows on
forms in wort, after one year at the room
temperature, 3'1 vol. per cent, of alcohol, and
trose in yeast
it
formed
O^S vol. per cent, of alcohol
and a half mouth.
The fungus is extraordinarily widely distributed in

nature, and is found everywhere on manure, decomposing
vegetable matter and in
soil.
a
Fii;.
59. .l/(v/r racenwsus,
Fres.
a,
piece of
solutiou, separating into splierical yeast
budding,
i^^.
sporangium carriers
branched, 2 to 3 cm. high
brownish and 30
columella
is
mycelium immersed
in sugar
Spherical yeast multiplying by
to
40
/a
in diameter.
yu.
and 57)
The spores are
long and 3 to 5
84
/t
ellip-
thick.
fi
thick, yellowish
with brown, lumpy or ridged thickenings.

displays a very abundant
59).
are, as a rule,
The
Zygospores occur very seldom,
pear-shaped.
spherical, 70 to
58 and
(Figs. 56, 57,
(Figs. 56
the sporangia (Fig. 57) spherical,
soidal or spherical, 5 to 8
and are
b.
(After Brefekl.)
Mucor racemosus, Fresenius
The
gemma
and provided
This species
formation (Fig. 58).
Hansen found the following temperature

12*
limits:
In
180
wort and on wort-agar-gelatine, the

development
for the
yeast
maximum
mycelium
of the
temperature
32 to 33 C, of
is
and gemmse 32 C, and the minimum tempera-
cells
ture for the mj^celium and
gemmse
The temperature
C.
development of sporangia on wort-agar-gela-
limits for the
tine are 31 to 32 C.
When Mucor
and
3 to i C.
racc7nosus is seeded on
wort gelatine,
its
felty appearance quickly changes, the surface becoming
At the ordinary room
and the gelatine liquefying.
clear
temperature after fourteen days,
it
forms 1'3
alcohol in wort, after one year 7'0 vol.
ments maltose.
At
25 C.
10 per cent, of dextrose,
it
vol.
per cent, of
per cent.
It fer-
forms, in yeast water containing
26
vol.
per cent, of alcohol after
one and a half month, and at 25 C, 2 3
vol.
per cent, of
alcohol in yeast water containing 10 per cent, of cane sugar

after one
one
and a half month.

hitherto
the
of
This fungus
investigated
the only
is
Miwor species
generates invertase, and, in consequence,
is
which
able to trans-
form cane sugar into invert sugar and to ferment the

latter.
This fungus
is
widely distributed and occurs particularly
on plums.
Mucor erectus, Bainier,
closely related to the fore-
is
going species, and was formerly confused with

not,
however, contain invertase
It does
it.
on the other hand,
it
can
transform starch into a reducing sugar.
When
which
is
it is
sown on wort
gelatine, a
growth develops
morphologically similar to M. racemosus.
At the
ordinary room temperature after two and a half months
forms 8
vol.
per cent, of alcohol after the same period.
In a 10 per cent,
solution of dextrose in yeast water at 25 C.

vol.
it
it
per cent, of alcohol in wort, and at 25 C. 7 vol.
per cent, of alcohol in fifteen days.
it
forms
35
In several respects
thus excels brewery yeast, being able to produce a higher
MUCOR
amount
of alcohol in wort
mentation,
it is
Mucor
18]
but, an regards rapidity of fer-
inferior.
Went and
oryzae,
Prinsen
spores)
are
This species
the
only
found in
is
known
"
and various organisms which

arrack in Java.
which
is
11.,
is
multiplication.
of
in a mixture of rice
i.e.,
used in the manufacture of
It transforms the rice starch into dextrose,
then fermented by the yeasts present.
cannot produce alcohol.
Fig. 60.
organs
raggi,"
Mnoii-
s/iiiii/sas,
Sporanguim
It
may
van Tiegheiii.
carrier with columella
raggi
Its
gemmae
possibly be identical
I.,
Sporangium
and loose
with
carrier with sporangia.
spores.
(After Gayon.)
the lihizopm oryzte to be described later, which

in "
The
Geerligs.
Gemmae (chlamydo-
mj^celium has rhizoid-like offshoots.
is
also
found
".
species
related to the above
same manner is
Mucor (Amylomyces)
and employed
Rouxii,
propagated
by chlamydospores and
sporangium
fructification
by Wehmer.
which is
by means of
was discovered
Calmette,
also
this latter fact
The growths are
in the
grey, light yellow to light
yellowish-brown on agar-agar, but on
rice
they are orange-
182
The
yellow.
about 50
columella
sporang'ia are colourless to yellowish, sphei-ical,
diameter, smooth and translucent.
in
/i
spherical,
is
smooth and
(5
x 28
smooth and shining.
The
are
shaped
long
the fungus
is
30 to 40" C.
is
made from
commerce
article of
Fr<;. 61.
these into
in
"
end of the
best
is
round,
colourless,
growing temperature for

found on
rice husk.s,
is
Mycelium witli underlying branched Larriers.

and the columella together with the expanded
form a pear-shaped body.
^l^.
(After Lichtheim.
sides this species, various saccharomycetes, aspergillre

Its
species, in
changing the
species has
rice
starch into sugar,
latter capable of fermentation.
can ferment sugar
but the process
is
begun
facture, but, as
and
function consists, like that of the above
bacteria.
making the
and
common
East Asia, and which contains, be-
at o have burst
carrier
The
and the spores
Chinese yeast," which
Mtiatr {^mi^mhi/er, Cohn.
The sporangia
seldom
fj.),
It
colourless,
it
itself, like
and thus
This fungus
most other species of Mucor,
best performed
to be used, of
by saccharomycetes. The
late years, in spirit manu-
seems, without real success.
MUCOR
Mucor
Tieghem
van
spinosus,
by the thorny,
tinguished
found on the columella
183
(Fig.
(Fig.
is
60),
irregular growths
these sometimes
60, II.);
appear only as bacterium -shaped growths, or they

be entirely absent.
dis-
frequently
may
sown on wort gelatine,

becomes covered with a brown felt, and is
the latter
If the fungus
Gemmae are formed.

The fungus forms 5 '5 vol. per
is
liquefied.
22 C. in a year.
of fermentation,
eight months.
cent, of alcohol in
In a maltose solution
and forms 34
vol.
it
per cent, of alcohol in
In yeast water, to which 10 per
dextrose has been added,
it
wort at
soon shows signs
produces 2
vol.
cent, of
per cent, of
alcohol in sixteen days at 25 C.
Mucor alternans, van Tieghem, according to Gayon

and Dubourg's researches, is able to change dextrin and
starch into sugars, and to ferment these substances.
It
forms alcohol up to 4 '2
vol.
per cent.
Mucor corymbifer, Cohn

in
(Fig. 61), differs considerably
appearance from the forms hitherto described.
The
mycelium
is
thick
the separate mycelium threads being very long.
felt,
white, later on light grey,
The sporangium
carriers
do not grow out
and forms a very

straight,
but form
decumbent umbellate raceme-like branches, the ends carrying

from one to twelve umbellate sporangia; below the end-
umbel these
fruit carriers develop, in addition, a
single, short-stalked, smaller,
number
of
racemose and to some extent
The sporangium carriers are expanded

below the sporangia. The latter are colourless, pear-shaped
and 10 to 70 /i in diameter. The columella is conical, broad
dwarfed sporangia.
and often warty and brownish. The spores are

very small and elliptical, being 3 /i long and 2 fi broad. The
optimum temperature is remarkably high (37 C). The
at the top
fungus
is
pathogenic to man, and has been found, for
example, in the eye.
Wort
gelatine
is
not liquefied by this
fungus at ordinary temperature even after three months.
184
(2)
Genus
This differs from the
Bhizopus, Ehrenberg.
genus in that
first
mycelium
tlie
threads develop stolon-like side branches (Fig. 62,
grow archwise through the
until their ends
air,
into contact with the substratum,
Fig. 62.
R/iirso/ms itignw.ns, Elireuberg.
carriers, there
figure.
About
(") is free
being usually more of

f.
(After
De Bary.)
f(.
when
which
peculiar adherent
Runners or stolons and sporangium
tlie latter (3 to 5)
i,
a),
come again
A columella (c)
the sporangium wall grew from
.9.
than are shown in the
the lower part of which
i;".
(After Fischer.)
c,
collapsed mushroom-like columella covered, like the previous one, with spores.
1-5--
(After Fischer.
oi'gans, rliizoids, are
i/,
Ilipe
warty zygo.spore.
developed.
',!'.
(After
De Bary.
The sporangium
appear where these rhizoids are produced.

Ehrenberg {Mucor
Rhizopus
nigricans,
Ehrenberg),
is
represented
in
Iîg.
62.
Two
carriers
stolonifer,
to
five
RHIZOPUS
185
spoi-angium carriers are found together;

2 to 4
mm.
they are about
and carry a globular blackish-brown
long,
sporangium, which encloses almost the upper half of the

columella (Fig. 62,
columella
is
b),
leaving the lower part
well-developed and dome-shaped
spores have been set free
The
free.
after
the
collapses into an umbrella or
it
mushroom shape
(Fig. 62, c). The spores are slightly angular

and provided with ridged thickenings, being about 9 to 17 /i
in diameter, and of a
fructification has
berries
grejdsh-brown colour.
been observed
and on earth-nut
(Fig. 62, d)
170 to 220
is
on unripe goose-
The suspensors are
cake.
bodied, whilst the zygospore, which

spherical warts,
Zygospore
is
thick-
covered with hemi-
barrel-shaped and has a diameter of from
fi.
This species sometimes
broken grains, and
it is
fruits, especially apples.
occurs in
large
also productive of
J.
quantity on
many
decay in
Behrens found that this fungus
secretes a protoplasm-poison,
i.e.,
a substance which has a
poisonous action on the protoplasm of the living fruit
cell.
It can also convert starch into sugar.
The
Rhizopus oryzae, Went and Prinsen Geerligs.
sporangia are blackish -brown with pear-shaped columellae,
and frequently a rim remains

sporangium wall. The size is
being about 175
fu,
after the bursting of the

variable, the length often
and the breadth about 100
/i.
The
spores themselves are somewhat angular, light grey, 7
long and 5
fj,
broad.
Gemmae
/.t
are produced in abundance,
whilst peculiar wreath-like branchings occur on the aerial

mycelia.
It changes rice starch into dextrose
and
is
a constituent
of the "raggi" mentioned above, employed in the
manu-
probable relationship to
facture of arrack in Java.
Its
Mucor oryzae has been already
stated.
186
The Higher Fungi (Mycomycetes).
II.
The mycelium
is
divided by septa.
Sac Fungi (Ascomycetes).
A.
The fungi belonging

a sporangium which
asci
may
is
to this division
form endospores in
an ascus or
called
sac.
Many
such
be enclosed in a distinct outer envelope.

Order
I.
Gymnoascea.
The
These are the simplest of the ascomycetes.
asci
have no outer coating.
True Yeast Fungi (Saccharomycetes).

General.
To
this
family belong
all
true alcoholic yeast fungi, on
the activity of which the alcoholic fermentation industry
depends
belong some of the most formidable
to it also
enemies with which this industry has to contend.

1.
The Saccharomycetes Distinct Fungi.
Since the year 1837,

is
a vegetable growth,
by various
it
when
it
became evident that yeast
has been asserted at different times
authorities that yeast
is
not an independent
organism, but only a separate stage of development of some
Experimental confirmation of this
higher fungus.
was
attempted, and, as the methods of that time were very

imperfect,
The mould
some very remarkable

fungi, especially,
parent growths
this
results
were obtained.
were accepted as the probable
assumption was apparently strength-
ened by the discovery of the formation of yeast-like

Miwor, for here were budding
were capable of forming
cells
alcohol.
cells in
which, like real yeast,
Sometimes
it
was
be-
lieved to have been proved that Penicillium or Mtocor were
the parent forms, sometimes Ustilago, Aspergillus, Sterigmatocystis,
Dematium,
etc.
Claims for the
have been put forward quite recently.
latter,
especially,
YEAST CELL STRUCTUKE
187
Whereas formerly only alcohol-forming yeast fungi were

understood under the denomination
extended later to include
became known that

forms budding cells,
and the
of Ustilago.
budding fungi.
term was
After
was asserted that the yeast
it
it
the smut found on grain,
Ustilago,
cells
alcoholic fermentation fungi of practice,
were morphologically
this that yeast is
all
" yeast," this
identical.
was again inferred from
It
derived from Ustilago.
Although
thi
was never proved, it was yet strong enough

cause confusion of ideas. Even now the word " yeast "
opinion
to
is
frequently used in text-books to designate not only true

saccharomycetes, but also
budding fungi.
all
There exists at the present time, meanwhile, no proof at

all
that saccharomycetes are a stage of development of other
On
fungi.
cetes
the contrary,
we must
equally as independent
regard them as aseoniy-
as,
exoascese, the
the
e.g.,
independence of which no one has doubted, and to which
group they are

forms (budding
and
exoascese,
closely connected, appearing in the
same
endospores and mycelium) as the
cells,
forms only.
in these
Structure and Shape of Yeast Cells.
2.
A Saccharomyoes growth
always consists of budding
under certain conditions also of
asci
cells,
with endospores and of
mycelium.
The budding
cell
mass of protoplasm
The
Cell
consists of a
in
Contents.The
cell
which was proved by Sehmitz

spherical
served
Hansen
flat nuclei.
nucleus.
and
body
To
see this
membrane
which there
nucleus, the existence of

in 1879,
has, however, in
In each yeast
it is
cell
as a rule, a
is,
some cases ob-
there
is
usually necessary to
stain the preparation (see p. 89).
cases
enclosing a
a cell nucleus.
is
Only
only one
fix,
harden
in exceptional
can they be seen without this preliminary treat-
188
The
ment.
cell
nucleus has been studied in recent times by
Jannsens, Leblanc and Wager.
Essential points with regard
and function have not yet been cleared up.
to its structure
According to Jannsens and Leblanc the protoplasm of

the yeast
its
cell
forms a
becomes granular, and
By
network
line
appearance changes
during fermentation
vacuoles appear, the protoplasm
and
fat
oil particles
may
vacuoles are understood cavities in the
filled
with
granule,
is
cell sap.
be formed.
which are
cells,
highly refractive body, the vacuole-
usually seen in vacuoles, which
constant
in
is
The
motion, the so-called Brownian molecular movement.
number
of these granules
usually one to three
is
only in
more than three or none
exceptional cases are there
at
all.
According to Kiister these bodies are decomposition products

derived from the plasma; they form a plastic, semi-fluid mass.
Using a weak aqueous solution of neutral red

1
(1 to 5,000 or
to 10,000) they become intensely red in a few minutes,
the material
colourless.
is
becoming
In the
in a sugar solution, the latter gradu-
red.
protoplasm
cell
may
aniline dyes, especially
numerous
often be seen
granules usually of angular shape.
by
remaining
suitable, all other parts of the cell
Vacuole-granules coloured in this manner give
up the colouring matter

ally
if
They
are easily stained
methyl green (Casagrandi). They
are soluble in alcohol, ether, chloroform, chloroform and

ether, caustic potash, caustic soda, petroleum
sometimes they
re(juire several
days to dissolve.
ing to Will they are of a fatty nature
observed that
when
ether, etc.
Accord-
he has further
the oily substance had been removed
by treatment with absolute
alcohol, small
faint
bubbles
appeared in the place of the granules, in the interior of
which a network could be seen.

If absolute alcohol
may
is
added
to a preparation the cells
be seen to shrink up, and are soon killed.
In general
YEAST CELL STRUCTURE

dead
cells
Section
189
are distinguished from living ones, as
II.,
b\'
we saw
the greater ease with which tliey take
in
up
colouring matter.
The Cell Wail and its Gelatinous Formation.

The
membrane of the yeast cell is very thin in young individuals
;
Fig. 63. Gelatinous network.
stained with methyl violet
Network formation and yeast
I.,
;
most of the
right) still lie in the meshes.
network forms complete walls

lying in the meshes.
in old cells,
and
various reagents
to Will's
It
(From Hansen's
it
may
which are
cells
have disappeared
some (on the
be seen in a, h and
there are in a 3
cells, in b 1,
and
that the
in c 2 cells
original drawing.)
in such as live
tions of nutrition,
micromillimetres.
II.,
;
cells
under unfavourable condi-
may become tolerably thick,
e.g.,
several
It can be shown distinctly by means of

{e.g.,
dilute acid
and
alkalis).
According
and Casagrandi's experiments the membrane con-
.FERMENTATION ORGANISMS
190
two or more layers
sists of
the
cells for
this
may
be shown by treating
a long time (days to weeks) with a
per cent,
The membrane dissolves easily in

more slowly in concentrated
solution of osmic acid.
concentrated chromic acid,
sulphuric acid (Casagrandi).
The membrane
ditions,
the
of
of the cell gives
a mucilage which
network
gelatinous
under certain con-
off,
takes part in the formation
by
described
Hansen
(Fig.
After hardening an ordinary microscope prepara-
63).
tion
it
shows
the form of strands and plates
in
itself
The granulations
originally present between the cells may be taken up into
the substance of the network, which may be stained by this
means. This formation may be readily obtained if a lump
between which the
of thick yeast, as
it
cells
usually occurs in breweries,
a glass, covered, and put
drying takes place.
are enclosed.
away
is
placed in
for a short time until partial
It usually occurs also in spore cultures
on gypsum blocks, on gelatine and in yeast ring formations.

Will considers the network, which appears,
e.g.,
during
the drying up of beer yeast, to be different from that which

occurs in film formations and in the yeast ring.
opinion
gelatinisation
the
of
takes part in bringing about
cell
membrane
In his
possibly
the former, the albumen
mixed up with the yeast, however, playing the chief

part.
The networks formed in the film and in the yeast
ring
since
may also,
according to him, be of different constitution,
some forms are found which give the albumen reaction
and some which do
not.
It
is
very
difficult to distinguish
here what, in the network formation, originates in the
what in the
surrounding medium this
wall
itself,
cell
is
contents,
and what
cell
in the
perhaps a problem which does
not as yet admit of a solution.
Shape of the
Cells.
Budding
cells
occur especially in or
upon nutrient liquids, but are also found on solid substrata.
MODES OF PROPAGATION
Their shape
is
exceedingly varied
191
they are spherical, egg-
shaped, oval, sausage-shaped, filament, dumb-bell, and lemonshaped,
besides occurring
etc.,
now and then
quite irregular and abnormal forms.
no
fact that
one species
It
is
in cultures in
a noteworthy
definite cell shape is absolutely peculiar to

it
true that the majority of the cells of
is
a species, under certain conditions of culture, occur in a

certain shape
will
but
these conditions are altered, the shape
if
alter also.
Saccharomyces species can seldom or
never be determined by microscopic
Hansen
observation
clearly demonstrated this multiplicity of
alone.
form
of
the species and the defectiveness of the classification which
had been employed before
showed
that,
shape of the
under
"
Variation
Vegetative
of budding.
for
all
new
true saccharomycetes by
This takes place by a small outgrowth
forming on the mother

ing in
characteristic
".)
increase (the formation of
vegetative cells) takes place in
means
the
culture,
Modes of Propagation.
3.
Budding.
233,
p.
conditions of
a good group
cells affords
(Compare
species.
Simultaneously he
his time.
definite
cell (Fig. 64)
and gradually
increas-
size.
The new
cell
may
separate from the mother
remain connected with
buds are formed
it
(Fig. 64).
cell,
or
may
in the latter case large colonies of
In the genus Schizosaccharomyces
vegetative increase takes place not by budding, but by
Near the middle of the mother cell is formed

a septum which splits up and thus sets the new cell free.
The budding of a single cell was studied by Mitscher-
splitting
lich in
off.
1843; he distributed a
as to obtain one or
paration,
(Fig. 64).
two
cells in
and gave a drawing
Under
little
yeast in beer wort, so
an ordinary sealed up preof the various generations
these circumstances thirteen hours passed
192
had formed a new cell of the same

When the preparation was three days old the number
before the seeded

size.
cell
of descendants of the original
cell
had increased
Kiitzing proceeded in the same way.
nine.
after,
twenty-
Pasteur also repeated this experiment, but with the
difference that, instead of placing
he used grape
yeast
to
Several years
cells
He
juice.
wort on the cover
glass,
then found that each of the two
present had formed three cells in the course of
two hours.
Fig.
64. Multiplication
9 A.M.
of top yeast:
IV., lOJ A.M.
28/5, 8 A.M.
XIII., 30/5;
V., 12
I.,
26/5, 7 r.M.
NOON;
VI., 3| P.M.
II.,
;
27/5, 8 a.m.
VII., 8 P.M.
IX., 10 A.M.
XI., 1 P.M.
XII.,
X., 11 A.M.
XIV., 2/6, 12 o'clock. (After Mitscherlich.)
;
III.,
VIII.,
29/5, 8 P.M.
In studying the budding of yeast by the above method

Ranvier's moist chambers (see pp. 69 and 93) are the most
convenient to employ.
may
be obtained by studying a series of drawings from
history, such as those

84,
Further information on this subject
from Hansen given
life
in Figs. 82, 83,
118 and 119, in which data with regard to time are
furnished.
193
Further thorough investigations of yeast multiplication
under various conditions have been made by Rasm. Peder-
and afterwards by Hansen, Hayduck and
sen,
method employed
the yeast
cells
others.
The
in these experiments consisted in counting
by means
of counting chambers.
Rasm.
Pedersen carried out his experiments with brewery bottom

yeast which had been cultivated in unhopped wort.
times required to complete the generation of a

for different temperatures, as follows
hrs. at 13-5
C, 65
hrs. at 23
C,
cell
The
were,
20 hrs. at 4 C, 10"5
5-8 hrs. at 28
C, and
9 hrs. at 34 C.
As one might
expect, the various species
increase at different rates.
first
and races
proved by Han-
comparative experiments on Saccharomyces apiculatus
sen's
and some bottom yeast
it
would be necessary
number
of
These will be more fully
species.
In order to characterise species in this
described later on.
way
This was
similar
to carry out an extremely large
experiments
this
has not yet been
done.
The vigour with which vegetative
increase proceeds
depends of course on the conditions under which the species

are cultivated, and the terms energy of multiplication and
power
of multiplication under certain conditions of cultiva-
tion are employed.
stood the
time,
number
and power
number
By
energy of multiplication
of cells produced
of
hy one
multiplication
cell in
is
under-
a certain
denotes the absolute
of cells which one cell is capable of generating.
Aeration of the culture liquid must be mentioned as one

of the foremost factors in accelerating vegetative increase.
Temperature also plays an important part, and as important,

of course,
is
the chemical composition of the culture liquid.
Examples of excellent culture liquids are those in general

use in the fermentation industry,
viz.,
wort, must,
lowest temperature at which budding
13
is
etc.
The
observed with
194
saccharomycetes
C.
but species
near 0 and the highest
lies
Sacch. Pastorianus
and Sacch. Pastorianus
I.
is
about 47
For example,
differ also in this particular.
II.
have con-
minimum temperatures than Sacch. cerevisia

The maximum temperature for Sacch. Pastorianus I. is
siderably lower
I.
about 34 C, whilst Sacch.
cerevisice
exhibits a vigorous
I.
increase at 40 C. and Sacch. Marxianus even at about 47

C.
is
The above holds for those
cases in
which the culture liquid
the ordinary hopped wort (14 per cent. Balling) as used in
Although the opinion ob-
bottom fermentation breweries.
tained formerly that top yeasts were able to produce buds

at higher temperatures than^bottom yeasts, this
is
case.
budding ceases
much lower temperature than
at a
certain bottom yeasts,
e.g.,
a yeast
to the bottom and forms a colony, as

foregoing; gradually the
and form new
cells
T he
colonies.
If it
we have
cells to
be carried about
cells rise in
large
and may form there a thick layer
When
of yeast on the froth.
sinks
separate from one another
a top yeast, the
is
quantities to the surface
it
seen in the
de velopment of^jiarbonic acid
gas during fermentation causes the

in the liquid.
that for
ellipsoideus I.
seeded in a nutrient liquid,
cell is
from
the top yeast Sacch. Pastorianus
III and the bottom yeast Sacch.
When
far
There are top yeasts in which
being generally the
fermentation
is
finished, the
yeast again sinks to the bottom and there forms a more or

less solid
sedimentary yeast.
Good brewery yeast usually
gives a dough-like deposit which
while on the other hand
sedimentary yeast which either
and lumps or
is
may
lies
on the bottom,
yeasts give a cheese-like
on the bottom in crumbs
partially distributed throughout the liquid
in a finely divided state.

species
lies close
many wild
By
a certain treatment several
be induced to form cheese yeast.
may^lumever,
after.flfiEmentation is over,
Single
cells
remain on the
surface of the liquid and form a film there just as jome
195
develop a yeast ring on the wall of the vessel along the
edge of the^rface"of Ifee Ir^md.
Film formation at the surface of fermenting liquids

a phenomenon of widespread character
microscopic
different
were not
As pure
fungoid forms.
is
occurs in manycultures
use long ago, the observations then
in
often related
common by
it
made
such film growths as were formed in
to
and
The exact study of this subject was
begun by Hansen, and it was chiefly with the following
several different species, saccharomycetes
non-saccharomycetes.
six species, to be described later, that he carried out his
experiments
and
III.,
Sacch. cerevisicB
and Sacch.
investigations
was
I.,
ellipsoideus
as follows
growth may form a
I.
Sacch. Pastorianus
and
The
11.
I., II.
result of his
In order that a Saccharomyces
film, the culture
must be allowed
to
remain at rest for a long time with an abundant supply
Now
there
are a few species, eg., Sacch. anomalus and Sacch.
memhow-
of
air,
the temperature being tolerably high.
branafaciens,
which form a
ever, is the usual
sedimentary yeast
form of growth in these individuals;

is
seldom or never formed directly by
them, but only by the film

also distinguished
cells
sinking.
These films are
from those of the typical saccharomycetes
between the
their dull appearance, air being present
by
cells as in a
are,
in
film at once; the latter,
Mycoderma film
films of typical saccharomycetes
The
on the other hand, slimy.
what follows.
The microscopic appearance
same
latter will be treated
of film cells belonging to the
species varies for difl'erent temperatures
the limiting
temperatures of film formation as well as the time of
appearance for different species at the
vary
also.
Thus not only are
details
its
same temperature
given about the most
important conditions for the appearance of the film form, but

also
new
characteristics of the species.
13*
196
Flu.
Q5.
SuCi-Jutnuni/res
Hanseu.
tiediment
(After Hansen.)
Flii. 67.
Stirc/urroiin/ri'.-i
Hansen.
/.,
cere rim'.
yeast.
J.,
J^tistnruntiis
Sediuipnt yeast.
^'^^.
cerevisiw 1.,
Film growth at 156

(After Hansen.)
('.
Sarc/iarmiiytr.s PastorianuH I.,

Hansen. Film growth at 153 C. ^p.
Fic. 68.
(After
Sacchaminyrex Paalm'iauus
Hansen. Sediment yeast. ^s.
69.
//.,
Sacduiromyres
Hansen.
A-a.
(After Hansen.)
Fig.
Fig. 66.
^}^\
Fig.
{After Hansen.
Holm
70.
in
âccltaromi/fes
11., Han.sen.
(J.
Han.sen's treatise.)
Ji^f.
Pastifrianus
3
Film growth at 15
(After
Hohn
in Han-seu'.-i
treatise.
t^K
?= (''''^-^
Fig.
1\.- Sacflwrimiycss
iôstorUinus IIL,
Hansen.
(After Hansen.)
Sediment
veast.
i<^'~.
Kli:.
72.
Srir,-lia,-i'i,n/cf.i '/'iiildri'i.an.s
iii.
197
Film growth
ffl., Hansen.
at
153^
C.
(After Hansen.)
^^G
Fig. 73.
/.,
Sacchid'omyce-^ elUjjsoUleus
Hansen.
Sediment
yeast. ^\^.
(After Hansen.
Fig. 74.
Sacdummiyces
ellipsoideiin J.,
Hansen.
Film growtli at 1513 C.
^" " (After Holm in Hansen's treatise.)
(j?<
Fig. I't..Saccliariimijces ellipsnidens II.,
Hansen. Sediment yeast.

Hansen.)
''1^.
(After
Fiu. 7&.Sncrliariimyce.s eUipsoii/evs

//.,
Hansen.
28 3
C.
--fi'.
Film
growth
at
(After Hansen.
198
With regard
cells,
to the microscopic appearance of the film
these are, as a rule, very long shaped in the older
growths, and the
and then they
cells
also develop a
film formation
now
Spores are found
mycelium.
an exception, namely, in that species
in the film cells as
of
tend to assume irregular forms
called
The
the yeast ring.
latter is
formed, as mentioned above, along the edge of the liquid

surface on the wall of the flask and
very marked in
is
certain species.
For instances of the varied appearance of the film

in different species as well as
cells
when compared with bottom
yeast, see figures 65 to 76.
The
difference in the film cells of these species
most
is
noticeable at 13 to 15 C.
The highest and lowest temperatures at which film forhas been observed by Hansen in the different
species were given by him in 1886 in the above-mentioned
mation
They
treatise.
are as follows
Sacch. cerevisia
I.
Sacch. Pastorianus
33 to 34 C. and 6 to 7
II.
I.,
and
III.
26 to 28 C.
and
3 to 5 C.
Sacch. eUipsoideus
Sacch. eUipsoideus
I.
II.
33 to 34 C.
As regards the time required

the film, Sacch. eUipsoideus
and
36 to 38 C.
II. is
6 to 7 C.
and
for the
3 to 5 C.
development of
especially noticeable, this
species developing a very vigorous film in about ten
often
earlier
require a
much
at
22 to 23 C.
The other
days
five species
longer time.
Will has shown that the different generations of cells
formed in the film exhibit
dissimilarities.
His
results,
however, cannot be further referred to in a limited book

like
to
the present.
We
therefore refer
those
who
desire
study these conditions more particularly to his papers
(1895 and
1899) mentioned in the literature review for
this section.
With regard
199
to the limiting temperatures
of film formation for four of the species of beer
yeasts
Fig. 77.
bottom
examined by him, a minimum temperature was
Saccharomyces Luduiigii, Hansen.
Mycelium and spore formation from
very old cultures in cherry juice and yeast water respectively.
I.
to IV.,
Mycelia or fragments of such with broad thick septa.

V., An irregular,
branched small mycelium completely devoid of septa. VI. Mycelium threads,
î>^-^.
(After Hansen.
also with broad septa
in each cell (asous) 4 spores.
,
found at 4 to
C. for two,
and at
the other two, while he found the
to
maximum
10 C.
for
temperature
200
for three species to be near 30
C,
and, for the fourth,
28 C.
When
a growth has formed
a film, special
changes take place in the nutrient
chemical
Thus wort
liquid.
usually assumes a light yellow colour under these conditions,
and develops a disagreeable smell and

of the film, however,
is
taste.
The formation
not always to blame for
this, since
beer which has stood for a long time, and on which no film
has appeared,
may
likewise assume the above properties.
According to Raymann and Kruis, the
Fro.
78. Restiug
Cells Germinating.
film cells, if allowed
(After Will.)
to remain for a long time, are capable of converting the
alcohol formed in the culture liquid into carhonic acid.aod.,

.
water by oxidation.
Hansen showed
that,
under certain conditions; saccharo-
mycetes can also develop a mycelium provided with septa.
He
observed such an one
Sacch. Ludwigii (Fig.

species.
77),
first in
Sacch.
and afterwards
It occurs chiefly in old films
on solid nutrient material.
and
mycelium of
Marxianus and
in
some other
in old cultures
this
kind then
frequently shows forms similar to Dematium or Monilia.
As an example
201
of the influence of temperature on the
above, an observation
made by Hansen may be mentioned
bottom yeast No. 1, which

had been for a long time in a saccharose solution, and
which gives a mycelium on cultivation in wort at a low
here, of a culture of Carlsberg
temperature, but not at a high one.

transitions in cell shape can be
Further,
possible
all
found from round
cells to
colonies with branched mycelia, consisting of very elongated

cells.
Of
late years
P.
Lindner, Will and others have also
observed such mycelium forms in various typical saccharomycetes.
In addition the resting
described
by Will
sometimes form, when germinating, mycelium -like
colonies,
the
cells of
cells
which are often furnished with septa (Fig. 78).

cells are to be found both in film and yeast
These resting
ring formations.
which
They have
consists of two,
a strong thickened
sometimes several, layers
shown by treatment with hydrochloric

glycogen and
latter that
oil
globules.
acid),
membrane
(this
can be
and are
rich in
Will says with respect to the
they are soluble in alcohol and become grayish-
green, later brownish-black
by the addition
of concentrated
sulphuric acid, in contradistinction to the fat globules of
sediment yeast
and
cells
which are
diflBcultly soluble in alcohol
by concentrated sulphuric acid. In

cultures in which all other cells have died these resting or
" durative " cells may still be found living.
The more unare not coloured
favourable the composition of the culture liquid
is
for the
multiplication of yeast the quicker are cells turned into

resting
cells.'
When these germinate, the
in the neighbourhood of the point at

is
formed, the quantity of fat dwindling
tion proceeds.
fat globules collect
which the daughter
specially characteristic
away
mode
cell
as germina-
of germina-
tion consists in the sprouting of club-like or sausage-shaped

cells
from the resting
cells,
septa then appearing in the
202
former.
Cells with
may
an ordinary thin wall
alive a very long time
and thus appear
also
keep
as resting cells.
Colonies formed from saccharomycetes on solid nutrient

substrata also present some marks in their appearance which
by no means
serve to distinguish between species, but
every
case,
Hansen
Fig. 79.
and here
Saccharomycetes
5.
I.
whicli form
3.
Siicxh. eitipsoideuft I.
cells
we have
ascospores.
6.
a, cells
cells
with septa
A,
with distinct indica-
drew attention
among them
be brought about by the culture
He
c,
2.
I.
Sacch. Pastorianua III.
{After Hansen.)
About'"?-.
Saccharomyces already mentioned,
perature.
4.
Sacch. ellipsfiihus II.
fact that differences exist
I.,
Sacch. cerefisicK
1.
Sacch. PastorUinns II,
with more than normal number of spores
tions of spore formation.
may
in
reckon on variations.
to
communication on the six species of
in his hrst
Sacch. Pastoriaiius
also
in this respect
to the
which
medium and by tem-
found, for instance, that Sacch. elUpsoideux
cultivated on wort gelatine at 25
from the other
five
species,
assuming a net-like structure
C,
is
very different
the surface of the colonies

;
further, that Sacch.
Pas-
torianus II. in streak cultures in yeast
15 C.
develops,
in sixteen
203
water gelatine at
colonies with
days,
smooth
edges, whereas the latter are hairy in Sacch. Pastorianus
under the same conditions of cultivation.
III.,
Aderhold,
Lindner and others have made subsequent communications

on differences among the species in the above respect. For
this
purpose Lindner sows drops of yeast on nutrient gela-
tine
and thus obtains the
Spore Formation.
so-called giant colonies.
Besides vegetative
increase
by bud-
ding, saccharomycetes, like all other ascomycetes, form endospores, the cell being transformed into an ascus (Fig. 79).
Schwann, in 1839, first observed spores in yeast. They are
not mentioned again until 1868, by de Seynes, and were
described for several species
by Reess
The most
in 1870.
contradictory views prevailed with regard to the conditions
Hansen published
of their formation until
For example, the
1883.
only enfeebled
cells
belief
washed
up
in
till
his researches in
then had been that
water were capable of
forming spores, and that the most favourable temperature

lay near the freezing point, whereas Hansen showed that
this
view
was
entirely
erroneous.
He
found, on the
contrary, that spore formation does not, as a rule, take

place under these conditions, and that young, well-nourished
cells
must be sown
obtained.
are:
a strong spore formation
if
is
to be
According to him, further essential conditions
abundant moisture, plenty
of fresh air
and a com-
paratively high temperature (for most species yet investigated,
25
C.
is
a good temperature).
He
carried out
thorough and comprehensive experiments, particularly on

temperature conditions
these will be described later on.
His methods of spore culture are described in Section IL,

p.
121.
There are species of which the young
under almost
all
cells
form spores
conditions of cultivation, even under such
204
conditions as are very unfavourable for the majoritj'' of them.
growth
eon,sisting of old cells gives a less vigorous spoi'e
formation
at
the growth
if
is
very old
does not sporulate
it
all.
Quite recently Hansen made additional researches on

this point,
and
has,
among
other things, defined
these investigations
vigorous
which
is
comprised
in
tlie differ-
The
ence between spore formation and budding.
the following
result of
If
young,
are bro>ight into a thin layer of water to
cells
air has free access, colonies are
formed (even
if
the culture liquid has been removed from the cells) at
by budding
Fit:.
hereafter spore formation takes place, begin-
Stii:chnn>tin/i:fy
SO.
all
first
ccrfrisiii
I.,
Hansen.
Spores at conimenceineut of
Formation of a septum niay be seen at
c and g.
In e,f
septum formed by
the coalescing of three spores into a three-winged spore body; the enclosing
i-","".
(After Hansen.)
wall of the latter is burst in tliree places,
êrmiuatioii.
and
ning
//
the walls of
first in
tlie
mother
the mother
younger members of the
cells liave burst
cell
f/
the yeast
cell
colony..
It
this to the
After several days spores
may
cells,
i.e.,
such
cells
bo seen from this that
can produce spores directly without previously
forming buds.
This also happened
when
yeast in a solution of calcium sulphate.
markable
(/,
and extending from
are generally also found in the youngest

as have not put forth buds.
a,
sltows a
fact
brought out by Hansen
cultivating a wine
Hut the most
in this
connection
that the spore itself can occur as a spore mother
happens when
tlie
spore, after
reis
This
cell.
being a short time in a
culture liquid containing sugar, has swelled
up and
is
then
transferred to an aqueous solution of
No
205
calcium
sulphate.
buds are then produced, and instead spores are formed
in the interior of the swollen spore.
Spores, like vegetative
consist
cells,
which encloses protoplasm and a
of a
nucleus.
cell
membrane
They have
the same soft consistency as the latter, but possess greater
power
to resist drying, heating, etc.
Their shape
they are most frequently spherical

L,
Figs. 79
(1),
80, 81, 82
and
tendency to an ellipsoidal shape

are kidney-shaped
"
shaped
(e.g.,
{e.g.,
89),
;
is
Sacch.
(e.g.,
varied
cerevisice
with greater or
less
in particular species they
Sacch. Marxianus), in others "hat-
Sacch. anomalus. Figs. 83
and
102),
i.e.,
shaped
a\''
Sacrlumimycea
Fig. 81.
LPU
^0
cerevisice I., Hansien.
Germination of old spores.
-i-\'^.
(After Hansen.)
like the
the edge.
segment of a sphere with a projecting rim round

In some a highly refractive body
middle of the spore
{e.g.,
of spores in a cell varies
Hansen found
that
is
.Sacch. hyalosporus).
from one to eleven

there
was a
found
in the
The number
(Fig. 79).
difference
between
and wild yeast in the structure of the spore

The spores of culture yeast appear to be empty,
culture yeast
plasma.
while,
on the other hand, the spores of wild yeast are
strongly refractive.
This difference
is
of importance in
the analysis of brewery yeast, although the details have
not yet been specified.
An
appearance often observed in the spore-bearing
cell
206
is
the growing together of the spore walls, a septum being
thus formed (Fig. 80,
The
g).
a many-winged spore
Sometimes
body,
cell
can thus be changed into
its
walls forming one unit.
also pseudo-septa are
formed between the spores
by the latter compressing the plasma which

them (Figs. 79, a, and 80, a, d, e).
lies
between
Hansen. Germinating spores. The series

/.
Temperature
was cultivated on wort gelatine, the others in wort.
about 20 C. a and b dried some time beforehand. Time data reckoned from
beginning of experi-: ent. u, Three spores connected, no mother-cell wall
Fic. 82.
Saccharomyces cererisur
e-e'""
a" after 22, a'" after 30. b, A cell witli four spores
with four spores c' after 9 hrs. i" after 10^. rf, A
a' after 19 hrs.,
18 hrs.
c,
three spores;
after 25.
e,
cell
rf'
after lOJ- hrs., t^" after 13,
cell
with two spores
t^'"
after 17,
d"" after
e'-e'"" after 7^, 85, 11,
b'
cell
after
with
21, d'""
20 and 50 hrs.
f and f/, Two cells with spores ,/', g' after 22 hrs., /", g'' after
25. A, A cell with two spores h' after 9 hrs. h" after 13 in It" the wall between
the two spores has disappeared, and both have grown into one. ^y^.
respectively,
(After Hansen.)
Spores free themselves from the mother-cell by swelling up and causing the wall of the mother-cell to burst.
Hansen found
the following two types of germination
First
type
germination takes
place
207
like
ordinary
budding, and can occur at any point of the surface of the

spore (Figs. 80, 81, 82 and 83).
Sometimes
it
begins while the spore
Septum
mother-cell.
when
Sometimes,
formation
the
Examples
parasite.
and
II.,
Sacch. cerevisice
I., II.
and
partially dried
at 28
C,
gypsum block
and
c at
b'-b'"" after 10, 21, 24,

c'" after
tion,
(Figs. 80, 81
I.
and
III., Sacch. ellipsoideus I.
21 hrs.
23 C.
culture.
Spores germinating from an old,
Cultivation took place in dilute wort
a'-a"" after 7, 12, 15
25 and 27 hrs. respectively
i-"/".
and 20
hrs. respectively
after 8, c" after 10,
c'
and
(After Hansen.
Second type (Figs. 84 and 85)

generally
place.
Sacch. anomalus (Fig. 83).
Fig. HZ.~~Saccka.romyces an&malus, Hansen.
in the
grow together, one takes

and accordingly acts as a
spores
nourishment from the others,

82), Sacch. Pastorianus
lies
takes
still
usually
grow together
Two
or
more spores
in the very first stages of germina-
yet old spores are able to germinate individually
without growing together.
Germination begins with a
wart-like or sausage-shaped lengthening which grows on

and often occurs as germ threads or bunches. Only from
this promycelium is the development of the yeast cells
effected later on, a partition wall being first
formed between
the promycelium and the young yeast
the latter
tached by fission of the wall.

(Figs.
84 and
85).
cell
Example
is
de-
Sacch. Ludwigii
208
By
most saccharomycetes belong to the
far the
first
type.
^^'
(S?
^'^S
nX'^J
Fig. 84.
Saccharomyces Ludwigii, Hausen.
gypsum block
culture
a, b
and
from a
e,f and g
The cultivation was made in
Grermination of
were \\ months old at the room temperature.

wort, a at 25
C,
after 8 hrs.
a'' after 25,
groups
after
the others at 18 to 20 C.
9^
a'" after 26.
hrs., b" after 12.
c'-c'"" after 12, 15, 20,
o,
b,
a,
A cell
cell
cell
and 33
rf,
with four spores
a'
with four spores in two
with four spores in two groups
24 and 27 hrs. respectively,
d'-d'""" after 18, 20, 26, 28, 29, 30^
f after 19 hrs.
spores
were 12 days old at 25 C,
d,
Two
hrs. respectively.
free
/,
spores
Four
free
group of three spores, of which the two lowermost, A, were connected, but are separated from one another by fission the
uppermost spore, ^, has separated itself in the same way from a fourth spore ;
g'h' after 17 hrs., g"h" after 21, g"'h"' after 23, j""A"" after 26^, (?'"" after 28.
spores
gh,
The lowermost spore
in this
group did not develop.
(After Hansen.)
Just as Hansen's investigations on film formation have
been incorporated in the system of yeast analysis, so also
have
his
above experiments on spore formation been fruitful
209
as regards important characteristics for distinguishing groups
and single species
he based on this his method of analysing
brewery yeast described
in Section
II., p.
134.
It
was found,
that in different species at the same temperature spores
first,
begin to form after different time intervals, and, secondly, that

the temperature limits of spore formation are different for
Hansen thus determined the spore curves

by observing for a series of different temperatures the times at which the formation of spores first began.
The cardinal points, viz., the maximum, optimum and minidifferent species.
for six species
mum
temperatures, have special significance, and of these
wtpâh
Flu. 85.
Germination of spores from an old

Sacchanrmyces Ludwigii, Hansen.
culture.
The germination took place in dilute wort, a and 6,
gypsum block
in which each spore has developed its
Groups of spores
represents the
c
first
various forms of coalescence
particularly the
may
own germ
a
group
(After Hansen.
stages of germination, b a further development
first
and
may
be seen.
last.
i|i
to
i-J-i.
Sacch. cerevisia I.
C.
35 C.
thread,
in the
The two following examples
serve as illustrations of such spore curves

At 37J
no spores develop.
210
Sacch. Pastorianus
At 31J
C.
I.
no spores develop.
29J-30J C. first indications appear after 30 hours.

29 C.
27
24
27J C.
26
23^ C.
35
,,
18 C.
,,
15 C.
10 C.
8J C.
7 C.
3-4 C.
,,
J C.
50
,,
89
,,
14
5 days.
no spores develop.
Other investigators have pubHshed similar curves for

other species.
Those given by Will for four species of
brewery bottom yeasts are especially noteworthy and
will
be considered more fully in the systematic description.
Fig. 86.
Schiziisaei:lmromyaôcUisporus,'Bei}eT\Ti<ik.
Formation of the ascus.
a round cell shortly before the formation of the septum.
and VI.,
after 1, 3, 6,
10 and 17
hrs. respectively..
from the commencement of the observation.
It
may
^"^J
II.,
The times
III.,
are
I.,
IV., V.
reckoned
(After SchiSnuing.
be seen from the above two series of numbers
that spore formation proceeds very slowly at low temperatures,
but more quickly as the temperature rises until a
certain point,
namely the optimum,
is
reached, after which
spore formation proceeds the more slowly the nearer the
maximum
temperature
is
approached.
Hansen's experiments on the
him
to state the general
effect of
law that the
temperature led
maximum temperature
for the formation of spores in Saccharomycetes
always
lies
several degrees lower than that for bud formation and the
minimum temperature a few
degrees higher.
He determined
the temperature limits for budding and spore formation in
CHEMICAL CONSTITUENTS OF THE CELL

maximum
tempera-
between 47 and 34 C, the
minimum
In these species the
eleven species.
tures for budding
lie
211
temperatures between 3 and
C, the maximum tempera-
between 37 and 28 C, and the

minimum temperatures between 11 and 3 G. Those species
tures for spore formation
which have the highest temperature maxima for budding

and spore formation have the same also for film formation.
Schionning has observed a peculiar mode of ascus formation, in a single species
charomyces,
belonging to the genus Schizosac-
Schizosacch. octosporus.
viz.,
This takes place in the following manner

(Fig. 86) enlarges in
The two new
cells
either touching one another or connected together at
lie
one point
form a
cell
cells
gradually
shaped like an hour glass
grows and the hour
cell
(IV.).
glass shape disappears (V.)
assumes an ellipsoidal shape and the ascus
finallj'^
it
formed
(VI.).
4.
The
In ^ further stage both
(III.)-
coalesce so as to
The
cell I.
(II.).
After a certain time fission takes place.
now
The
one direction and forms a septum
cell
The spores
are then formed in the latter.
The Chemical Constituents of
membrane
is
the Cell.
consists, according to Casagrandi,
probably of pectose or perhaps of an analogous pectin sub.stance,
and the
chief constituents of the protoplasm consist
of albuminoids.
Glycogen and
fat are frequently present
in large quantity.
showed that yeast cells contain glycogen. Kayser and Boullanger found that, with a plentiful
air supply, there is always less glycogen formed than with
Errera (1885)
a small air supply.
first
The greater the amount
of sugar present
or the weaker the acidity of the substratum the more gly-
cogen
is
formed.
Large doses of tartaric acid are said to
be very effective in preventing formation of glycogen.
Otherwise the constituents of yeast
14*
cells
are substantially
212
the same as those of the higher fungi,
and yeast gum belong
carbohydrates,
viz.,
nitrogenous and mineral substances.
fat,
Pectose, glycogen
and the
to the carbohydrates,
al-
buminoids, peptones and various enzymes to the group of
The mineral substances are
nitrogenous bodies.
phoric acid, sulphuric acid,
silicic acid,
sodium, magnesium and calcium
phos-
chlorine, potassium,
and
phosphoric acid
potassium are present in largest quantity.

Formerly, too
much weight was
attached to the chemical
investigation of yeast, and thus quite unsupported conclusions were draMoi as to the
There are
still
some who
all
of the yeast in practice.

'^
Fermentation Pheiwmena.
5.
Nearly
work
cling to these fallacies.
the saccharomycetes produce a fermentation
in nutrient liquids containing sugar.
This can be recog-
nised by the froth which forms on the surface of the liquid,
and which
is
caused
by the
carbo_nic acid gas
developed by
the breaking up of the sugar.
Ferments.
sugars was
species.
species
The
first
He
action of the alcoholic yeast fungi on
studied
by Hansen by means
of pure culture
investigated the action of forty different fungoid
and races on four
different kinds of sugar.
With
regard to the saccharomycetes he established the following

three types
(1)
and saccharose,
species
e.g.,
which ferment maltose, dextrose
brewery yeast species and the other
culture yeasts, as well as most of the wild yeasts
which ferment dextrose and saccharose,

anus, Sacch. exiguus, Sacch. Ludwigii,
e.g.,
and
(3) species
ferment neither dextrose, maltose nor saccharose,

'
The contents
of the yeast cell are of
(2)
species
Sacch. Marxi-
e.g.,
which
Sacch.
such a composition that yeast
refuse has in recent times been found of value, sometimes as forage
and
has also begun to be used in the

preparation of a, substitute for coffee and in the manufacture of soap.
This is, of course, of great economic importance to the brewer.
as a food, sometimes as a medicine.
It
FERMENTATION PHENOMENA
membrancefaciens.
yOi
213
the sugars, dextrose, d-mannose and
d-galactose are fermented direct]j% the others only after
previous hydrolysis
by
recently,
by means
of enzymes.) E. Fischer has
his chemical researches,
made important
con-
tributions towards the solution of these difficult questions.
Whereas formerly only one enzyme was known

present in yeast,
viz.,
to be
or invertase, which has

up saccharose into dextrose and
into melibiose and fructose, Fischer
invertin
the power of breaking

fructose,
and
raelitriose
found (1894) another enzyme,
viz.,
yeast glucase or yeast
The reason
enzyme was not discovered sooner may be explained by the fact that it cannot be extracted from the
maltase, which turns maltose into dextrose.
why
this
uninjured yeast
the yeast
among
cell.
According to Fischer this conver-
maltose into dextrose goes on in the interior of
sion of
cell.
third enzyme, which
species of bottom yeast,
is
up melibiose into dextrose and galactose.

/
is
melibiase,
Fischer's investigations that hydrolysis
found chiefly
which breaks
from
It follows
precedes
all
fer-
mentations of polysaccharides, and that the action of yeast
on sugar
is
of a purely chemical nature.
the following law
For a yeast
He
further states
to be able to attack a sugar
the stereo-chemical structure of the albumen molecule of the
yeast
cell
must not
differ to
any great extent from that
of
the sugar molecule.
These discoveries preceded those on the alcohol-forming

ferment by E. Buchner (1897),
zymase.
By
who gave
the latter the
name
subjecting the yeast to great pressure he
succeeded in obtaining a juice which was able to cause

fermentation in sugar solutions.
The enzyme lactase, which breaks up lactose into

and dextrose, seldom occurs in saccharomycetes.
Only those yeasts which contain this enzyme are capable
Duclaux (1887) was the first to
of fermenting lactose.
d-galactose
214
find a yeast
which ferments
have since described
and
lactose,
But
others.
in
later investigators
most cases
it
cannot
be decided whether these are typical saccharomycetes, as

it
is
not stated whether the species form spores or not.
Jorgensen has quite recently described a species of this

kind, Sacoh.
fragilis.
Besides the above sugar-transforming ferments,
sac-
charomycetes also contain a ferment similar to trypsin,
which can peptouise
gelatine,
the
This
may
liquefied in the process.

cells
latter
substance being
be observed
when
yeast
are seeded on culture gelatine and the culture has
attained a certain age.
This proteolysis, as
yeast varies in different species.
it is
study of
it
called, of
has been
made by Beijerinck, Wehmer and Will.

One of the most constant characteristics of the saccharomycetes is the enzyme content. A yeast species cannot,
as Dubourg states, be brought by culture to such a state as
to ferment a sugar
It
is
which
also impossible
species to lose those

cell can,
it
could not previously ferment.
by any treatment
to cause the various
enzymes which they contain.
The
however, by means of a certain nutrition, be made
to ferment
more or
less of a particular sugar.
The author
has carried out experiments on different yeast species,

following Dubourg's method of procedure, but always with
negative results.
Influence of Air and Temperature on Fermentation.
Among
the factors
which exert a considerable influence on
the progress of fermentation, besides that of the chemical
composition of the liquid, those of temperature and the
amount of oxygen present in the liquid may be mentioned.

The usual limits of temperature within which fermentation
may take place at all are 0 and 40 C. This much is
known of the effect of oxygen on the progress of brewing
in practice
that, in
order to get a good result,
it
is
necessity to atirate the wort,
from the
to let
i.e.,
it
215
take up oxygen
Pasteur's researches on this subject led
air.
to state the theory that fermentation
is
life
him
without air
and that yeast can only decompose sugar by taking the

necessary oxygen from the sugar molecule.
His theory
proved to be wrong, as we have seen.
has, however,
found that by supplying oxygen multiplication

but fermentation
restrained,
is
up oxygen from the
is
He
favoured,
and further that wort takes
two ways, viz., partly by forming a mechanical mixture with it and partly by entering
air in
into chemical combination with
it.
deeper insight into
the influence of aeration on fermentation in breweries could
by the study
only be obtained
and
races,
practice
to
for,
as
of
oxygen
(see, for instance, his
He
and
2).
pure culture species
investigations
have shown, they behave
bottom yeast Nos.

results.
Hansen's
in
brewery
differently with respect
experiments on Carlsberg
Korff'recently obtained similar
experimented on the three yeast races Saaz,
Frohberg and Logos, cultivating them in a 10 per
cent,
solution of saccharose with an addition of yeast water or
and hydrogen
By
air,
oxygen
respectively, the three species then
showed
Hayduck's asparagine
solution.
passing in
very different behaviours as regards their energy of
fer-
mentation, fermenting power, energy of multiplication and
power
of multiplication.
In this respect there are three things which must be

considered,
the chemical condition of the wort, the
viz.,
on the yeast species in question, and the

demands which the finished product must satisfy. Every
brewery must therefore find out by trial that method of
aerating the wort which gives the best result under the
effect of aeration
prevailing circumstances.
rules for this.
There are no
definite general
Hansen mentions a remarkable experiment,
which showed that wort can be in such a condition that
it
216
need not be subjected to the usual aeration in the brewery,

which was formerly considered quite indispensable to obtain
a good fermentation and a clear beer.
But such a chemical

The experiment
was made on Carlsberg bottom yeast Nos. 1 and 2 in
ordinary lager beer wort. The variation thus caused in the
condition of the wort
yeast
cells will
be treated
In course of time
theoretical,
very exceptional.
is
later.
many
investigations, practical
have been made into
in the old Carlsberg
No. 1
may
be
As an
behaviour.
this
example of the former, the experiments
of
and
Anton Petersen
brewery on Carlsberg bottom yeast
cited, the result
of
which was that those
brews which contained a large amount of oxygen showed

on an average a greater attenuation after the primary
fermentation than those which contained
The numerous
theoretical
less
oxygen.
investigations
been undertaken since Pasteur
{e.g.,
by
which have
Rasmus
Niigeli,
Pedersen, Hansen and others) on the effect of aeration on the
yeast
cell
and on fermentation, show that aeration
exercises
a favourable influence on the total energy of the yeast in
consequence of the increase of the multiplying power, but
under these circumstances, the individual
that,
less alcohol
cell
forms
when no aeration takes place. Hansen

that when the yeast cells have free access to
the air, or even when surrounded by it, they
than
found further,
the oxygen of
yet produce an active fermentation, an observation
can
which contradicts the theory of Pasteur mentioned above.
The
latter also holds for the
Giltay and Aberson.
experiments of Adr.
Brown observed
J.
Brown,
that a plentiful
supply of oxygen increases the fermentative activity of the

single
cell,
even when the
cells
are in such circumstances
as prevent multiplication.
Excess of oxygen slows the fermentation

has already reached the
maximum
if
the yeast
of its multiplication, or
if
217
the nutrient solution was charged from the beginning
with a quantity of yeast exceeding the
maximum amount
(Prior).
Finally,
may be added that when
it
as to cause a violent commotion,
its
aeration
is
so strong
influence then becomes
very markedly so with defective
disadvantageous, and
nutrimental conditions and yeast species of small ferment-
ing power (Buchner and Rapp).
Energy of
By
Fermentation and
Fermenting Power.
activity or energy of fermentation
understood the
is
intensity with which a yeast can decompose a sugar within

It of course varies in the different yeast
a certain time.
Prior has
species.
Meissl's
method
carbonic acid which

-sugar solution of
30 C.
determined
for
it
some
species
by
the latter consists in noting the weight of

is
liberated
by
of yeast from a
gram
a certain composition
in six hours at
According to Meissl, a normal yeast
is
one which
liberates 175 gram of carbonic acid gas under the above
conditions
the energy of fermentation of this
-down as 100.
îven species
Pastorianus
136'40
106-13
155'48
I.
II.
III.
elliiKoideus I.
No. 2
then put
Carlsberg bottom yeast No. 1
Saccli.
is
Prior found the following values for the
...
.
II.
Permeability of the Cell iWembrane
280-72
202-20
285-76
219-03
Since the trans-
formation of the sugar into fermentable sugar takes place in

the interior of the
a.lso
cells,
the energy of fermentation
is
a measure of the permeability of the cell wall (Prior).
This varies according to the age and condition of the

1
4-5
c.c.
cells,
of a mixture of 400 grams of candy sugar, 25 grams of

phosphate and 25 grams of potassium phosphate are dissolved
of tap water.
grams
ammonium
in 50
thus
218
and depends,
on the power of the latter to form
besides,
fungous mucilage, since the permeability diminishes the
more
this substance is
given
off.
The permeabihty
of course, with the different kinds of sugar.
varies,,
For instance,
Prior obtained the following result for Carlsberg bottom
yeast No. 2
It
Saccharose.
Dextrose.
Fructose.
Maltose.
106-1.3
87-09
73-67
69-71
may
be seen from this that saccharose had the greatest
diffusing power.
Products of Fermentation,
Besides
ethyl alcohol
and
carbonic acid gas, the saccharomycetes form, during fermentation, other substances also, although in smaller amounts,.
viz.,
glycerine and succinic acid.
Sacch. Hanscnii,
Zopf discovered a species,
which forms oxalic
Kruis have proved
that,
acid.
Raymann and
under certain conditions, the cul-
ture yeasts employed in the manufacture of spirits form
amyl
In addition to these, volatile organic
alcohol.
are formed,
e.g.,
The quantity
substances.
and
acetic acid
volatile, ester-like
acid.s
bouquet
of these substances varies accord-
ing to the conditions under which the fermentation takesplace,
and
and
their quality also varies in the different species
races, so that
the product
extremely variable.
formed by the
Having regard
latter is
to this, the necessity
urged by Hansen for the systematic selection of races in

practice will be recognised.
According to Prior, the wild
yeast species {Sacch. Pastorianus L,

ellipsoideus
I.
and
of fixed acids,
is
II.)
II.
and
form larger amounts of
III.,
Sacch.
volatile
usually the case.
The
ester-like substances
produce a
strong taste and smell in the finished product, even

present
in
than
whereas with culture yeasts the opposite
when
small quantities, these substances, which the
various species produce, being widely different from one

another.
Thus Sacch. anomalus brings out a strong
taste
21&
and smell
of fruit ester, while
some
discovered
by Hansen develop
a very strong bitter taste
and disagreeable
of the disease yeasts
smell.
There are formed in wine, by the different wine yeasts,
Wortmann calls secondary

bouquet substances (fermentation bouquet), and which are
those volatile compounds which
the substantial cause of the special taste of wines from
These by-products are therefore of no
particular places.
importance.
little
Many
yeasts are capable of a reducing action, forming
sulphuretted hydrogen
acid in
when sulphur
is
present during
Other species are able to form sulphurous
fermentation.
must
W. Seifert) and also in wort

Many wine yeasts have an acid-
Haas,
(B.
(Schwackhofer, Will).
consuming action (Schukow, Wortmann), gradually using
up
the organic acids present in the wines.
{Of.
W.
Seifert's
researches mentioned later on.)
Auto-Fermentation
When
no culture
liquid
are formed.
It
is
thick liquid yeast
is
be seen that, although
present, alcohol
and carbonic acid gas
it
This phenomenon
is
called auto-fermentation.
takes place through the yeast transforming
contained food
stuffs.
in the yeast cell

first
into sugar,
and
alcohol.
is,
set aside
may
at a favourable temperature,
its
self-
According to Lintner, the glycogen
in auto-fermentation, apparently turned
and
this then
fermented into carbonic acid
smell,
During auto-fermentation yeast gives off a

more or less strong, of fruit ether, which probably
arises
from
esters of the higher alcohols.
Yeast Types.
species occur
tation.
We
of view.
by
In fermentation industries various yeast
which exhibit
shall
The
now
different actions during
intensity with which the sugars are attacked
we have seen. At the very

when Hansen introduced his pure culture system
the yeast species varies, as
beginning,
fermen-
consider yeast types from this point
220
into the brewery, he proved that there
was a
named by him Carlsberg bottom
yeast No.
Several such types were found later

established the following three,
and No.
2.
the Berlin station has
inz.,
Saaz, Frohberg and
These are thus characterised by Prior
Logos.
difference
between the two brewery bottom yeasts
in this respect
The Saaz
yeasts in a fermentation leave unfermented most achroodextrin III.,
and consequently
also
more maltose than those
of
the Frohberg type, which again leave more than the Logos
Saaz and
Prior, however, does not recognise the
yeast.
Frohberg types in the physiological sense of fermentation.

According to this author the same degree of fermentation
finally reached
with both,
if
the fermentation
is
is
conducted
under the most favourable conditions (large yeast supply,
high temperature, strong aeration).
.^ Top
face
is
fermentation
is
fermentation this layer
is
never thick, and
in
the really only noticeable point of difference,
prominent.
definite
bottom
sometimes
is
In typical top and bottom fermentations
entirely absent.
this,
one in which the froth on the sur-
often covered with a thick layer of yeast
is
very
Various investigators have attempted to find
pronounced characteristics for each of these groups
but just as there are species which, with respect to the
phenomenon
gories, so
is
of
it
fermentation, stand between both cate-
also with those properties
which have been
classified as special characteristics.
A. Bau considered he
had found a distinctive property
the bottom yeasts in
x)f
the fact that they completely ferment melitriose (raffinose),
whereas top yeasts are unable to do

generally, so far as
his
included under Sacch. cerevisia
by
himself
The
but recent
test applies
go,
to
forms
experiments
Schukow have shown that most

bottom yeast forms among the wine yeasts
and
of the typical
this.
own experiments
by
cannot completely ferment melitriose, and the above char-
acteristic
The
has thus undergone a considerable limitation.
action
vertase
221
is
therefore
as
follows
The enzyme
in-
present in yeast decomposes the melitriose into
melibiose and fructose
the top yeasts especially are only
capable of fermenting the latter of these (Bau).
On the other
hand, most bottom yeasts contain the enzyme melibiase which
breaks up melibiose into dextrose and galactose, both of which

are fermented
by most bottom yeasts
(E. Fischer).
Sometimes a bottom yeast may for
a time exhibit feeble
signs of top fermentation (Hansen, Kiihle).
In this respect,
no absolute boundary can be drawn between top

and bottom yeast. It is certain, however, that no one has
therefore,
hitherto been able to transform a typical top yeast into a
permanent typical bottom
yeast,
and
vice versa.
It
used to
be a general belief that by the cultivation of a top yeast

at a
low temperature
it
could be transformed into a bottom
But Hansen has cultivated such typical top yeasts

Sacch. cerevisicB I. and Sacch. Pastorianus III. for several
yeast.
as
years at a temperature of 5 to 7 C. without anything
happening except that, as one might expect, the fermentation
was
feebler
but as soon as the cultivation was continued
again at a high temperature, the signs of top fermentation
became as prominent as
before.
Conversely, Hansen has
cultivated typical bottom yeasts such as Sacch. Pastorianus
L, Sacch. ellipsoideus
1
and No.
2,
I.
and
II.,
and several others
temperature,
i.e.,
Carlsberg bottom yeast No.

for years at ordinary
room
at a temperature considerably higher than
that usually employed in bottom fermentation breweries,
without any signs of top fermentation ever appearing.
In
a bottom yeast
was
earlier times the
view was held that
allowed to form a
film, it
if
was then turned
into a top yeast.
Hansen has shown, by exact experiments, that
this also is
entirely false.
Culture yeasts
is
the
name given
to such yeast species
222
as have for long been cultivated in fermentation industries,
though only recently
in a systematic
a certain amount of control.

is
now extremely
As
manner and under
the consumption of beer
widespread, culture yeasts
be met with in nature
most yeast
will, of course,
however, which
species,
occur either on fruits or in the earth belong to so-called

wild yeast species.
Culture yeasts
are, like
partly top yeasts and partly bottom
yeasts.
wild yeasts,
Their use
depends on certain special qualities for which they are

prized,
good
in breweries,
e.g.,
when they
give a stable beer, a
A yeast which
must not only multiply comparatively
the finished beer, but must also be able to suppress
clarification, a particular flavour, etc.
gives a stable beer
slowly in
rival yeasts during fermentation.
certain species are
may,
in
some
cases,
The immediate cause why
more pre-eminent
in the latter respect
be sought in the fact that they can
themselves to nutrimental
conditions,
and
oxygen more powerfully than
assimilate
other cases
it is
their rivals
due chiefly to them giving
fit
can
especially
;
in
off substances
during multiplication which act as poisons on the intruders.

Since
ties
tlie
demands made regarding
taste
and other
quali-
vary so widely the choice of available species and races
must
necessarily adjust itself accordingly, whether
wine manufacture, or for breweries,

yeast factories
distilleries or
In the
branches of industry the demands are more for
and
be for
pressed
thus the number of species and races intro-
duced into practice continually increases.

taste, smell
it
stability
first
two
clarification,
in the latter a large production
of alcohol and great multiplying
power are more particularly
sought.
Injurious and Stimulating Influence of Chemical
6.
and
Physical Factors.
Influence of Chemical Factors,
as with
all
With saccharomycetes,
other organisms, the decomposition products act as
CHEMICAL AND PHYSICAL FACTORS

a poison
Here
to the organism in question.
formed, and the organic acids,
among
it is
223
the alcohol
other things, which
The
exercise an injurious influence on their growth.
dele-
terious effect of carbonic acid, however, does not appear to
be great.
As regards the
of Hansen on the
mentioned
effect of tartaric acid
(see p. 135).
very quickly
which
action of organic acids, the experiments
if
He found
have already been
that beer yeasts die
off"
they are cultivated in a sugar solution to
tartaric acid has been added, an effect not
produced
on wild yeasts.
The stimulating effect of antiseptics on yeast has been
studied by earlier investigators, e.g., by Biernacki and Schulz
(antiseptics in general), by Hay duck (sulphuric and lactic
acids) and by Heinzelman (salicylic acid).
Agents were
thus found, by means of which not only could the development of bacteria be prevented, but the energy of fer-
Now
mentation also increased.

practice
were
also instituted.
quite recently
fluoric acid
is,
and then experiments in
In this direction Effront has
recommended the use
of fluorides.
Hydro-
however, a strong poison for yeast which can
hardly withstand
to 2
grams per
hectolitre
adaptation, the dose can be raised to 200 grams.
but,
by
yeast
habituated to such large quantities of hydrofluoric acid

possesses indeed but feeble powers of budding, although
has great fermenting power.

in distilleries.
According to
The method
Holm and
is
it
only applicable
Jorgensen's experi-
ments, this addition of fluorides hastens the development of
Mycoderma, and, at the same time, Bacterium aceti is not there-
by
destroyed.
In mixtures of brewery yeast with wild yeast
the same authors state that the development of the wild yeast
is
favoured, the effect being thus the same as that of tar-
taric acid.
This method also
is,
therefore, quite inapplicable
to the " purification " of beer yeast.
224
Sulphurous
sublimate and several other
acid, corrosive
substances are likewise strong yeast poisons.

holic fermentation
is,
following solutions
gallol 1
sium hydroxide
chlorine 1
if
beer
is
solution
10,000,
Phenol
Bokorny gives
50.
Thus
200, resorcin 1
sulphuric acid
5,000, potassium
and iodine
100, pyro-
5,000, potas-
permanganate
10,000,
Siebel found that
10,000.
treated with a solution of formalin (40 per cent,
formaldehyde) in the proportion
of
neither yeast, Mycoderma nor bacteria develop

tions of
alco-
according to Yabe, prevented by the
10,000,
in solu-
and Mycoderma develop but not
50,000, yeast
bacteria.
Influence of Physical Factors.

of physical causes, yeast cells
As regards the influence
do not usually withstand 50 to
60 C. moist heat, but die off between these
Hansen found
young
that strong
two temperatures.
cells of Sacch. ellipsoideus
die after live minutes' heating in distilled water at a
II.
temperature between 54 and 56
C.
Old
cells of
species could withstand a temperature of 60
the
same
Ave
for
minutes under the same conditions without being
killed.
Quite ripe spores of the same species, which had been par-
on a gypsum block for about a week, withstood

minutes heating in water at 62 but not at 66 C.
tially dried
five
Similar experiments were
strong young
cells
made with
Sacch. cerevisia
I.
could withstand five minutes heating at
52 C. under the same conditions, but not at 54
spores treated in the same
manner
C, and
as those of the foregoing
species could withstand five minutes heating at 58, but not

at 62 C.
down
to
Yeast
- 130
cells are said to
from experiments
be able to stand coolingis
known
in breweries that yeast cells can
remain
C. for
about 200 hours, and
it
frozen in ice for months at a time without being killed

(Prior).
Drying and the
effect
of temperature are
related to
CHEMICAL AND PHYSICAL FACTORS

one another.
225
Experiments by Hansen and others have
shown that yeast cells die comparatively quickly when

dried up, and that this occurs in nature, e.g., when the cells
are on the iminjured surface of fruit.
Under these circumsomewhat longer life than the
stances the spores have a
vegetative
cells,
but the difference
able treatment, however,

cell
life
is
By
not great.
a suit-
can be preserved in the dried
This will be mentioned under the
for a long time.
heading following.
The injurious
effect of violent
already been touched upon
Experiments on the
shaking on yeast
cells
has
(p. 217).
effect of light
have been made from
Kny
time to time by various investigators.
used as the
source of light five flat gas flames, and carried out his
experiments on pressed yeast (Sacch.
cerevisiat)
placed in an artificial culture solution in

dishes
which was
flat crystallising
and at the same temperature, part exposed
action of light,
and part
away
set
in the dark.
to the
The heat
from the source of light was removed by means of water.
He
arrived at the result that the budding of Sacch.
cerevisicB
takes place in this moderate light with the same activity as

in darkness.
Marshall
Ward remarked
a destructive effect
of light on the spores of Sacch. pyriformis.
Lohmann made
experiments with intense light (arc lamps and sunlight) and

excluded the heat radiated from the sources of light by
means
of water
cells.
A distillery yeast, called Race II., was
seeded in wort gelatine on glass slips and for eight hours
was subjected to light from

was set away in the dark.
above 18) it was found that
at constant temperature part
the arc lamp, and another part
For high temperatures
(i.e.,
budding was retarded in the cultures subjected
to the Hght.
The same yeast was also seeded on agar-agar plates in Petri

dishes, a part exposed to sunlight and another part kept in
darkness.
Illumination for several hours in this case killed

15
226
Diffused daylight also had a retarding influence
the yeast.
on budding, but only after prolonged
It resulted
action.
from experiments with Sacch. Pastorianus
I.
that this species
has greater resisting power than the above one against the
both from the sun and from the arc lamp.
effect of light,
It has long
been
known
in the
brewing world that beer
sensitive to light in a high degree.
is
Special experiments
this point were made by Ney, Beck and W. Schultze.

The question put before one here is whether the disagreeable
smell and taste, which beer gets when exposed to sunlight,
on
originates in the effect of light on the yeast or on the beer
At the time when the
itself.
first
two named
carried out
experiments (1878 and 1882), Sacch. exiguus was
their
always looked upon

wrong, and
it
as the cause of everything that
was found,
went
in concordance with the above,
that in those flasks least protected from light, and the contents of
which had therefore acquired a very bad smell and
was a more or less copious development of

cells," which they set down as the abovenamed species without further ado, and also of lactic acid
From this it would seem then that light was
bacteria.
the cause of the good beer yeast, with which the beer
there
taste,
"
abnormal yeast
was
treated,
being restrained and the supposed
exiguus being furthered in its development.
communications
Pastorianus
I.
it
Sacch.
But from the
appears rather to have been Sacch.
or an allied species, and since the latter shows
a greater power of resistance to the effect of light than

Sacch. cerevisicB, as
ments
was
of
Lohmann,
we have
it is
seen from the above experi-
not impossible that this species
responsible for the disagreeable smell and taste in the
cases cited, since the action of the light in the experiments
was very prolonged (three weeks).

what the rdle played by the yeast
transformations really
is.
But
it is
not yet clear
cells in these
undesirable
VITALITY OF YEAST
Vitality of Yeast in Nutrient Solutions
7.
227
and in
the
Dry
State.
Hansen's researches have likewise furnished explanations

of the vitality of saccharomycetes in nutrient solutions.
The
results
were the following
for these fungi
is,
years' observations,
and
The
best preserving liquid
as stated in Section
a 10 per cent.
II.,
In the course of more than twenty
solution of saccharose.
species
and
in
experiments with forty-four
only ones which died in this
varieties, the
solution were Sacch. Ludwigii, Carlsberg bottom yeast No.
and
asporogenous variety, in several
its
only after some years,
growths; as regards
dying
off
all
and,
cases,
only some of
indeed,
2,
however,
the
the other species and varieties no
was observed, although the majority
of
them had
been sixteen to seventeen years and several of them more

than twenty years in the sugar
saccharomycetes
The behaviour
solution.
is essentially different in wort,
great irregularities prevail.
it still
water the extent of the seeding
species
it is
a small or a large one
stronger
cells,
resisting
much
of great
also
still
to
living after ten
in fact, live at the cost of the
power
this
moment,
i.e.,
in the first case the
were dead after one and a half
the latter they were
The
is
lived after
In preservation
twelve years usually death occurred early.
whether
and here
In one case the same species
died frequently in five months, in another
in
of
two
years, in
The
years.
weaker
ones.
of yeast towards drying varies very
depends mainly on whether the single
subjected to the drying or the cells
cell
lie
together in large
quantity and thus form a thick layer.
Hansen drew
is
attention to this in 1885.
Later on he made experiments
on the resistance of the isolated
cell to
drying by dipping a
piece of platinum wire into a yeast mass, placing the wire in
an empty Freudenreich flask and shaking the

a
way
latter in such
that the small quantity of yeast on the wire was
spread over the bottom and walls of the
15*
flask.
He
thus
228
obtained as thin a layer of yeast as possible.
then
It
resulted that under these circumstances the saccharomycetes
Some
remain alive only for a short time.

than
less
Marxianus held out longest,
Sacch.
days.
five
species died in
being alive for three months, also Sacch. anomalus and Sacch.
membranafaciens, which lived eighty and sixty-five days respectively.
These facts refer to the vegetative
cells.
Under
the same circumstances spores lived at least five months.
In addition, Hansen made numerous experiments relating

to the
drying of yeast
in layers
on
filter
cells in
other ways,
viz.,
when put on
paper and
small flasks with a wadding plug (Section
In the
117).
two years
wool, the species tested lived for
even more than three years

spores,
and
it is
pp. 62
II.,
and
under the same circumstances the
spores lived for one to
formed
placed
as a rule, the vegetative cells died
first case,
in the course of a year
when
cotton wool in
On
longer.
the cotton
more than a
year,
some
under these conditions they
probable that this had something to
do with their longer period of
life.
Experiments
in practice
on the preservation of brewery yeast by drying have been

described in Section
8.
II., p.
118.
Disease Yeasts and Mixed Fermentations.
Diseases caused by Fermentation Products.
We have
seen from the above that yeasts occur which are the cause
of diseases in beer.
Pastorianus
may
I.
As an example
be
mentioned,
of such a yeast, Sacch.
which, according
to
Hansen's experiments (1882), produces a bitter taste and

disagreeable smell in bottom fermentation beer.
If
the
quantity of this yeast amounts to ^ that of the stock yeast,

the disease
is
very noticeable
to ^V ^he disease
time,
is still
even when
appreciable.
It
have an undesirable action on the
cause turbidity.
it
only amounts
may, at the same

clarification, and
Will has isolated two similar disease yeasts
DISEASE YEASTS
229
in Bavarian bottom fermentation breweries; the one gives the
beer a sharp, bitter after-taste and produces strong turbidity,
somewhat sweet disagreeable aromatic
the other causes a

taste,
and a
bitter astringent after-taste, as well as turbidity.
Diseases
caused
by Turbidity.
Several
wild
yeast
species produce in beer the disease called yeast turbidity
by
multiplying rapidly during storage, particularly in bottles, the

beer becoming
with
filled
cells,
the specific gravity of which
such that they remain suspended.
This was demonby Hansen in his experiments with Sacch. Pastorianus III. and Sacch. elUpsoideus II. (1883). The same holds
is
strated
for these disease yeasts as for the foregoing, namely, that
they must be present at the beginning of the primary

fermentation in order to be able to cause the disease.
An
which takes place only at the end
infection, therefore,
of the primary fermentation, while bringing the beer into
the storage cellar,

to
îj
of the latter
is
without
effect.
and the beer
is
If it here
amounts
also casked
with an
extract of 7 '5 per cent. Balling, the storage being interrupted

after
two and a third months, the disease will make its

However, this will not happen if the quantity
appearance.
of extract
is
is
reduced to 67 per cent. Balling, and the beer
stored for at least three months.
Wortmann has
drawn
recently
that turbidity of a particular kind
ance in wine, by the
cell
and the contents of the
attention to the fact
may make
wall of dead
cells
its
appear-
dissolving,
latter being distributed.
Diseases similar to the above have also been observed in
top fermentation breweries (de Bavay, Frew).
Competitive Relations,
When two or more species are
present together in a nutrient solution they have, as a rule,
an injurious action on one another as regards multiplication,
among them.
make experiments
a state of competition arising
Hansen was the
first
to
in this direc-
230
In his paper of 1881 he describes the competitive
tion.
relations in beer wort
bottom
between Sacch. apiculatus and brewery
It resulted that
yeasts.
when the same number
of
was seeded in the same flask, the

was smaller than in the correspondwhich the same number of cells of each
of both species
cells
multiplication of both
ing flasks in
The increase of Sacch.

was considerably lessened, but the amount of
alcohol formed was the same both in the flask with the
had been seeded separately.
species
apiculatus
mixed
seeding, and in that which contained Sacch. cerevisicB
only.
In another series of experiments, carried out in a
similar,
manner, but with the difference that the seeding of
Sacch. apiculatus contained twice the
that of Sacch.
cerevisice,
number
of cells in
the result was the same as regards
the mutual retarding action which the two species exercised
on
flask
containing Sacch.
was
cerevisice
the'
but here somewhat
multiplication,
formed in the
of
alcohol
The increase
alone.
cerevisice
also restrained
series
first
carried
less
with the two species than
was
in that
of Sacch.
to a greater extent than in
experiments.
The experiments were
out at various temperatures, and with different
species of
brewery bottom
yeast.
The main
result
was that
Sacch. apiculatus, as the
weaker probably at the
primary fermentation,
checked in the struggle with Sacch.
cerevisice,
but that
the increase of
its
tion of the latter.
it
is
close of the
can also exercise a restraining action on
stronger rival and on the alcohol produc-
When
Sacch. apiculatus increased
each species was in a separate flask
more rapidly than
Sacch. cerevisice
with equally numerous seedings the proportion was 3:1.
very remarkable result was obtained by Hansen in
his experiments in practice with
brewery yeast
and No.
2.
mixed fermentations
species, viz., Carlsberg
The
chief result
gives a beer of less stability
was
when
bottom yeasts No.
of
1
that the pitching yeast

it
consists of a
mixture
MIXED FERMENTATIONS
of
two brewery yeast
when
species than
it
231
consists of one
In these mixtures the species present
of the species only.
disease yeast, making

The experiments showed that this
happened not only when the two species were mixed in the
proportion 9:1, but even when the proportion was 19:1.
in smallest proportion acted as a
the beer less stable.
The
disease then appeared only
beer
was interrupted
when
the storage of the
month after three

it was noticeable.
after IJ to It
months' storage only a faint indication of
In his experiments on mixed seeding, Vuylsteke found

that
when
a mixture
Pastorianus
I.
of
was seeded
Sacch.
cerevisice
in wort, the
I.
number
and Sacch.
of cells of the
former species per unit of volume increased from the

to the second
the
number
13"3
and
day from
to 4-81
of cells of Sacch. Pastorianus
12'2
respectively.
Sacch. Pastorianus
I.
had been
From
|^ =
I.
this
rose from 1 to
the increase of
2-76 and
times greater than that of Sacch. cerevisia
mixture of Sacch.
was taken
cerevisicB I.
twenty- four hours, and the
502 and
1 to
The increase
compared with the
Syree,
I.
was thus
in a research
j^
III.
I. 'in-
4"62 in the
cells of Sacch. Pastoriamos III.
of Sacch. cerevisicB
first
Pastorianus III. in
cells of Sacch.
to 3-01.
2-35
When
and Sacch. Pastorianus
the proportion 1 to 3'57 and
G.
1^ =
I.
for pitching, the cells of Sacch. cerevisicB
creased in the proportion 1 to
first
and 5"18 respectively;
O'Tl
cell
of the
increase
and -^^ =
0'65.
on the competitive struggle
between the culture yeast Frohberg and Sacch. Pastorianus

He emIII., has recently obtained the following results
:
ployed, as culture substratum in his experiments, yeast
water with 10 per
cent, of saccharose added.
the Frohberg yeast
when by
itself
At
25 C.
exhibited an energy of
multiplication of 1110 (after four days) and a multiplying
power
of 1659 (after four weeks);
5 to 6 C. the
at
a temperature of
numbers were respectively 383 and 474.
232
As regards
Sacch. Pastorianus III. he found, with the
conditions, the following
When
was
as
same
numbers: 921, 1310, 824 and 843.
the two yeast species were together the behaviour
shown
in the following table
Proportion of colls in the
mixed
yeasts.
VARIATION OF YEAST
233
He
important basis to Delbriick's natural pure culture.
shown, namely,
as
may
be remembered, that great
has
differ-
among the species and races.

Immediately after the introduction of the pure culture
ences exist in this relation
system, Hansen also communicated the scientific principles

of a method for improving stock yeast, which had long been
known
in breweries.
This method consists in taking the
fermenting wort at the commencement of the primary fer-
mentation instead of sedimentary yeast during a
The reason why
fermentations.
thus obtained
lies in
series of
a really better yeast can be
the fact that, according to Hansen's ex-
periments, the culture yeast preponderates at the beginning
of the primary fermentation and the wild yeast at the end.
In this matter there are two things between which

proper distinction must be made, the preparation of the
pure culture and the maintenance of the

brewery.
only in the
latter
in the
The natural pure culture has its use and value

last named direction
it must not be forgotten,
;
however, that
its
sphere of action
still lies
for the greater
part in darkness.
9.
Saccharomycetes
is
are, like all
The greater part
to variation.
subject
Variation.
other organisms, subject
knowledge
of our
of this
due to Hansen, and, in the following, a risuwA
of his investigations on this point
is
given.
The changes caused by variation are

or permanent
temporary
either
between both extremes intermediate forms
are met with.
Temporary
Varieties.
On
the introduction of the pure
culture system the researches in connection with
laboratory and in practice gave
many
it
in the
opportunities for
the observation of different variation phenomena.
Such
observations are mentioned in several places in Hansen's

papers.
If a yeast
is
taken from the brewery and cultivated
234
some time under laboratory
easily
assumes
qualities (greater attenuation, impaired clarification,
unusual
for
taste) other
than those
mena were frequent
in
conditions,
it
formerly possessed.
it
former times
as,
Such pheno-
in preparing pure
yeast in the laboratory for use in breweries, the conditions

in practice
more
in
were not always
difficult cases
sufficiently well imitated.
Also-
the same wort was not used as in
the brewery for which the yeast was prepared.
new
properties acquired in
But the
manner soon disappear
this
again under normal working conditions.
Of other appearances of temporary variation, the followmay be mentioned Hansen sometimes found in a culture
Carlsberg bottom yeast No. 1 on gelatine, colonies which
ing
of
consisted of normal egg-shaped
which contained sausage-shaped

vated by
itself,
shaped
sometimes colonies
Each colony,
gave a progeny which retained
acteristic cell form.
finally in a
cells,
cells.
was only
It
culti-
its
char-
after repeated cultures,,
fermenting vat in the brewery, that the sausage-
cells
disappeared, and the yeast again contained
only normal egg-shaped
cells.
cultivating Sacch. Pastorianus
Hansen further found by

II.
on wort gelatine at 25
that vegetations form partly with the appearance of Sacch.

ellipsoideus,
and partly with that of Sacch. Pastorianus.
In general, vegetative
as regards size
number
in the
cells
and shape, the

mother
spores produce small
cell.
and spores vaiy very much
latter also
with regard to the
Feeble vegetative
cells
and
Other peculiar changes of shape
cells.
have been already mentioned in the description of the

growths of film formation.
Hansen's experiments made in 1883-84 with Carlsberg
bottom yeast Nos.
and 2 have shown that a previous
culture in unaerated wort
may have
an important influence
on the clearing power of a brewery yeast in

thereby cause a precursory variation.
The
practice,
first
and
species
VARIATION OF YEAST
in particular, after
235
had passed through repeated
it
fer-
mentations in unaerated wort in the brewery, gave a very

poor clarification and an extraordinarily strong fermentation.
Under the same
but returned tolerably quickly to
clarification,
condition,
conditions yeast No. 2 also gave poor
viz.,
fermentations
in
the fermenting vats of
The
influence
of
chemical
here.
This
example
is
substances
expressed more plainly
Hansen observed that the
Pastorianus
I.,
smell in wort, loses this power
if
brewery^
the
disease yeast
Sacch..
taste
and
kept for a time in a
Another example of
variation, which, however, continued

erations,
normal
makes itself felt

by the following
which produces a disagreeable
saccharose solution.
its
had passed through two primary-
after it
precursory
through a few gen-
was communicated by Hansen
in 1886,
when he
showed that the film cells of certain species and the cells,
of old growths which had been developed in a saccharose
solution could form, in wort cultures, a loose-lying and
often
cheese-like
yeast
sediment quite different to
the
This change only disappeared
normal dough-like sediment.
after repeated cultures in wort.
Cheese-like yeast can
also-
be formed after prolonged drying.
In his
first
drew attention
this function
viduals of the
on external
torianus
I.,
spores with
paper on spore formation (1883) Hansen

to the
fluctuations
which
may
appear in
these depend partly on the fact that indi-
same
factors.
species
behave
differently,
For example the
and partly
cells of Sacch.
Pas-
produced in wort at 27 C. in two days, form
more
than those produced at the
difficulty
same temperature in one day the difference is still greater if

the cultures are one and seven days old respectively. About
;
Hansen published the discovery of asporoHe had noted that Sacch. Ludwigii when allowed
six years later

genesis.
to stand in culture
media could form
cells
which had
lost-
236
He
the power of developing spores.
under these conditions the isolated

vegetative forms
the
first
further observed that
developed three
cells
possessed the power of vigorous
spore formation, the second had almost lost this power, and
the third formed no spores at
The observed deviation
all.
from normal behaviour was found
two forms
to be hereditary in
But by
cultures for a long time.^
wort
cultivation of the latter
containing dextrose, these
in a culture liquid
The same
quickly resumed their normal condition.
be-
haviour was noted afterwards by the author as regards

Sacch. Marxianus.
It appeared later that all asporogenous
varieties of Sacch. Ludwigii are not
The above variation
in this way.
by dextrose
afiected
of Sacch. Ludwigii
forms
an example of transition from an entirely temporary variation to a

offers,
permanent one.
The
effect of
dextrose in this case
on the other hand, an instance of the
specific action
of a single chemical substance.
Permanent
Varieties.
Hansen
prepared in the same
year a permanent asporogenous variety showing that Sacch.

Pastorianus
I.
completely loses
its
power
of forming spores
on being cultivated for several generations

at a temperature
maximum
which
is
maximum
and only a
for
little
lower than
bud formation.
His later
experiments have shown that this law holds for

saccharomycetes (culture and wild yeasts).
Hansen
also observed the rise of asporogenous
all
typical
The peculiar
species Sacch. viambranafaciens, Sacch. aiwmalus

'
wort
higher than the temperature
for spore formation,
the temperature
in aerated
and Sacch.
forms in other species
when
allowed to remain for a long time on culture gelatine and in wort.

Beijerinck and P. Lindner have recently made similar observations. Thus
Beijerinek has separated two forms of Schizosacch. octosporus, an asporogenous and a sporogenous. With the first he found that the formation of
trypsin had been very much restricted, and that the formation of acid was
greater than in the sporogenous form.
Alfred Jorgensen states that top
yeasts preserved on gelatine gave a slower clearing and a greater attenuation than under normal conditions.
VARIATION OF YEAST
Ludwigii appear to be the
species
deviate so
also
exceptions
only
much from
very
mycetes that they ought probably to be

of
new
237
but these
saecharo-
true
up
set
as types
genera.
Quite recently Hansen has published detailed descrip-
permanent
tions of his investigations on the formation of
asporogenous saccharomycetes by the above mode of cul-
The
ture.
starting point of his experiments
from a single
To
a spore.
cell,
some
in
cases a vegetative
was always
cell,
in others
find out the conditions of spore formation at
various stages during treatment he employed surface plate

cultures on wort gelatine (see p. 106).
colonies
were then, as soon
directly on moist
colonies
gypsum
The grown up
as their size permitted, placed
Those
blocks for sporulation.
which were too small
for this
mode
of treatment
were put in wort and the sediment yeast formed was

placed on gypsum blocks. The chief experiments were
made
partly with Sacch. Pastorianus
with Johannisberg
result of
II.
at 36 C.
I.
at 32
As an example
partly
of
the
the treatment of the first-named yeast at the
different stages the following table is given

In the 2nd stage
But
C, and
4th
7th
,,
besides
per cent, permanent asporogenous cells were found.
60
100
these
permanent asporogenous
transition forms were found,

for a time the power
i.e.,
cells,
other
some which had only
of sporulating;
lost
after culture for a
shorter or longer time in wort they again became sporogenous.
It requires cultivation for
be decided whether a vegetation
two years before

is
it
can
a permanent asporo-
genous one or not.

Whilst nutrient liquids were employed in the foregoing
experiments, Hansen also carried out numerous experi-
ments with
solid
substrata as the cultivating
medium.
-238
Sacch.
cerevisia
considerable
I.
and Sacch. Pastorimius
number
gelatine at 25 C.
wort
formed very few, and
Sacch. ellipsoideiis
I.,
hrancBfaciens
I.,
on
cells
further became evident that
it
and Johannis-
Sacch. Pastorianus III., Sacch. ellipsoide.us II.

berij II.
formed a
II.
permanent asporogenous
of
finally that Sacch. Pastorianus
Sacch.
Litdivigii
and Sacch. mem-
formed no permanent asporogenous
cells
what-
permanent asporogenous variety was formed by

ever.
Sacch. anomalus on agar-wort gelatine at 34 C. and on the
same substratum
at 32 C.
by
Sacch. Pastorianus
I.
In order to decide the fundamental question whether, in

the formation of these asporogenous varieties,
of selection or transformation,
ments with the Johannisherg

it
was absolutely impossible
Hansen made
II.
was a
it
case
special experi-
In a normal growth
yeast.
to find a single cell
which by
normal culture did not produce a sporogenous growth.
was a single cell, either a vegetative

The vegetation with which the expericell or a spore.
ment had been begun was analysed in such a way that
The
at
starting point
least
1,000
cells
were
isolated
and the vegetations
produced by them tested for spore formation.
they
sporulated
showed further
in
that,
menced, the transition
forms
appear.
Hansen's
abundance.
as soon as the
formsthe
In
all
cases
experiments
treatment
is
com-
temporarily asporogenous
But such were never found
at the start-
ing point, and their inception must be ascribed to the

treatment.
Finally, the variation described is a general
phenomenon which always makes
its
appearance
if
the cells
As a result, both
point and of those at the
are subjected to the above treatment.

of the analyses at the starting
different stages of the treatment, it is plainly to be inferred
that the variation which appears during the treatment originates in a transformation.
The following behaviour, which is
also treated experimentally here,
is
extremely peculiar,
viz..
VARIATION OF YEAST
that even
when
239
the starting point of the experiment
single vegetative
cell
temporary asporogenous
cells
sporogenous
cells,
and permanent asporogenous
can be again taken from the
kinds which will once more reproduce
As regards the
or_a spore, yet the following three
categories appear during the treatment
cells; a single cell
is
all
first
two
three categories.
conditions for transformation the follow-
ing factors come to be considered
the chemical composition
of the culture liquid, the vibrations caused by shaking the

flask,
the aeration of the culture liquid and the temperature.
The following can be
set
down
as the result of Hansen's
experiments: a culture liquid of definite chemical composition

is as little required as are vibrations.
high temperature
But
it
may
is
be said of the culture liquid, the vibrations and
the aeration, that each

.so
Also aeration without the
incapable of causing the transformation.
far as they all
may
more or
have an indirect significance in

less aid multiplication.
But a
high temperature was shown to be the most important and

absolutely essential factor.
On
culture gelatine
we are able
by means
to produce the transformations described, not only
of high temperature, but also
by means
of chemical factors.
Hansen has prepared asporogenous varieties from

numerous other species besides those of Sacch. Pastorianus
The oldest of these are more than
I. and Johannisberg II.
twelve years old, and they have always maintained their
numerous
As a rule the
saccharomycetes treated in the above manner lose simIn
ultaneously the power of forming spores and films.
permanent asporogenous character in
cultivations and very varied
some,
also, a
spite of
conditions.
multiplying power of the
cells
in wort has
been observed greater than that of the parent forms, and
with regard to alcohol production greater deviations from

In this respect
the parent forms have been presented.
Hansen has
also
brought about differences by other methods.
240
obtaining both an increased and also a diminished production
Thus Carlsberg bottom yeast No.
of alcohol.
was
culti-
vated at 32" C. in eight successive wort cultures, the suc-
ceeding one being inoculated from the preceding without
any aeration
by cultivation
then,
The ninth culture
of the Hasks taking place.
saccharose, gave
wort containing 10 per
in
cent,
of
to 2 vol. per cent, less alcohol than the
normal Carlsberg bottom yeast No.
1,
a better clarification in practice.
Hansen obtained from
spores of
Sacch. cerevisia
and, in addition, also
which were seeded on yeast
I.
gelatine, a vegetation which gave up to 3 vol. per

more alcohol than the corresponding vegetation which
water
cent,
had been cultivated only

In the latter
time.
wort during the whole
in beer
case,
however,
it
is
than transformation which takes place.
selection rather
The
single in-
dividuals of a species purely cultivated in wort may,
show
e.g.,
a great difference in fermenting power, even although
they have been produced
same nutrient solution and
in the
under the same favourable conditions.

main, for the most part,
still
in the
dark
These matters
;
re-
the same remark
applies also to variation as regards clarifying power.

If the culture of a sporeless non-film-forming variety be
allowed to remain for a long time in the fermented nutrient

solution, the latter exhibits
an alcohol content far greater
than that of the corresponding film-forming parent form

reared under the same conditions.
in six
months, there were 55
Thus, Hansen found that,
vol.
the nutrient liquid of the variety, while there was only 1'5
vol.
per cent, in that of the corresponding parent form,
although the last-named flask also contained 5 5

cent,
when
It
fact already
the
alcohol
was only a month
mentioned that
old.
The cause
lies in
film cells are capable of
formed into carbonic acid
varieties here
vol.
mentioned have
lost this
per
the
turning
and water; the
power
of
trans-
VARIATION OF YEAST
forming alcohol simultaneously with the
241
loss of the
power
of film formation.
With regard to these varieties which have lost their

power of forming spores and films, it was found that these
new properties are quite permanent and heritable. Such
have been cultivated under the most widely varied
varieties
circumstances for long times (up to twelve years) without

exhibiting the slightest indication of returning to the parent
forms.
The number
of transient variations
But
are easily produced.

varieties the
above
is
of those
the only one
greatest interest attaches to
is
and they
legion
which lead
to
permanent
we know
The
of.
for several reasons.
it
In the
constant varieties mentioned the most important factor in
transformation
formation
may
temperature
is
must be subjected
are at
hand
variation
but in order that the trans-
take place, a certain number of generations

Since the same factors
to its influence.
in nature as well as in the laboratory, the
may
appear there
Practical Results and Variation in Practice.
asked what practical

following answer
the
life
and the
therefore employ
We
hitherto.
choice from
utility
If
it is
such investigations can have, the
may be given
they deepen our insight into
efficiency of these organisms,
them
same
also.
and we can
in our service to a fuller extent than
make our
already known
shall not be content as before to
among
the species and varieties
in nature or in the brewery,
we shall
rather begin ourselves
to introduce improvements and transform species according

to
our desire.
The
practical application to the
yeast consists in being able to
analysis of brewery
make such an analysis with

One of our most
greater ease and certainty than formerly.
important researches on wild yeast
and
species, as for
is
based on spore culture,
example Carlsberg bottom yeast No.

16
1,
242
which gives extremely few spores or none at

respect, easy to control.
If,
all, are,
in this
on the other hand, a brewery
yeast with strong spore formation has to be dealt with, the
matter
is
somewhat more
remains that
we
From
difficult.
the above
are able to take from the cells their
it
power
thereby making the analysis easier.
of forming spores,
Hansen has done
this several
bottom yeast No.
and has brewed good normal beer with
new
the
2,
years ago for Carlsberg
variety formed in this manner.
kept in mind that the transformed species
Yet
it
must be
may also undergo

The sporeless
manner to
a simultaneous change in other respects.
variety will often act in a brewery in a different

its
parent form.
The experiments
berg bottom yeast No.

practical application of
variation.
carried out with Carls-
are additional examples of the
the results of the researches on
This yeast gives a beer that
is stable,
attenuated during the primary fermentation.

has,
by the method
but greatly
Now Hansen
of culture described, prepared from
it
at
a high temperature a permanent variety with less attenuat-
ing power which gives a fuller beer than the parent form.
This variety has, however, one failing
Numerous
found
in the
articles
it
acts too slowly.
on the variation of beer yeast are to be
brewery journals both before and after the
duction of the pure culture system
intro-
the most of these treat
of their degeneration in practice, others of their improve-
The attainment of the latter was sought in one way

by their cultivation in any culture liquid of definite chemical
The best known researches in this direction
composition.
made
by Hay duck. According to him the enrichare those
ment.
ment
of the yeast with nitrogen
degeneration
is
one of the causes of
in such cases, in order to regenerate the yeast,
he recommends that
it
should
first,
before pitching, be
allowed to ferment in a cane sugar solution.
Seyffert
found recently that a yeast which had hitherto given a
VARIATION OF YEAST
good
bad
clarification in the
brewery, but which suddenly went
in that respect, could be
good
clarification
water.
243
brought round again to give a
by the addition of
gypsum
to the
brewing
The experiments with stimulating antiseptics already
mentioned made by Biernacki, Effront, Hayduck, Heinzel-
mann and
Schulz come under this head.
Besides Hansen,
Kukla and Will have published papers

Hansen's two new
brewery yeast.
beer yeast and his experiments with them in
Delbriick, Jcirgensen,
on the variation
varieties of
of
These experiments assume a
practice have been described.
from the
special interest
formed
fact that the
two
species
which
the starting points for the transformation are well
known, and are present
in
most laboratories, and
the fact that a definite method
for the experiments described below

object of attaining a racial
also
from
This does not hold
given.
is
which
start
improvement by
with the
selection
in
seeding.
The
application of the pure culture system in practice
consists, as
we have
seen, not only in the preparation of
pure cultures of a certain species or race, but at the same

time in a selection from among the vegetations produced by
Thus with the introduction of the pure

the brewery a racial improvement
the same time striven after. It was a case here of
the individuals.
culture system
is at
into
always selecting the
best, since practical
men always made
greater demands and forced us to experiment,
i.e.,
compelled
us to seek for such individuals as satisfied these demands.

It
all
is
not only required that the species or race shall keep
those properties which are of value for the practical
man, but
it is
desired that at the
same time such individuals
manner serviceable
shall be selected as will vary in a
brewery concerned,
i.e.,
possess
good
degree and lose undesirable ones.

best cases this
may
qualities in a
high
Of course even in the

The yeast
be only partially attained.
16*
to the
244
by Hansen for the two Carlsberg breweries

was the one which became known as Carlsberg bottom
species selected
yeast No.
stability),
tion,
to
1,
which has
but also
its
good
qualities (good taste, great
its leas desirable
ones (sluggish clarifica-
somewhat too great attenuation). He then began at once
improve the race by continual
selection,
and
his successors
have, later on, worked in the same direction, outside the
Carlsberg laboratory as well, not only with this species but
with other species and
races.
Racial improvement thus consists in repeated selection

of the best individuals.
It
may
be seen from the papers
published by Hansen and other investigators on experiments

in this direction that it is not possible to set
rules here.
perimenter will
desirable
which
result
not to be regulated.
is
up
definite
The exoften be deceived and arrive at an unhe is here confronted by something
Experiments must be carried
It is a
out.
matter quite different
from the mere preparation of the above asporogenous races

in the latter case the conditions are
in
on
mind
that,
racial
even
when
improvement
brewery fermenting
race
known
Finally
transformation can be regulated.
exactty and the

it
must be borne
the material for such experiments
is
taken from the contents of the
vats, there is
no guarantee that the
taken out stands in genetic connection with that
They
formerly selected.
blood
relations
because
are not therefore of necessity
both
agree
in
char-
botanical
acteristics.
Similar communications on racial improvement have
been made by the technologists of wine fermentation, but

here also there
In
is
no mention made
of definite methods.
these communications also, information about the
all
species
from which the
start
was made
is
as
rule
wanting.
culture yeast
may
also be
subject in
practice
to
VARIATION OF YEAST
variation in a harmful direction.
245
Hansen
in his Practical
when in 1892 summing up his

experiments on this subject during the course of years,
Studies
in
Fermentation,
says as follows
"
When we
regard the variations of yeast
brewery practice from a biological point of view, we are

inclined to look upon them as quite insignificant for the
in
practical brewer, however, the matter
quite different.
is
The changes
can, indeed, occur in a very disagreeable

manner, and sometimes cause an appreciable irregularity.
In the course of a year they pass like a wave through
the brewery, and in most cases
we have no
idea of their
cause."
Investigations on all that occurs in practice are
difficult
in a high
work here
culture
with
yeast
the
pure
absolutely
concerned,
we do not
not only because
degree,
but
the
culture
of
the
composition
of
the
wort and other external factors are so very complicated
and variable that they frequently escape our

spite of
control.
In
very extended and arduous attempts on the part
of the author
by experimental means
to
shed light on
the most important questions which have cropped up in

this region of late years,
he
is
still
unable to record com-
pletely satisfactory results, but hopes later to be able to
give some elucidations.
This may, however, be
said, that
those variations which come to light in actual practice
under the influence of the factors there predominant are

only transient, which shows that the pure culture system
has not only gained a firm foothold in the brewing system
of the
whole world, but daily spreads further into
other alcohol fermentation industries.
Some of
all
the
the culture
yeasts are particularly permanent, whilst others are more inclined to variation.
to the first of these.
instance, there
Carlsberg bottom yeast No. 1 belongs
In the
New
was a pure culture
Carlsberg Brewery, for
of this species in the fer-
mentation cylinder of the pure culture apparatus which
246
had been introduced more than

tions of course occurred, but
years before.
Fluctua-
no fixed changes.
Various
five
made communications on a special constancy

yeasts. The experiments on this point by Irmisch,
authors have
in culture
Jorgensen and
P.
practice a yeast
in
are
an adverse
now
Lindner are noteworthy.
growth
easily accessible
now
will
if
in
present which exhibits a variation
means
direction, a degeneration, the

;
new
culture
is
of cure
then introduced.
Circulation in Nature.
10.
We
is
Finally,
shortly describe
what
is
so far
known
of
the circulation of the saccharomycetes in nature.
In the years 1880-81 Hansen published the results of
one of the most important and most interesting biological

researches on yeast cells which
had carried out

yeast
named Saccharomyces
find that this
autumn on
juice of
we
apiculatus,
fungus occurs and
which
it
multiplies
fructifies in
From it we
summer and
during winter and spring

fruit
ceptional cases in other places.
partly through
means
Reess.
the injured parts of sweet juicy fruits, in the
found in the ground under the
cells
and which he
possess,
in the preceding years with the cells of the
the
of the rain
falling
which
and only
of the
in quite ex-
into the
It gets
it is
ground
and partly by
fruit
trickles over the fruit.
Those
which pass into the intestines of birds and insects
and are given
oflT
with the excrements also pass
finally into
the earth.
Hansen has likewise shown that Sacch.
apiculatus appears
normally only on sweet juicy fruits and under the latter in

the ground
the cause of this
resisting drying
when
which
this
is
the very slight power of
fungus possesses.
Therefore,
present on the surface of unripe whole fruit, on
leaves, twigs, etc.,
where
it
cannot multiply,
it
'a comparatively short time through drying.
dies off after
Otherwise
it
CIRCULATION IN NATURE
maintains
its life in fruit
the soil where
it is
its
where
other small animals.
by
in
Direct experi-
this.
winter abode in the ground
the sweet juicy fruit
and
multiplies,
it
protected from drying.
ments have demonstrated
From
juice
247
it
again reaches
aid of the wind, and of insects
Rain
may
spect, viz., in the case of fruit
and
also be effectual in this re-
growing near the ground.
As
soon as multiplication goes on in the fruit juice, insects again
play an important part in distribution by carrying the
from
fruit to fruit
according to
Wortmann wasps
cells
are par-
ticularly efficacious in this respect in vineyards.
Insects
however, the distributing agents only during a small
are,
part of the year, and then only on the sunny days of that
period
through the year the wind carries the
all
about with the dust and
finally
deposits
them
cells
in large
quantities on the fruit.
With regard to typical saccharomycetes recent investigaHansen have shown that they likewise pass the
tions of
winter in the ground, and that sweet juicy fruits are essential
breeding places for them, so that they pass through the
same
Miiller-Thurgau and
vestigations of
not spend
ments,
winter underground.
the
however,
living yeast
producing
has,
Wortmann
in-
confirm
Pasteur was of the opinion that wine yeasts do
this.
i.e.,
The
circulation in nature as Sacch. apiculatus.
in
speak
the
soil
besides,
when
seeded
Hansen's experi-
this,
he
since
found
under the vines in the wine-
Germany
districts of
at a time
against
in
spring and summer,
Hansen
there are no ripe grapes.
saccharomycetes in the
soil
under
natural conditions, and has found them living there more
than three years

places
of
after.
After finding that the breeding
saccharomycetes are on sweet juicy fruit he
proceeded from this and followed the
way during
cells
farther on their
the different seasons of the year.
The
sac-
248
may
charomycetes
of
course follow, besides the normal
some other accidental and exceptional
circulation,
Only the regular annual repetition
one.
the circulation
of
is
of interest in relation to a knowledge of this portion of
the
economy
Some
of nature.
writers have expressed the view that the breed-
ing places of the saccharomycetes are in the nectar of

flowers until the time of ripe fruit, and that the winter
is
passed in the intestines and excrements of insects and
Hansen's investigations have shown, how-
other animals.
ever, that
if
they are found in these places at
insects
have
also
all,
it is
The author's experiments on
quite a matter of accident.
demonstrated
this.
Hansen's researches on the circulation of saccharomycetes

in nature,
and on the amount
of micro-organisms in the air
at various seasons of the year, have led to the following

results,
very important to the brewer
insects are
of
yeast cells in nature,
clouds
cells,
in
(1)
Wind and
the most important means of transportation

especially
the
first
(2)
dust
the harvest months are rich in strong yeast
produced on sweet juicy fruit; and
the year the open
cooling
by which wild yeast
vessels
species
make
(3) all
through
are the chief means

their
way
into the
brewery.
The
New
practical
outcome of
this
was
that, in the
Old and
Carlsberg Breweries in Copenhagen, the ways in
the neighbourhood of the cooling vessels were sprinkled
with water, so that the dust might not infect the wort,
a procedure which
after the pure
cooling vessels
was followed
in other places.
Later,
was introduced, the open

were removed from the above breweries and
culture system
replaced by closed holders for cooling and aerating the

wort.
The cask deposit and likewise impure pitching yeast may
SYSTEMATIC
249
also give rise to infection; before introducing the pure culture
system the
latter was probably the worst source of infection.

by any means, wild yeast, etc., makes its way into
If,
practice,
it
may
connections,
collect in the
and these
water
pipes, especially at the
according to Will's
are, therefore,
experiments, the most dangerous sources of infection
the wort has been once infected
if
the cracks and crevices
in fermenting vessels are also dangerous in this respect.
very important to keep these places
It is therefore
clean.
shown that the filter

But the wind with the dust
the year round, the chief means by
Will's experiments have also clearly
bag
a source of danger.
is
remains,
it carries
all
which uninvited intruders make their way into the brewery.

Combined with this we have the insects in the summer
months on sunny days. Now that impure pitching yeast
has become rare, the open cooling vessels therefore form
most
the
important
most
and
dangerous
source
of
infection.
Systematic.
1.
Genus
(Synonyms
Saccharomyces, (Meyen)
Mycoderma, Persoon
Beess.
Cryptococcus, Kiitz-
ing; Torula, Turpin; Hormiscium, Bail.)

Single
place
cell
fungi in which vegetative increase takes
by budding and which develop endospores
interior
under certain conditions.
in their
Sometimes they
may
form a typical mycelium.
The various
be grouped
The
related species here
may, as before
following classification
is
of this kind,
to be found in Hansen's paper of 1888

1.
stated,
according to their action on the various sugars.
and
its
basis
is
Those species which ferment maltose, dextrose and
.saccharose.
25a
Examples
Saccli. cerevisice
Hansen.
7.,
Fastm-iaims
I.,
III.,
ellipsoldeiis I.,
II-,
besides
brewery yeast
species
and
in general the culture
yeasts hitherto investigated.
Those species which ferment dextrose and saccharose
2.
but not maltose.

Examples:
SaccJi.
Hansen.
Marxianus,
,,
exiguus (Reess),
Ludwigii,
,,
,,
Saturnus,
Kloeker.
Those species which ferment dextrose, but neither
i.
saccharose nor maltose.

Example
Sacch. mali Diiclauxi, Kayser.
Those species which ferment dextrose and maltose,
4.
but not saccharose.

Example;
Saccli n.
sji.,
isolated
from the stomach
of
a bee by the
author.
5.
Those species which ferment neither maltose, dextrose
nor saccharose.
Examples
Sacch. niembrancefaciens,
,,
6.
,,
farinosus,
,,
,,
anomaliis var. belgwus,
Those species which can ferment

Example
The
descriptions
in question,
lactose.
Sncch. frngilis, Jorgensen.
and investigations
below are from the authors
name
who have
of the species given
established the species
and whose names are added
of the species.
gators are given this
Hansen.
Lindner.
)tyalosporus,
Where
is
to the systematic
the researches of other investi-
specially mentioned.
SACCHAROMYCES
Saccharomyces
87,
88 and
89), was
The
Hansen
(Figs.
79 1, 80, 81, 82,
London
later also in the yeast of a
cells in
and round
1.,
from a top yeast in an Edinburgli
isolated
and found
brewery,
brewery.
large
cerevisise
251
the yeast sediment are, as a rule,
In film growths at 6 to 15
(Fig. 87).
same shape
as in the
sediment yeast, with only isolated abnormal forms
(Fig. 88).
The
fi;
C. they are for the
most part
the spores
size of
of the
from
varies
are usually one to four in each
79
and
The germination
89).
in Figs. 80, 81
At
At
At
At
At
and
30 C.
seldom
five
there
(Figs.
of the spores is represented
82.
20
to 12 C.
10 days.
9 C.
no spores develop.
Saccharaniycea
Han.sen.
Sediment
o-rei'lsin
yeast,
I.,
Fig. 88.
^î.
i?i.
liotxhanii-niices reremsui- /.
spores.
Sacelutrunii/ces cereoisiti: I.,
Hansen.
(After Hansen.)
C.
2^ to
37^ C. no spores develop.

36 to 37 C. the first indications are seen after 29 hours.
Fig. 87.
Fig. 89.
cell,
Hansen.
Film growtli at
15-6''
C.
(After Hansen.
First stages of
development
of the
(After Hansen.)
The temperature limits for film formation are 33'^ to 34

and 6 to 7 C. The species is a vigorous top beer yeast.
We shall describe some of the numerous foi'ms which are
employed in the industry.
252
The
may
cell
form of Carlsberg Bottom Yeast No.
The
be seen from Fig. 90.
extreme
difficulty
species
Fig.
In practice
all.
\iO.C'(i,ishi',-rj
Hansen,
after a long time (five to six days at
25 C.) they are only to be found singly.
are none at
1,
forms spores with
it is
BoUinn Ymxt Nn.
indifferent claritication
1,
Very often there
inclined to give only an
Hansen.
(After Hansen.)
and a strong attenuation,
but,
on the
As seen
in Fig.
other hand, produces a tine stable beer.
Carlsberg Bottom Yeast No. 2, Hansen.

91, the
shape of the
ceding species
it
cells is
also
more regular than
in the pre-
forms spores somewhat more
Beer prepared with this yeast
is
easily.
not so stable, but clears
better in practice.
\r.
91.
Ijarlshrrij JSutlmn
yeast
i-"/s.
Nil. 2,
Hansen.
Some
cells
with spores.
(After Hansen.)
Of four culture yeasts of the Munich station described

by Will, Tribe 93 and Tribe 2 belong to the strongly fermenting species, Tribe 6 to the yeasts of medium fermentation,
while Tribe 7
species.
The
last
is
to be classed with the feebly fermenting
forms spores with great
difficulty,
while
SACCHAROMYCES
the
cardinal
points
for
Tribe
Limits for spore

mation.
Optimum
them abundantly and easily. The

spore and film formation are the
three produce
first
following
^
253
Tribe
2.
Tribe 93.
6.
Tribe
7.
for-
|31 to 11 C. 31
to 11 C.
30 to 10 C. 30 to 13
for spore 1
formation.
' ^^
^^ ^^
^^
2^
^^ ^-
25 to 31
Limits for film for-\ 28 to 31
30 to 31
mation.
7 to 10 C.
4 to 7 C.
/ 7 to 10 C.
' ^^ ^'
25 to 28
4 to 7 C.
Film formation goes on most quickly in Tribe 7. The

cells of all four species are round or oval
in Tribe 7 giant
;
cells
occur regularly.
The three types
of brewery yeasts, Saaz, Frohberg
Logos, have been mentioned on
p.
and
219 with regard to their
action on the sugars.
There
is
number
also a very large
Species and Races.
two such
description of
Top Yeast
of Beer
Hansen, in his paper of 1882, gives a
by pure
species prepared
culture
the one was isolated from a Burton yeast, the other from
an Edinburgh one.
another.
They were
plainly different from one
detailed description of
Sacch. cerevisicB
I.,
Hansen's top yeast,
has already been given.
have been prepared by pure culture

Jorgensen and Schonfeld.
Several others
by
later, especially
Jorgensen groups the forms
present in his institute, including the bottom yeast species

as well, from the practical point of view,
how
according to
viz.,
quickly or slowly they clear and at the same time
ferment strongly or weakly.
The
distillery yeast.
Race
II.,
isolated
at
originated in a distillery in
of the Frohberg type,
large cells
and
obtained from
its
it
and
is
West
Prussia,
the Berlin
Germany
Station, has attained a wide distribution in
is
in practice are very
adapted for the fermentation of
good
difficultly
it
a top yeast
distinguished chiefly
great fermentative power.
The
it is
by
its
results
especially
fermentable and
254
highly concentrated mashew,
its
power
of resistance to high
alcoholic content being great.
The
station at Berlin has also isolated various yeasts for
use in pressed yeast manufacture, and Race IX. especially
has given satisfactory results.
We
shall
now
describe several wild yeasts.
Saccharomyces Pastorianus
93),
was
first
I.,
Hansen
brewery, and later also in diseased beer.
wort consists
Fl(.. 92.
J'u.iluriuiun.
Sediment yeast,
Fig. 93.
j^".
Hansen.
(After Hansen.)
number
IX,
elongated
The
in
Hansen's paper.)
size of the spores is
they seldom attain a diameter of 5
oftenest
to
4,
in
round and
Haccharoinyces Paslmiunna J.,

Film growth at 15 3 C. ij^.
Holm
(After
oval cells are also found (Fig. 92).
li to 31
92 and
The growth
chiefly of sausage-shaped cells, but
"itnr.lnii-iiiiiyces
/..Hansen,
(Figs. 792,
found in dust in the air of a Copenhagen
They
fi.
sometimes also 5 to 10 in very
cells.
At 31i C. no spores develop.

,,
29^ to .301 C. the
'
271 C.
3 to 4 C.
,,
i C.
and
are seen after
.30
hours.
24
13 days.
no spores develop.
The temperature
C.
first indicatioiis
8 to 5 C.
limits for film formation are 26 to 28
This species
is
a bottom yeast form and,
SACCHAROMYCES
255
as already stated, a dangerous disease yeast in breweries,

since
causes a disagreeable smell and a
it
taste in the beer.
strong bitter
It usually has a detrimental effect
clarification as well.
While thus possessing bad
for beer manufacture,
it
on the
qualities
can yet give a good product in the
preparation of wine (Mach and Portele).
SaccharomycesPastorianus
fl5),
was
also
brewerj'.
The
cells are
usually
94.
Hansen
somewhat
Fig.
Saccharoiiiyces Pustoriau'Ufi
Hansen. Sediment yeast.

(After Hansen.
//.,
(Figs. 79
air in a
3,
94 and
Copenhagen
larger than those
This yeast produces a feeble top
of the preceding species.
V\G.
11.,
found by Hansen in the
^-".
Saccharoinyces
95.
Pasturiaau.'i
Film growth at 15 3
(After Holm in Hansen's
//.,
Hansen.
C.
i""-.
paper.
fermentation.
to 5
The
size of the spores is 2 to 5
/i,
seldom 4
/x.
At 29 C. no spores develop.
,,
27" to 28 C. the
25 C.
3 to 4
,,
first
indications are seen after 34 hours.
J C. no spores develop.
C'.
,,
25
17 days.
The temperature limits for film formation are 26 to

C. and 3 to 5 C. The cells of the young film at 13 to
C.
28
15
are distinguished from the corresponding cells of the
succeeding species by generally being round or oval, whereas

in Sacch. Pastorianus III.
found under these
many
conditions.
sausage-shaped
cells
are
Streak cultures on yeast-
256
water gelatine at 15 C. show colonies with smooth borders

in sixteen days, the species differing
from Sacch. Pastoriantcs
III. in this respect also.
Saccharomyces Pastorianus
and
Copenhagen beer
Fig.
96.
111.,
Hansen
(Figs.
79
4,
96
This species was found in bottom fermentation
97).
The shape
affected with yeast turbidity.
Skucharomyces Pastorianus III., Hansen.
Sediment
ijs.
yeast.
(After Hansen.
of the cells
is,
The spores
the two preceding species.

size,
same as those of
for a culture in wort, the
seldom 3i to 4
are 2 to 4
/x
in
/a.
^^^
Fig. 97.
,'iaecharormjces
Pastorianus III., Hansen.

ao.
Film growtli at 15-3
C.
(After Hansen).
25 C.
8J C.
4 C. no spores develop.
,,
C.
,,
28
9 days.
The temperature limits for film formation are 26 to 28"

and 3 to 5 C. The cells of the young film at 13 to 15
C. are distinguished
Pastorianus
II.
from the corresponding
by many
of
cells
of Sacch.
them being very long and
SACCHAROMYCES
sausage-shaped (Fig. 97)
257
in the latter species the cells are
frequently round or oval.
Streak cultures on yeast-water gelatine at 15 C. give,

in sixteen days, colonies
with distinctly hairy borders.
This species produces a more vigorous top fermentation
than Sacch. Pastorianus
It
II.
is
which causes turbidity in beer.
Saccharomyces
Fig. 98.
ellipsoidei/s I.,
HanseD. Sediment yeast,
-i-fi.
a dangerous disease yeast
But a small addition
Fig.
99. /Saccharomyces
Hansen.
(After
^^.
Hansen.
ellipsoideus
I.
Film growth at 15-13 C.
(After
Holm in
Han.sen's paper.
some circumstances, make
species to stock yeast can, in
opalescent beer clear, probably
of this
by removing during
after-
fermentation the substances which cause the opalescence.
Saccharomyces ellipsoideus
99).
I., Hansen (Figs. 79 5, 98 and

found
This species was
by Hansen on the surface of
ripe grapes in the Vosges district.
dal shape, but
2 to 4
/i
may
The
cells
also be sausage shaped.
in size, seldom 3| to 4
17
fi.
are of ellipsoi-
The spores are
258
At 32i
no spores develop.
C.
30J to 31 J C. the
25 C.
,,
,,
first
,,
î C.
4 C. no spores develop.
,,
21
,,
11 days.
The temperature limits of film formation are 33 to 34

C. and 6 to 7 C. The cells of the young film at 13 to 15
C. are distinguished from the corresponding ones of Sacch.
ellipsoideus II., which are round or oval, by the large number
of long, sausage-shaped cells (Fig. 99).
Streak cultures on wort gelatine at 25 C. after eleven

to fourteen days give colonies with a peculiar reticulated
structure (here dififering from
and from Sacch.

This species
Numerous
is
Hansen's foregoing species
ellipsoideus II.).
one
of the
many vphich
are active in wine fermentation.
forms, closely related to this species, have been isolated in the
experimental stations for wine culture by Aderhold, Hotter, Marx, Muller-
Thurgau, W.
Seifert,
Wortmann and
Among
others.
these species there
are some in which the cells are vigorous spore formers, e.g., the species
" Johannisberg II.," which has become so well known through Aderhold
which 99 to 100 per cent, of the cells

The maximum temperature for spore
formation is, in this species, according to Hansen, between 33 and 34J
C, and the minimum temperature between 3 and 2 C. Another wine
and Wortmann's
researches,
and
of
develop spores on gypsum blocks.
yeast is the Walimrzheini yeast. According to Aderhold this yeast is distinguished, in one respect, by the rapidity with which it forms a film
in
which a large quantity
of spore-bearing cells appear.
Saccharomyces ellipsoideus
and
in
101), is
II.,
in size, seldom 4 to 5
At 35
Hansen
(Figs. 79
100
C.
The spores
are 2 to 5
fi
/a.
no spores develop.
29 C.
8 C.
4 C.
no
spores develop.
The temperature
C, and
The
6,
a very dangerous disease yeast (yeast turbidity)
22
9 days.
limits for film formation are 36 to 38
3 to 5 C.
cells of
the
young
film at 13 to 15 C. are distin-
SACCHAROMYCES
259
guished from the corresponding ones of Saooh. elUpsoideiis
by being
Two
disease yeasts isolated
The
related to this species.
by Will
are very closely
cardinal points in the spore
formation of these two yeasts are as follows

At 41"
And
C.
I.
round or oval in shape.
chiefly
For one
no spores develop.
39 C. the
34 C.
first
11 hours.
8to9C.
4 to 5 C.
9 days.
for the other
no spores develop.
,,
30 to 31 C. the
first
23-5 to 24 C.
3 C.
0"5 to 1 C. no spores develop.
29
21 days.
OQoô
Hansen. Sediment Yeast.
//.,
//..Hansen. Film Grovfth at 28 to

3 C.
(After Hansen.
"f"-.
"J-"-.
(After Hansen.)
Saccharomyces
of Ilex Aquifolium.
The
Ilicis,
The
Saccharumi/ces eUipsoideus
FiG. 101.
Saccharoiiiyces ellipsoideuK
100.
Fii;.
Gronlund, was found on the fruit
cells
are mostly spherical in shape.
cardinal points for spore formation are the following
,,
36 to 37 C. the
first
32 C.
18
9i C.
,,
20 days.
,,
8 C.
no spores develop.
Streak cultures on wort gelatine have a mealy appearance.
Wort fermented with
able bitter taste.
It
is
this species
assumes a
disagi'ee-
a bottom yeast which ferments
17*
260
saccharose, dextrose
and maltose
in
wort
it
produces
278
per cent, of alcohol.
vol.
Saccharomyces
the same
It
fruit.
Aquifolii, GrSniund,
is
was discovered on
a top yeast and, judging from the
The cardinal
appearance of the spores, a culture yeast.
points for spore formation are the following,;

At 30i to 31 C. no spores develop.
27^ to 28^" C. the
27 G.
10 to 10^ C.
,,
fiist

,,
,,
8 to 8i C. no spores develop.
28
15 days.
Streak cultures on wort gelatine have a shiny appearThis species gives to wort a sweet taste with a bitter
ance.
after-taste
and ferments saccharose, dextrose and maltose.
In wort
forms 371
it
vol.
Saccharomyces Vordermanni, Went and Prinsen GeerThe cells are

ligs, is, in appearance, similar to wine yeast.
rounded
gated
four
it
like a pear or onion;
a film
not formed.
is
sometimes angular or elon-
The number
Like
all
of spores
is
usually
the preceding species
ferments maltose, dextrose and saccharose, the latter after
inversion.
is
are found.
cells
It
forms 9 to 10 per
in "
present
Raggi," which
cent, of alcohol.
is
employed
in
The fungus
Java in
thlT
manufacture of arrack.
"Raggi
"
is
made
in the form of balls or cakes which cona|^ of rice,

and other vegetable substances, and are saturated
Among the latter are bacteria, yeast cells and mould
pieces of sugar cane
with organisms.
The presence
advantageous to fermentaThe yeast

they are in large quantity.
cells belong partly to Sacch. Vordermanni and partly to Monilia javanica.
The mould fungi are the Mucor oryza and Rhizopus oryzce already described they both turn rice starch into sugar, which is then fermented
fungi.
tion, but rather
of the first of these is not
unfavourable
if
The first of these two

both by Sacch. Vordermanni and Monilia javanica.
fungi gives a very fine arrack, whilst the latter produces an alcohol with a
bad taste. Here, therefore, a pure culture of Sacch. Vordermanni could
be employed to great advantage.
SACCHAROMYCES
261
Saccharomyces pyriformis, Marshall Ward, is active in

the fermentation of ginger beer.
The cells are similar in
shape to those of the Sacch.
ellipsoideus group.
On wort it
forms a film composed of pear or sausage-shaped cells. In
conjunction with the Bacterium vermiforme, also found by
Marshall Ward, it produces, from a sugar solution con-
taining ginger, an acid frothing beverage, ginger beer, which

is
prepared in
many
Saccharomyces
grapes.
The
cells
cottages in England.
Marxlanus,
Hansen,
was found
on
are small, oval or egg shaped, or elongated
and sausage shaped, often in colonies. When cultures have

been in wort for some time, bodies form which consist of
myceHum-like colonies. When the wort cultures are two
to three
months old a film
is
just visible, consisting partly
of short, sausage-shaped cells and partly of oval ones.
On
Hansen observed that this species develops

a mycelium which is similar, for instance, to that formed
by Monilia Candida. The spores are more or less kidney
solid substrata
shaped, sometimes, however, round or oval, and usually
about 3'5
/i
long.
After cultivation in wort they are not
formed particularly
easily,
but are formed
much more
quickly and in large quantity after cultivation in a nutrient

solution containing dextrose (the author).
The cardinal points
for spore formation are, according
maximum between 34 and 32 C,

and 22 C, and minimum between
to the author, as follows
optimum
8 and 4 C.
4^*rding
between 25
to
Hansen
it
gives only 1 to 1 3 vol. per cent,
of alcohol after standing for a long time in wort.
It is
capable of fermenting dextrose and saccharose, the latter

after inversion, but not maltose.
In a 15 per cent, solution
of saccharose in yeast water 3'75 vol. per cent, of alcohol
were formed
at 25* C. in eighteen days,
in thirty-eight days.
and
7 vol. per cent,
In yeast water containing 10 and
262
15 per cent, of dextrose

vol.
it
formed, in a month, 6"5 and 8
per cent, of alcohol respectively.
Saccharomyces exiguus, (Reess) Hansen, was found

general in pressed
yeast.
This species
from the preceding one in that

celium-like colonies in
formation of spores
of a film
wort nor mycelia on

scanty
it is
in
distinguished
myThe
does not develop

gelatine.
here also only the semblance
formed after several months.
is
ing species
is
it
is
Like the preced-
incapable of fermenting maltose.
formed
It
6 vol. per cent, of alcohol in a 15 per cent, solution of
saccharose in yeast water.
In a 15 per
dextrose
cent,
solution 8 vol. per cent, of alcohol were formed in fourteen
days at 25 C.
Formerly the opinion was held that
Fig. 102.
s'oi,'c/(('OwiycfsoiiMs,
Hansen.
this species
Spore-bearing
cells.
was the
^y-K
(After
Hansen.
cause of turbidity in beer.
Hansen
has,
however, shown
that even a large addition of this fungus at the beginning

or end of the primary fermentation or during storage causes
no kind of disease
in lager beer.
Saccharomyces anomalus, Hansen
(Figs. 83
and 102),
was found by Hansen in an impure brewery yeast from

Bavaria.
This fungus was also found later in EngHsh and
Belgian beer, on green malt, on bran, in syrup of althea,
in soil
and on
In wort
fruit,
it
e.g.,
plums.
produces fermentation very soon, both at
ordinary room temperature and at 25 C.
ginning of the fermentation a dull grey film

fermentation the liquor
is
developed.
is
At the very
is
be-
formed; during
turbid and a smell like fruit ether
The microscopic appearance
of the vegetative
SACCHAROMYCES
reminiscent of a
cells is
Toriila.
sometimes sausage shaped
They
and
are small, oval
of these are found parti-
After some time
cularly in old cultures.
may
Two
many
263
with spores
cells
be found both in the film and in the sediment yeast.

to four spores are formed in each cell
tinguished from the spores of
They
their shape.
are, in fact,
ing rim round the edge of the
diameter of the
all
these are dis-
other saccharomycetes by
hemispherical with a projectflat side,
i.e.,
hat-shaped.
not including the rim,
flat side,
is
The
2 to 3
/.t.
Nielsen found the following cardinal points for spore
development
,,
32^ to 32 C. the
first
30 C.
7i to 6 C.
3 to 2^ C.
,,
indications are seen after 19 to 21 hours.
found
in
those of
Endomyces
13 to 14 days.
The shape possessed by the spores

also
17 to 19
no spores develop.
of this species
decipiens,
is
a fungus
which appears as a parasite on a mushroom (Agaricus

melleus),
on the lamellae of which
whitish layer.
it
spreads itself as a
In spite of the similarity of the spores,
however, Endomyces decipiens has no genetic connection
with Sacch. anomalus.
Further, the spores of the latter
on germinating form buds, whilst those of Endomyces form
germ threads
at
in this latter species
no budding takes place
all.
According to Nielsen's experiments, Sacch. anomalus in
wort forms
days.
ethyl
species
W.
0'9 vol. per cent, of alcohol
Seifert states that the
acetate,
and that
in
and
ester
ester in eleven
formed here
is
wort containing alcohol the
forms acetic acid and decomposes the alcohol into
carbonic acid and water

destroys the acetic
ester.
a prolonged action of the fungus
According to Nielsen,
it
causes no
fermentation in maltose solutions, and secretes hardly any

invertase.
264
Belated forms have been found by Holm, Jorgensen, Lindner, Will,

Lindner found one of this kind, among others, in
Zeidler and the author.
an Armenian beverage,
"
Mazun
"
Jorgensen states that he has found a
which had yeast turbidity.

In bottom fermentation beer it has been observed several times, but never
under such conditions that it could be looked upon as a disease yeast.
A species which also has hat-shaped spores has been named Sacch.
atiomalus, var. belgicm, by Lindner it ferments neither maltose, dextrose
nor saccharose, and produces no smell of fruit ester. Beijerinck describes
a typical Sacch. muyinalus under the name Mycoder>na pulverulenta ; others
have also isolated new related species.
related form in English top fermentation beer
Saccharomyces membranaefaciens, Hansen, was found

in a gelatinous mass
roots of an elm tree.

in the water
which had developed on the injured

It was also found later by Koehler
from a polluted stream, and by Jtirgensen
in
white wines.
The
species quickly forms,
on the whole surface of the
wort, a strongly developed, light gray corrugated film, consisting chiefly of sausage-shaped
rich in vacuoles
it
wort gelatine
is
with a reddish
tinge.
They
Myooderma cerevism and M.
The shape
and
cells
liquefied very quickly
by
of the spores
are similar to the colonies of
vini.
is
very variable
rounded and sometimes inclined
They
and elongated, oval
forms on this substratum dull gray colonies, often
it
they are often
to a hemispherical shape.
are formed in large quantity both on
gypsum
blocks
in films.
This species
alcohol
it
is
distinguished
by
inability to
its
form
produces fermentation neither in saccharose,
dextrose, maltose nor lactose solutions
it also
secretes
invertase.
Nielsen found that

At 35 no spores developed.
334 to 33 G. the
31 to 30J C.
7i to 6 C.
first
3 to 2J C. no spores developed.
17 to 18
6 to
7 days.
no
SACCHAROMYCES
W. Seifert found that Sacch.
265
membrancefaciens grows even
in the presence of 12-2 vol. per cent, of alcohol.

artificial
nutrient liquid (Pasteur's solution) with 4'8 vol.
per cent, of alcohol and an addition of malic acid
0110
it
formed
per cent, of glycerine in fourteen weeks; during the
same time 3'8
vol.
per cent, of alcohol disappeared.
hardly attacks tartaric acid at
all,
nor
citric acid,
It
but malic
Acetic and succinic acids are completely consumed
acid.
hy
In an
The
it.
acetic acid
and glycerine formed are again used
In dextrose solutions (not in those of saccharose or
up.
maltose)
it
forms fixed and volatile
acids.
It destroys the
bouquet of wine, the esters present being broken up, while
new and
less
favourable ones are formed.
found in Crimean wine a variety which he calls Sacch. meinThe latter stops growing in the presence of
12'2 vol. per cent, of alcohol. The maximum temperature for spore formation is 34 C, the minimum temperature 5 to 6 C.
Seifert also isolated another form from Californian claret, viz., Sacch.
membrancefaciens var. californicus, which is capable of growing in the
presence of 12-2 vol. per cent, of alcohol. The temperature maximum for
spore formation is 33 C, the minimum 7 to 12 C.
Both of the latter forms are further distinguished from Sacch. memiraneefaciens, Hansen, by their forming much less glycerine in the above
Pasteur solution and being able only to attack alcohol to a small extent.
Other related forms are described by Pichi under the names Sacch.
viembrancefaciens II. and III., and by Lindner under the names Sacch.
Seifert
braruBfaciens var. tauricus.
hyalosporus and Sacch. farinosus.
Saccharomyces
is
Lindner, was isolated from Dan-
It ferments dextrose, inverts saccharose,
zig Jopen beer.
and
Bailii,
distinguished by
able amcebiform
cells in
its
developing extremely remark-
old cultures.
It
forms no
film.
Saccharomyces mali Duclauxi, Kayser, which was found

in cider, is described as follows
long and 4 to 7
sediment.
off at
They
/i
The
cells
are 6 to 12
/u.
broad and form an easily disturbed
are moderately sensitive to acids, and die
about 55 C.
Spores appear at 15 C. in thirty hours.
This yeast ferments neither saccharose nor maltose, but
266
ferments invert sugar and imparts a distinct bouquet to the
fermented
liquid.
Saccharomyces cartilaginosus, Lindner, was found in

Kefir.
The
species ferments in
what smoky
taste
wort and gives to
like Sacch. Pastorianus III.
frilled giant colonies
and streak
On
peculiarly granular.
cultures.
it
a some-
forms
it
much
The plasma
is
the surface of the wort after some
weeks, small well-defined island colonies are formed, of some-
what compact, almost
gristly consistency.
Coalescence of
the islands into one single layer does not take place.
sediment yeast
is
following species
It
does not ferment milk sugar.
it
Saccharomyces
Kefir.
fragilis,
Jorgensen, was also found in
ferments milk sugar.
ing description
The growth
oval and elongated
cells.
Jorgensen gives the follow-
consists of comparatively small
In cultures on gypsum blocks
spore formation appears distinctly at 25

hours, at 15
of the spores
The
In contradistinction to the
flocculent.
The
in forty hours.
long,
in
C.
twenty
rounded shape
These form both in ferment-
is characteristic.
ing liquids and on gelatine.
In 10 per cent, lactose-yeast-
water the species gave about
alcohol at the
months
room temperature
gave 4 parts per
it
part per cent, by weight of
cent,
hopped wort (about 11 per cent.
in eight days
by weight
Ball.) it
after four
of alcohol.
In
produced about 1 part
per cent, by weight of alcohol at room temperature in ten days.
By
Kefir, as is well known, is understood the beverage which originated

Caucasus and is prepared there by fermenting milk. This fermentation is brought about by the so-called Kefir grains, which are yellow,
in the
hard granules of the size of a pea. The mode of preparation in the

Caucasus consists in placing the milk in goatskins to which the above
corns are next added it is then all shaken up from time to time. After
;
few days the drink is ready. The Kefir grains are taken out and kept
During fermentation, alcohol, lactic acid and
for another fermentation.
u,
carbonic acid are formed

co-existence
'
{e.g.,
'
of various
these products are formed by the accidental

organisms of which the number and species are
;
In literature Kefir fermentation and other similar fermentations

that of ginger beer) are represented and explained as the results of
SACCHAROMYCES
267
very variable.
Preudenreich found, in the Kefir grains examined by him,

a Torula and three bacteria, among these the Bacillus caucasicus. The
last appears to be always present with an alcoholic yeast fungus.
Besides
the two saccharomyeetes mentioned, several other species of Snccharomyces are cited as being found in Kefir.
In Armenia a beverage called Mazun, similar to Kefir, is prepared from
milk. According to Emmerling the exciter of fermentation consists of a
white, fatty, cheese-like mass which can be preserved for a long time.
After a short time it produces alcoholic fermentation in the milk simul;
taneously the casein coagulates, acid being formed, and a smell of fatty
acid ester
In this mass Emmerling found the following

among which were a number of coloured species,
some other mould fungi, a yellow Sarcina and the common
developed.
is
micro-organisms
yeasts
Oidium lactis,
hay bacillus {Bacillus subtilis), also some cocci and the Bacillus acidi
lactici, Hueppe.
The last-named and the cocci turn the milk sugar into
lactic acid
the milk sugar
is
also hydrolysed
ceptible to the attacks of the yeasts.

result of
tt
and thus rendered susis
thus also the
yeasts.
As already
This fermentation
fortuitous co-existence of bacteria
and
mentioned, Lindner found Sacck. anomalus in this beverage.
we
Finally,
romyces which
shall describe another species of Saccha-
was found
in the exudations from oak trees,
namely,
Saccharomyces Ludwigii, Hansen
This
with
it
species
is
it
77,84 and 85).
something between a typical budding and a simple
division takes place.
that
(Figs.
the transitional form to the next genus, as
deserves a
It is in
somewhat
many
respects so remarkable
closer description.
It is pro-
bably the only Saccharomyces which can be immediately

recognised
by microscopic examination.
Although the
vegetative
cells possess all possible shapes,
the lemon shape
is
the most prominent
it
reminds one somewhat of that
This does not agree with the original signification of the

word, since in the cases cited it is only a question of a casual co-existence,
a mixed fermentation otherwise every fermentation in which two or
more species appeared would be a symbiotic fermentation. By symbiosis
a symbiosis.
was formerly understood an intimate relation which is, physiologically,

beneficial to both participants, and the lichens were given as an example
But there is nothing analogous in the above ferof a real symbiosis.
mentations.
There is really no longer a definable idea attached to the

it would therefore be best to cease using the word.
word symbiosis;
268
of
Sacch.
apiculatus,
but the
cells
are
of
new growth
at a point
and,
far larger,
The
besides, the appearance is quite different.
first
stages
are as in the preceding saccharomycetes
on the
cell
a wart makes
its
appearance; but
by means
The genus Schizo-
instead of a regular budding a septum appears
new
of which the
cell will
cut itself
off!
saccharomyces has no budding whatever
but inside the
cell
Thus Sacch. Litdwigii is in this

respect related partly, and in fact most nearly, to the
real saccharomycetes, and partly to the schizosaccharomythis
septum
is
formed.
Schizosacducromyces Pombe, Liudner. Vegetative and apore bearing

A|a. (After Lindner.
n, Germinating spores.
cells,
Fig. 103.
cetes on account of the
conditions
cultures
(p.
it
septum formation.
Under
certain
forms a typical mycelium, especially in old
199, Fig. 77).
Hansen's researches on spore germination (Figs. 84 and

85)
have been already alluded
species
.and
is
also distinguished
might well be
to.
In this respect the
from the other saccharomycetes
classified as a separate genus.
germination a promycelium
are developed from the
is
latter.
During
formed and the yeast
cells
Coalescence of the young
spores usually takes place in the early stages of germination.
SCHIZOSACCHAROMYCES
269
This species forms spores very easily on g3rpsum blocks
and
gelatine,
10 per
3 to 4
and
also in various nutrient liquids,
/x
e.g.,
in a
The spores are round, and
cent, saccharose solution.
According to Nielsen the cardinal
in diameter.
points of spore formation are the following
At 34 no spores are formed.

32 to 32J C. the
30 C.
6^ to 11 C.
2J to 3 C.
first
18 to 19
13 to 14 days.
no spores are formed.
Sacch. Ludivigii, according to Hansen, ferments dextrose
and saccharose but not maltose.

can produce up to 10
vol.
wort only 12 voL per
In dextrose-yeast- water
it
per cent, of alcohol, but in beer
In a saccharose solution
cent.
it
sometimes dies off rather quickly, a behaviour contrary to

that of most of the other Saccharomyces species.
2.
Genus
Schizosaccharomyces, P. Lindner.
and not by
This fission occurs through a septum being
budding.
formed nearly in the middle of the cell the septum splits
and the cell divides into two cells often hanging together as
Vegetative increase takes place
by
fission
if
by a
hinge.
Schizosaccharomyces Pombe, P. Lindner

found by Saare in
The two ends

the other
is
Pombe (negro
of the cells are often difierent
encircled
encloses the
by a
newly formed
(Fig. 103),
millet beer)
from
one
is
was
Africa.
rounded,
well-defined circular ridge which

conical
membrane.
In exhausted
culture solution the cells become shorter.
Spores are formed to the number of

ascus
to 4 in each
they form more easily in culture liquids than on
gypsum
blocks.
germinating tube.
Germination takes place by means of a
film
is
temperature for the growth
forms much
alcohol, the
not formed.
is
large
30 to 36 0.
amount
of
The optimum
This species
which has no
270
deleterious influence on the growth.
yeast,
It is a
form of top
and ferments maltose, dextrose, saccharose and also
dextrin
This fungus
contains invertin.
it
advantage in South American

stand the
warm
is
employed to
distilleries, as it is
able to
climate.
Schizosaccharomyces octosporus, Beijerinck
(Figs. 86,
104 and 105), was found by Beijerinck on currants from

Oreeee, and by Schionning on Italian raisins.
cells are cylindrical,
they are 4-5 to 6
Fig. 104.
/i
some
oval,
broad and 7 to 13
/i
iV-ii.
of the
long.
Sch rjismxharomyces uctuspuras, Beijerinck.

at 2,5 C.
Some
and in fresh wort cultures
young growth
in
wort
(After Schionning.)
The remarkable ascus formation in this species (Fig. 86)

observed by Schionning is described on page 211.
The
number of spores is eight as a rule, but somewhat frequently only four are found
from two
does
20'5
to seven.
its size
fi.
The
the
The shape
the breadth
is
number may
also
of the ascus varies
6 to 10'5
/x,
size of the spores is 3 to 5
vary
so also
and length 14 to
fx.
The
latter are
stained blue with a solution of iodine in potassium iodide

(Lindner).
strata,
more
They
are formed copiously on solid nutrient sub-
scantily on
gypsum
blocks and in wort cultures.
PERISPORACE^
271
According to Seiter they are formed on gypsum blocks at

25 in six to seven hours.
This species develops no film on
wort, but at room temperature a

in a
weak
yeast ring
is
formed
month.
It
ferments maltose and dextrose, but not saccharose.
Schionning states that

<about 14 per
fungus forms in beer wort
this
cent. Ball.) 4-6
three weeks at 25
C, and
vol.
6*56 vol. per cent, of alcohol in
five months.
xj
Fig.
105. Schizosaccliarorivyces
gelatine
some
C^^
octosporusj Beijerinck.
cells contain spores.
A young
growth on wort
After Schionning.
Schizosaccharomyces mellacei, Jorgensen, was isolated

by Greg from Jamaica rum. The spores of this species are
stained blue by iodine in potassium iodide (Holm). Altogether Greg
is
said to have found eight species of Schizo-
saccharomyces in the mashes in
Order
The
106
6).
PerisporacecB.
asei are enclosed in the so-called perithecium
latter is
closed,
II.
rum manufacture.
an envelope more or
less spherical,
and consisting of one or several layers of
the
completely
cells (Fig.
272
The conditions governing the formation of these periby Hansen and Klebs. Hansen
made most of his experiments with Anixiopsis stercoraria^
Hansen, a fungus which grows in the open air on manure,
and which also thrives in wort and on wort gelatine. He
found that in cultures on wort gelatine and wort agar
gelatine the limits for mycelium formation lie in the
neighbourhood of 36 C, and 2 C. At 25 C. a vigorousthecia have been studied
formation of perithecia takes place, but

fore 35 C.
It
is
reached, and
is
it is
minimum
its
thus seen that the temperature
maximum
the mycelium and gemmae
velopment of
over long be-
limit is near 8 C.
for the de-
remarkably
lies
higher than for the development of perithecia, and also
lower
of
mycelia
development of
the
that
minimum
to
behaviour
is
wish,
with
exactly
same
the
may
without
or
as
which, on the one hand

lower,
place.
than those at which
He
puts this
down
be
prepared,
This
perithecia.
(p.
namely,
210),
proceeds at temperatures-
still
higher, on
are
development
he observed in the
saccharomycetes as described already

that the budding of yeast
gemmae has
the
growths
Therefore,
perithecia.
according
and
than
temperature
spore
the other are
can take
formation
as a general rule for
most
fungi.
Klebs found that perithecium formation can always be

brought about with absolute certainty in Aspergillus repens,
de Bary, on
new bread between
25 C.J the result becomes

22 C. no fructification
vating this species on
is
and 32
25
cent,
tivation takes place at 16, but that

is
culti-
dextrin he further
found that only conidiophores are developed

28 C.
On
in general observed.
80 per
Below
C.
between 12 and
uncertain;
if
if
the
cul-
a temperature of
employed, the growth consists almost entirely of
perithecia.
At
15 C. formation of perithecia also takes
PERISPORACE^
but only
place,
if
273
the formation of conidia
is
in
some way
restricted.
Vegetative increase occurs in
by means
term
many
of the allied forms
Several species form sclerotia.
of conidia.
applied to certain hard tubercular bodies which
is
They
have a rind, and are often dark coloured.

thick
web of mycelium
food
stuffs.
varies,
This
threads,
and serve
After a period of
rest,
consist of a
for storing reserve
the length of which
they germinate into fruit carriers or fruit bodies.
The
Perisporacese are, with the following order, the
Sphseriacese, divisions of the Pyrenomycetes.
Several Aspergillus and Penicillium species cause
damage
in breweries
ticularly the
broken
by attacking the barley and

grains.
J.
much
malt, par-
Kauscher has carried out
comparative experiments with wort from mouldy and non-
mouldy malt and Lott later obtained the same results, viz.
(1) that mouldy malt gives less extract than normal malt (2)
the ratio between sugar and non- sugar diminishes (3) the
:
quantity of fermentable material decreases so that beer
prepared from mouldy malt contains

quantity of acid in the wort increases.
less alcohol
On
(4) the
the other hand
he found, in contradiction to previous observers, that the

malt attacked by the mould does not cause a mouldy smell
or taste in the wort nor in the beer prepared with
mentions that beer can of

is
itself
merely lying in a mouldy
assume a mouldy
it.
Prior
taste, if it
cellar.
In laboratories these fungi
may
be kept, either in the
10 per cent, saccharose
dry state
in filter paper, or in a
solution.
In both eases they live for a great number of
years.
In the case of Aspergillus glaucus, Hansen found
that this species kept alive in
paper preparation for
more than sixteen years, and Anixiopsis stercoraria lived,

under the same conditions, for more than twenty-two
years.
18
274
Brush Moulds (Aspergille^).
On
the conidiophores are flask-shaped bodies, sterigmata
or basidia, from which the conidia are formed

tion (Figs. 106
i, 2,
1.
The end
this
107 and 108
Genus
by
abstric-
A).
Aspergillus, Micheli.
of the conidiophore
is
inflated into a head.
On
head the numerous small flask-shaped sterigmata are
found from which the conidia are either directly detached,

or the former
all bear several smaller sterigmata from

which the conidia are developed. Those species, in which
the latter form of development takes place, are often
classified in a genus by themselves
Sterigmatocystis, van
Tieghem. Species are found, however, which exhibit both
:
forms.
Aecus fructification
species
(Figs.
106
5,
e) is
which were formerly reckoned
and we really ought therefore

the ascomycetes
till
known only
in the
in a
few
genus Eurotium,
to class only these species with
something further
is
known, while the
among the Fungi imperfecti. It

was first demonstrated by de Bary that Aspergillus is the
conidial stage of Eurotium.
In some species a formation of
others would be placed
sclerotia takes place
single forms
without any appearance of
gemma
formation
certain conditions of culture.
is
said
asci,
and in
appear under
to
Contrary to the following
genus, Penicillium, in which growth goes on at very low
temperatures,
e.g.,
near
0,
the aspergillae favour higher
temperatures.
Aspergillus glaucus, de Bary (Figs. 106

designation for several species
e-s), is
among which
a general
A. repens, de
Bary (Fig. 107 i-s), may be mentioned specially. The

growth forms on the substratum a covering at first bluishgreen and later brownish. The conidia are 6 to 15 /a in
ASPERGILLUS
diameter,
yellow
set
spherical
The
warty.
balls,
away.
somewhat
and
elHptical
slightly
which are in the form of
when
appear in large quantity
(On the
little
the cultures are
temperature on their formation,
effect of
According to de Bary they are formed in the
see p. 272.)
following
or
perithecia,
275
manner
From
the mycelium, side branches are
developed which, after having stopped growing, become

spiral
this
shaped at the point (Fig. 106
screw or spiral the ascogonium.
at the base of the latter

of the spiral
first
and grow up
De Bary
3,4).
calls
Side branches develop

;
one reaches the end
and the two grow together
(Fig.
106
4)
so
that the plasma masses unite and fructification takes place
by
The
this means.
side branches all ramify and become

The result of this is that finally a cell layer
divided by septa.
is
formed round the ascogonium
(Fig.
106
5)
the former be-
comes yellow and forms the wall of the perithecium.
Small
branches grow out on the inside and also ramify so much

that the whole space between the wall and the ascogonium
is filled
with a delicate texture (Fig. 106
s).
The
spirals of
the ascogonium are thus separated, these having meanwhile
been divided up by septa, and buds
The
parts of them.
ends (Fig. 106
6,
about 8 to 10
/i,
7),
in
latter
at various
asci at their
which are produced 8 colourless spores
in diameter, lens-shaped
a grooved rim (Fig. 106
Duclaux
now grow
branch and form
and provided with
s).
states that this
fungus forms diastase which
changes starch into dextrin and maltose.

It is extremely widespread in nature
on dead plants and
animal matter.
In maltings
damaged
grains.
it
J.
occurs,
as
already stated, mostly on
Behrens mentions that these and re-
lated forms can be found on hops, which then have a
colour
it
brown
probably attacks the constituents of hops, trans-
forming the
salts of organic acids into carbonates.
18*
276
P'lG,
106.
As2>eirjilh/,s rejtehs^
de Bary
and .-I. ijlavais, de Bary (6-8).

Another without the spore chains,
{!->),
Coiiidiophores with spore cliains.
2.
order to show the sterigraata.
Process of fnir.tification.
section
3, 4.
5.
of a young peritheciutii, the spiral ascogonium in the middle
outer shaded part represents a piece of the external yellow covering.
Longitudinal section of an almost ripe perithecium

spores, can be easily recognised.
different sides.
(After de Bary.)
7.
ripe ascus.
in
Longitudinal
the
6.
the asci, some containing

8.
Asoospores seen from
ASPERGILLUS
277
Aspergillus oryzae, Ahiburg. The growth
yellow or yellowish-green and later brown.
is
at
are either smooth or slightly warted, and are 6 to 7
diameter.
certain
Perithecia
above 30 C.
lies
The
sclerotia are
for the
growth
forms both maltase and diastase.
species plays au important part in East Asia,
on account
in
formed (Schiiinning
The optimum temperature

It
/i
have not yet been found; under
unknown conditions
and the author).
first
The conidia
where
it is
employed
powerful diastatic action in the manufacture of sake.

Sake, or rice beer, has been the national drink of the Japanese for thousands
of its
of years.
Its preparation is carried out in the four following stages (1) Preparation of " koji " (2) preparation of " moto " (3) the true fermentation
:
and
(4)
and clearing. The whole process lasts from November till

is begun by the above fungus transforming the rice starch
pressing
February.
It
into sugar by its diastatic action
the sugar
is
then fermented by one or
more
alcoholic yeasts either taken from the air or accidentally present in

the material. It is thus an impure fermentation. " Koji " consists of rice
grains which are grown over and penetrated by the mycelium of the
fungus.
It is prepared by sowing the conidia of the fungus, a yellow-green

powder, " tane-koji," on steamed rice, the former then being allowed to
germinate at 20 to 25 C. One vol. of conidia is said to turn about 40,000
The process
completed in the course of a few

rice with water and koji to
form a thick soup. After some days the whole mass begins to liquefy, the
diastase of the fungus turning the starch into a solution of sugar.
During
this process the temperature is only a little above 0.
Fermentation then
begins of itself, whereupon the temperature is raised to about 20" C, and
later to 30-35 C.
After fourteen days the "moto" is ready; it is a
of rice into koji.
vol.
days.
"Moto"
is
is
made by mixing steamed
and lactic acid and particularly yeast

which are used in the true sake fermentation. For the latter a large
quantity of steamed rice, koji, moto and water is mixed up, and the whole is
The
stirred up into a soup, placed in a fermenting vat and left to itself.
process finishes in two weeks. The fermented liquid is then pressed out.
liquid containing sugar, alcohol
cells
In the year 1888-89, 7-2 million hectolitres (4-4 million barrels) of sake
were manufactured in Japan from this the extensive use of the fungus
;
may
be seen.
Aspergillus fumigatus, Fresenius, has conidia which are

bluish-green and later brown.
According to Cohn
at
first
is
the cause of germinating barley, which has not been
properly turned, becoming

diseases in the air passages of
warm.
it
This species causes
mammals and
birds.
Aspergillus niger, van Tieghem, belongs to the sub-genus
278
The colour
Steriymatocystis, its ateriginata being branched.
of the
growth
in diameter,
is black.
The conidia measure
and are furnished with small warts.
3'6 to 4'5
It
/a
forms
spherical or cylindrical brownish-yellow or reddish-brown
which are
sclerotia,
0"5 to
The fungus contains
15 mm.
in diameter.
diastase, maltase,
invertase and
emulsin, and, according to van Tieghem, breaks up tan-
nin into gallic acid and glucose.
Wehmer
one of the most active producers of oxalic

of the sugar exposed to
it
into oxalic acid.
capable of doing this, however
two
similar species concerned.
states that
acid,
it is
turning half
It is not
always
perhaps in this there are
It belongs, like the preceding
one, to the pathogenic species.
2.
Genua
Penicillium, Link.
The conidiophore has septa and has short branches near

On the latter, as well as on the main axis, are
the top.
formed flask-shaped sterigmata, on which are chains of

conidia (Fig. 108
ciculated
this
A).
Sometimes the conidiophores are
fas-
form of development was formerly classed
in a separate genus,
fructification has
which was called Coremium.
Ascus
been observed only in a very few species
was found by Brefeld in P. glaucum and by Zukal in

P. luteum, and is a somewhat rare occurrence in these two
species.
But it is said by Ray to occur frequently in P.
it
The conditions of its formation are not known

with exactness, and it is thus always a matter of unsacchari.
certainty whether this fructification will be obtained.
Wortmann found living Penicillium conidia in wines

which were many years old and contained a high percentage
of alcohol
their resisting
power thus seems under these
circumstances to be very great.

Penicillium glaucum, Link (Figs. 107 and 108)
taceum),
is
(P. crus-
a collective species under which are grouped a
PENICILLIUM
large
number
of forms
which
differ
279
only slightly, and which
can scarcely be distinguished from one another by means of

the characteristics at present at our disposal.
The
colour of
the growth, initially white, then bluish-green, later grayishgreen,
and
The conidia are
Fig. 107.
spherical,
and 2 5
Penicillium glaucum, Link,
Mycelium,
The
common
finally often grayish-brown, is
c,
Conidiophores.
a,
to 4
/x
The conidium
iî.
to
all.
in diameter.
origisally seeded,
b.
(After Brefeld.
ascus fructification discovered by Brefeld proceeds,
according to him, in the following

sclerotium
is first
formed,
1 to 1'5
way
mm.
yellow or brown
in diameter
this is
produced by a screw-shaped ascogonium developing on a
mycelium thread and being surrounded by a growth con-
280
sisting of branched
mycelium threads, which grow up partly
from the base of the ascogonium and partly from the myceThis growth becomes gradually denser and harder
lium.
the ascogonium enlarges
placed on
rate
its
branches force themselves
between the middle growth, consisting of
in all directions
thinner-walled
and
When
cells.
damp
filter
such a sclerotium, fully ripe,
is
paper, the ascogenous threads sepa-
and push out thick side branches, the links of which

change into
finally
At the same time
asci.
thin threads
develop fi'om the ascogenous hyphae which penetrate into

the sterile growth and dissolve
FeniciUivm tfhtucum, Link.
Fig. 108.
Beginning of sclerotium
youug sclerotium
it
A, Conidiophore.
ascogenons hyplue
(a,
The
up.
foodstuffs thus
B, Sexual organs.
sterile threads).
b,
C,
D, Very
in section.
obtained are taken by the fine threads to the ascogenous

Finally, the dissolving process progresses so far
hj'phse.
that only the peripheral rind remains, while
appears
filled
up with spore masses.
tion takes place, according to Brefeld, only
nourishment on
ascus, are
7
fj.
bread.
yellowish,
The
elliptical,
the inside
Perithecium forma-
ascospores,
5 to 9
/x
by abundant
eight
in
each
long and 4 to
broad.
The cardinal temperatures
for the
said to
grow
at 35 to 36 C.
growth vary, accord-
Thus the fungus is

if glycerine and sodium formate
ing to Thiele, with the substratum.
PENICILLIUM
281
are present, whereas 4 per cent, of grape sugar impedes
growth
at a temperature above 31 C.
minimum
is,
The temperature
on the contrary, unchanged by the substances
named.
Pasteur states that the conidia are killed
if
exposed for
half an hour to a temperature of 127 to 132 C.

to 121 C. they retain
at 119
According to Lesage they are
life.
somewhat quickly by alcohol vapour, the times being

days, nearljr one day, and two hours when subjected
killed
six
vapour from 22-5 per
to the
cent.,
45 per
cent.,
and 90
per cent, solutions of alcohol respectively.
glaucum contains diastase and maltase (Bourquelot),
P.
also
cane sugar, and emulsin
ferment which inverts
The
(Gerard).
diastatic action
is,
however, weakened
content of the substratum in sugar increases
if
the
in a 10 to 15
per cent, cane sugar solution this fungus forms no diastase

(Katz).
glucose
It,
further, breaks
up tannin
its
two
this
is
by
said
certain conditions
it is
cells
however, to be
Pfeffer,
dependent for the most part on external
optically
up the
active isomers, using the levo-acid in building
(Lewkowitsch)
and
into gallic acid
Tieghem) and mandelic acid into
(v.
Under
influences.
said to be capable of forming mannite
Rotten grapes
as the product of decomposition (Muntz).
with PenicilUum growth
wine containing mannite.
may
therefore
produce a sick
Calcium oxalate
deposited
is
in the perithecia.
Mention
action on
injurious
p.
of its occurrence
wort and
beer
is
to be
its
found on
273.
When
it
is
Miiller-Thurgau
present in grape must, fermentation

is of
the opinion that the cause of this
injurious substances, for fermentation

is
on barley and malt, and
is
also restrained
much
delayed.
the formation of
when
the fungus
Miyoshi
forms a specific protoplasm poison, and J. Behrens has
experimentally its poisonous action on yeast as regards both
removed from the liquor before the addition
found that
shown
is
is
it
of the
yeast.
282
fermentation and fermentative power.

By a stronger nutrition of the
yeast, e.g., by addition of peptone to the nutrient solution, he succeeded
The fungus causes the mouldy taste in
in stopping this injurious action.
wine, as well as a cork or stopper taste, by growing through the cork of
the bottle (Wortmann). Like Aspergillus^ it attacks hops, colouring them
brown.
P. glaiKum
is
very widespread in nature, and the air
full of its conidia.
It
is,
ous guests in the laboratory,
the more a
all
it
only requires
a very small quantity of nutriment for the support of
but
it
amount
also requires a certain
regard to temperature, too,

well at about
it
is
as well as stone fruit,
and
it is
met with on
herring pickle, and isolated
it
on which substratum
It betrays its presence
by
Order III.
grew
it
thrives
it
fruit,
Wehmer found
by means
kernel
it
in
of plate cultures
on nutrient gelatine, containing 10 per

salt,
said to be able to penetrate
uninjured surface of grapes.
the
life
With
of moisture.
not particular
It is frequently
0.
is
most danger-
therefore, one of the
cent, of
common
easily.
its characteristic smell.
Sphariacece.
The fungi belonging to this order have, with one exception and contrary to the Perisporacece an opening in
,
the dark-coloured perithecium.

place.
This
order
includes
Conidia fructification takes

a
number
very large
of
parasitical species.
Spbjeriem.
Genus
The almost
Sphmrella, Ch. and de Not.
spherical perithecia are set in the epidermis
or in the uppermost layers of the
of the host
a simple, uncommon, papilla-shaped opening
end
in
fine
skin-like
bundles
web
coloured).
consistency.
The
asci
are
and
of
connected in
the spores are two-celled and colourless (seldom
SPH^RELLA (CLADOSPORIUM)
283
Sphasrella Tulasnei, Janczewski (Fig. 109), forms dark7),
03
At
its
coloured perithecia, frequently pear-shaped (Fig. 109
mm. long and 01 5
to 0-4
to
020 mm.
in diameter.
numerous mycelium threads are

often found which develop conidiophores (Fig. 109 7).
The
surface, chiefly at the neck,
perithecia contain asci with eight spores (Fig. 109
which the topmost

in length
The
and
is
6 "5 /a
larger than the others
name has been
glaiwus
and
is
28
/x
one of those fungi which were

(Fig.
109
1, 2, 3).
used, like Fenicillium glaucum, Aspergillus
others, to designate several fungi.
impossible to say which species Link meant.

the mycelium, at
all is
of
9),
in diameter.
conidial form
formerly called Cladosporium herbarum, Link

This
s,
its size is
first clear like
It
is
now
Common
to
water, and later olive
green or brown, which sends out conidiophores that abstrict
brown conidia often many-celled.
The form named
Cladosporium herbarum by Janczewski corresponds with that

usually so called (Fig. 109
1-4).
He
describes the conidia
as oval, either undivided or 2 to 5 celled (Fig. 109
the largest varieties they are 25
/x
long and 10
the smaller forms they are only half as large.
wall
is
olive
fi
In
s).
broad
Their
in
cell
brown, often warty, but smooth in the smaller
The young mycelium is uncoloured, and only

assumes the olive brown colour gradually. For the rest, the
variety.
appearance
may be made
out from Fig. 109.
Janczewski showed, by experiments with seedings on

barley and rye, that this fungus
is
the conidial form of the
above Sphcerella Tulasnei. It grows here as a saprophyte,

As soon as the conidia were brought to
not as a parasite.
germination on nutrient gelatine, a piece of the gelatine

with germinating conidia was placed on a cut rye leaf which
was kept in a very damp atmosphere. The mycelium then

grew up; as soon as the point of the latter reached one of the
stomata in the leaf,
it
forced
its
way through this into the leaf
couidial
sjiurii'iii Jierhanriii,
tlie
rye leaf sheath,
Link.
^f^'.
^]^.
5.
thread in a rye leaf sheatli.

H.
4.
The
Conidiii.
i-\^.
7.
form
.same growini^' out tliroiigh a
"1^.
6.
Clailo-
stoma
in
Bclerotiiim with a myt:elium
Perithecia witli couidiophores.
''i".
Longitudinal section of perithecium containing two asci with endospores.
^^-^.
9,
Ascus with ripe endospores,
from endospores.
^]^*.
^'^'>.
(After Janczewyki.)
10.
Conidiophores developed
UISCOMYCETES
where
in
When
continued to grow.
it
winter (and only
285
the seeding was done
when done during
the latter season),
the mycelium often developed sclerotium-like bodies (Fig.
109
li),
which
were
then
transformed
Janczewski never found these bodies
into
perithecia.
nature on the
in
any kind of corn. Placed on nutrient gelatine

were covered by mycelium threads extending radially
leaves of
the}'
and often
large
in
mycelium formed
After a few
quantity.
days this
The transformation of sclerotia

into perithecia takes place quickly.
The ascospores germinate easily on nutrient gelatine, and, in a few days, develop
conidia.
mycelia with the conidial form Gladosporium herbarum

the same time certain peculiar organs (Fig. 109
the function of which
is
lo)
at
appear,
not known, and which are often
found in the immediate neighbourhood of the conidiophores.

Gladosporium herbarwm
is
very
common on both dead and
In breweries this fungus appears sometimes
living plants.
in large quantity
on the walls of
malt, hops, etc.
Like some other mould fungi
cellars
cork taste to wine (Wortmann).
how
far
is
it
(Fres.) Sacc,
and the
identical with
it is
also
it
found on
can give a
It is not defined yet in
Hormodendron
cladosporioides
yet both fungi are extremely like one another
latter is
probably often called Gladosporium.
czewski says that he never succeeded

Gladosporium into Hormodetidron
in
Jan-
transforming
but the mycelium devel-
oped on the above sclerotia sometimes exhibited a develop-
ment
of conidiophores similar to those of Hormodendron.
Another similar fungus
by Zopf, which
who
is
is
Fumago, very thoroughly studied
specially to be
to those
intend taking up this question.

Order IV.
The ascophores
ance
recommended
they
may
Discomycetes.
are open
and have a very varied appear-
be either cup,
disc,
mussel or hat shaped.
286
The
asci generally contain 8 spores,
16, 32, 64,
but some species have
128 and sometimes more.
In manj% conidia
and
in some, sclerotium
fructification
is
found
in addition,
formation.
Cup Fungi
Genus
1.
(Pezizace^).
Sclerotinia, Fuchel.
The mycelium produces
sclerotia
from which
ascophores grow under certain conditions.
stipitate
Conidia fructi-
fication takes place.
Sclerotinia Fuckeliana, de Bary (Figs. 110
This species
is
best
under the name
as the conidia
and
111).
fructification
Botrytis cinerea, Persoon {B. r.ulgaris, Fr.).
This fructification
The
known
is
conidiophores,
top like panicles
usually developed
1
to 2
mm.
first
on the mycelium.
long, are branched at the
the ends of the branches have bulbous
swellings which bear numerous fine sterigmata which again
When
abstrict large conidia.
branches bearing them
die, so
the latter
ai'e ripe,
the side
new branches can then
that
take their place.

If the conidia are seeded out in
tum,
e.g.,
an unfavourable substra-
in a thin layer of water, they germinate
a very short
germ tube from the

;
latter or
and form
from small
flask-
shaped carriers small conidia (spermatia) are again abstricted,
which, however, cannot be made to germinate.
the other hand,
substratum, a typical mycelium
is
developed which then
forms either the usual large conidiophores

conditions, black
sclerotia, a
such a sclerotium
is
mediately after
but,
if it is
it
On
the large conidia are put into a good
if
or,
under certain
few millimetres
thick.
If
brought into a damp atmosphere im-
gets ripe, conidiophores again develop;
kept at rest for a year at
least,
the ascophore de-
velops in the shape of long-stalked cup fruits (Fig. 110^9).
SCLEROTINIA (BOTRYTIS)
The
287
spores of these asci may, like the large conidia, give
either mycelia with large conidiophores or small conidio-
phores of which the conidia do not germinate.
Fig. 110.
Sclerotinia Fuckeliana, de Bary.
conidiophores have grown

of the Botrytis form
b,
m, Mycelium.
branches and sterigmata.
^{-2.
l:,
phenomenon
cinerea, P.
Solerotium from which the Botrytls
^f\
C",
End
much
enlarged)
C, Conidiophore
of a conidiophore with
Germinating conidium.
of sclerotium with ascophore, p, (not

spores,
^f 2.. (After de Bary.
In Botrytis
a,
Sclerotium with two cup fruits
/;,
An
^^.
s.
Section
ascus with eight
Lindner frequently observed the
of inter-growth.
According to his investiga-
288
an irregular distribution of the plasma takes place in
tions,
the older mycelia,

ties of
some
The phenomenon
solely
of the cells storing
up
large quanti-
the contents, while others are completely emptied.
by the
of inter-growth
is
fact that the only cells
connected with this
which germinate inside
the old mycelium are almost always rich in protoplasm.

Fig. Ill represents a special case of this
in
kind of germination,
which small conidia or spermatia are formed inside the
At the same time spermatia have

mycelium thread.
cell.
also
grown out
laterally on the
Oxalic acid
and
which
Fm.
111.
Kissling showed that
kills living
de Bary.
it
by the mycelia
forms a poison
According to
The
Jiulrytis form.
J.
Behrens
Phenomenon
Abstriction of small eonidia (apermatia) inside a
of
(After
cell.
Lindner.)
this poison
sugar,
is
not an enzyme.
Botrytis turns starch into
and contains emulsin (Gerard).
This species
all
protoplasm.
Srlei-dtiniii Fuckelifiiui,
inter-growth.
P.
secreted in large quantity
is
sclerotia.
is
very widespread in nature, and occurs on
putrefying plant matter.
It occurs as a
parasite on
vines, both on the leaves and grapes.

Botrytis cinerea
manufacture.
stances
it
may sometimes
According
induces
the
to
play an important part in
Miiller-Thurgau,
so-called
under
" Edelfaule "
in
the
certain
grapes,
wine
circum-
which
forms the basis for attaining the highest concentration of the grape
juice, and especially for the appearance of peculiar bouquet substances, the so-called sherry bouquet, in wines
this occurs when it
attacks quite ripe grapes, consumes the acid and, by rotting the skin,
;
TORULA
289
allows the water to evaporate, and thus increases in a very high degree
the concentration and sugar content of the berry.
Wines thus made

ferment very slowly. The cause of this is
the separation of the protoplasm poison mentioned above, which reacts
injuriously on the yeast, as was shown by J. Behrens. This injurious
action can be prevented to some extent by a more vigorous nourishment
from over-ripe
of
(edelfaul) grapes
the yeast.
Grape must, in which Botrytis ciiierea has been cultivated, contains an

oxydase which, according to some investigators, causes the disease of wine
which
is
known as
" maladie de la casse," and which consists in a precipita-
Culture solutions of Botrytis when mixed

with equal quantities of sound wine cause the colouring matter to precipitate completely in about four hours.
The disease may be prevented
tion of the colouring material.
by heating up to 70 C, when the oxydase becomes inactive (Laborde).

The opinion obtained formerly that this fungus was responsible
for the smoky flavour of wine, but this is not the case (Mach, MiillerThurgau).
Imperfect fungi (Fungi imperfecti).
B.
A large
number of the fungi which have been discovered

one by one by mycologists cannot yet be classified in the
system set up by them these fungi are therefore grouped
;
for the present

to
under the above name.
the following organisms,
which
This applies also
are of interest for the
fermentation industry.
The Torula
Originally the
name
Species.
Torula was given to hyphomycetes
which had necklace-like, single or branched chains, of which

the round or oval members were separable from one another.
Later, however, the name included a number of diflferent
fungus
cerevisicB
species.
Torula
Thus Turpin
cerevisiae,
in
1838
while the
calls
name
Saccharomyces
Torula
was
after-
wards given by Cohn to the necklace-like chains formed by

the bacterium genus Micrococcus. Torula was understood by
Pasteui- to include yeast fungi with a very
formation
or not
weak
alcohol
he did not mention whether they formed spores
The
species of this
kind might, therefore, be true

19
290
saccharomycetes; this
Torula.
By
Torula
however, not the case with Hansen's
is,
Hansen understands yeast
similar to Saccharomyces, but do not
As regards
develop typical mould growths.
may
of alcohol they
to the
cells
which are
form endospores nor

the production
According
exhibit this in all degrees.
view held by the same investigator they will some
time in the future probably be ranked with forms in the
system widely separated from one another.
we
As, however,
are ignorant on this point as yet, these organisms are
for the time placed together in a
After Hansen had
made
group by themselves.
clear the conditions for the
formation of asporogenous varieties in the saccharomycetes,
and
since such asporogenous varieties can be
nature,
it is
formed
in
comprehensible that several forms which appear
under the name Torula possibly originate in saccharomycetes.

There are Torula species which have
characteristics
common
all
the physiological
to the saccharomycetes
this is true
also as regards the morphological features, but of course
with the exception of the property of greatest importance

to the saccharomycetes,
viz.,
that of endospore formation.
Torula species are very widely distributed in
Hansen found them always present
in the
nature.
ground and in
large quantity after winter in the hairy coats of bees and
wasps as well as
constantly
Some
in their nests.
when investigating a
The author also found them

large numberof these insects.
of these fungi cause a disagreeable taste
and smell in
wort and, according to Wortmann, also in wines. The latter
author found forms, in old bottled wines, which

.slimy.
species
make must
Rich. Meissner has in recent years isolated several
which cause wine to become viscous
(see below).
Hansen has thoroughly investigated, among others, seven

different species, which have, however, received no systematic
name.
These are as follows
Torula No.
(Fig.
112).The
cells
are 1-5 to 4'5
fi
in
TORULA
size.
291
After long standing in wort, this fungus forms a
scarcely appreciable
of frothing;
amount
Torula No. 2 (Fig. 113). The

diameter.
any
of alcohol without
trace
does not secrete any invertase.
it
The
protoplasm
cells
becomes
are 3 to 8
/^
in
granular in wort.
Otherwise this Torula behaves essentially like No. 1.

Torula No. 3 is similar to No. 2.
In wort it yields
\ vol. per cent, of alcohol
of froth,
with a small but distinct production
and does not form invertase.
Torula No.
114). The
(Fig.
are 2 to 6
cells
/a
in
4)8
diameter.
more than
'Pig.
\V1. Torula No.
\.
Fig.
113. Torula No.
2.
'-%><>.
i^.
(After Hansen.
(After Hansen.
It inverts saccharose and, in wort,

1 vol.
Torula No.
5,
forms a Kttle
per cent, of alcohol with vigorous frothing.
This species soon forms a grey film over
the whole surface of wort, yeast-water and lager beer

film is only slight
on a saccharose
but in wort it
is inverted by it
worthy fermentation and correspondingly only a
sugar
the
The latter
produces no note-
solution.
trace of
alcohol.
Torula No. 6 (Fig. 115) exhibits a distinct fermentation

in
wort and generates in
it 1"3 vol.
fermentation takes place in a maltose solution.
19*
No
It inverts
292
saccharose and in fifteen days at 25 C. generates 8'8 vol.
per cent, of alcohol in a 15 per cent, dextrose solution.
Torula No. 7 (Figs. 116 and 117) was found in the
under
vines.
wort
on the contrary
of saccharose,
It
1 vol.
per cent, of alcohol in beer
excites
no fermentation in solutions
produces
which
it
soil
it is
In yeast water
unable to invert.
ôo
\\i. Torula Xo.
FiQ.
4.
Fig.
J-",fi.
115. Tirrula No.
6.
i-V^-
(After Hansen.)
(After Hansen.
containing 15 per cent, of dextrose
it
formed 5 3
vol.
per
cent, of alcohol.
The
last
named
species
are
perhaps active in wine
manufacture, but hardly so in breweries and

Rich. Meissner isolated eleven Torula species which
disease called the ropiness (" Ziihewerden ") of wines
distilleries.
all
cause that
he has shown by
experiments that must as well as wine becomes slimy, oily and thick
Fig.
116. rorafa
iVo. 7.
Sedimentary yeast.
i-V~-
(After Hansen.)
when seeded with these. Most of these forms do not produce films, but
must is decolourised by all of them. In the few
only a yeast ring
species which form a film the latter was in some cases white, and in a
Only two of the eleven species referred to
single instance olive green.
;
bring about alcoholic fermentation.

vrithout
which they cannot grow.
Common to all
If
is
the need of oxygen,
the nutrient liquid contains more
than 5 vol. per cent, of alcohol, growth as a rule ceases, but at the same
time the organisms are not killed. These slime yeasts check the fer-
TORULA
293
mentation, not of the strong yeasts, but only of feebly fermenting yeasts
in the first few days of fermentation.
The ropiness of wine occurs
chiefly in those wines which are poor in tannin the disease can therefore
;
Torula No.
Fig. 117.
Film growth on a wort culture ten months
7.
old.
ô^.
(After Hansen.)
â
"O
^r"
^CXD
^ "(W ^
(a) A cell which has begun to develop

and a" the same cell after the lapse of IJ and 3J hours 6
another budding cell, b' after two hours, h" after three hours & is J-hour
older than c d was observed at 2J p.m., d' at 3J, d" at 3| e 10| o'clock,
i, /'" 5, /'" 5^ o'clock.
About 4|ji.
e"' 1
<f 12, e"
f 2^, f H,
Fig. 118.
a bud
Saccharmnynes apiculalxis, Keess.
"/
12J,
(After Hansen.)
be prevented by the addition of tannin, which latter checks the growth of

The addition of a pure wine yeast is an especially
the slime yeasts.
favourable means for suppressing the slime yeasts so completely that the
disease does not appear.
294
budding fungi are
Various red-coloured
with the Torula species
containing media, and the so-called pink yeast

of medical bacteriology
also
classed
these occur especially on starch-
is
Rosahef'e ")
("
classed along with them.
Saccharomyces Apiculatus, Reess.
budding fungus which does not form endospores and
which generally occurs in vineyards and orchards was

given this name by Reess.
It
has been thoroughly studied
by Hansen and we owe the following
'9
'yy
''C]
'#^5
'
to his investigations.
o'
^.
^'-
'0
*e
'D
\
rA
O,
c>-^
119. >Saccharoint/ces apiculatus,
Fig.
The
budding.
series
contain a refractive ball

IJ,
h" 2J
j lOi,/ IJ
8i, I"" 9J, l'""
10
III
Reess.
Most of the cells are in the act of
show abnormal cells.
The two cells i each
g was observed at 3j P.M., g' 5J, g" 6^ h lOJ, k'
and
ni
L lOh,
64,
III'
k' 12A,
6^1,
k" IJ,
/('"
2^; 17,
in" 7, i"' 1\, in"" 7J.
I'
8, /"
s'h. 5 m., I'"
About ^^.
(After
Hansen.
The
8
fx
cells (Figs.
118 and 119), which are generally 6 to
long and 2 to 3
ya
wide, are in some cases pointed at
both ends like lemons, in others oval.
both kinds of buds.
The fungus forms
In order to change into lemon-shaped
must grow through one or more buddings.

The lemon form is produced more especially at the beginning
of the budding and has then the preponderance
later the
cells the
oval buds
oval
cells
predominate.
SACCHAROMYCES APICULATUS
It is a
and
295
bottom yeast form which does not secrete invertase
in consequence cannot ferment cane sugar
incapable of fermenting maltose.
form more than

other hand
it
it
per cent, of alcohol in wort
1 vol.
forms
is
also
It therefore does not

;
on the
4-3 vol. per cent, of alcohol in yeast
water containing 10 per

Sacch. apiculatus, like
cent, of dextrose.
many
other fungi, undergoes a
remarkable variation.
Thus Hansen found that of two

growths investigated by him, one gave 3 and the other 43
Amthor investigated two varieties,

which one furnished 3-25 and the other 4-56 vol. per cent,
of alcohol, and Miiller-Thurgau found that in seven cultivavol. per cent, of alcohol.
of
tions in sterilised grape juice the alcohol production varied
between
2-5
and 3"8 per
cent,
by weight.
Will had two
growths, one of which evolved a mouldy smell, the other
an amyl
But whether such variations

or on what they depend, has not
ester-like bouquet.
are permanent or not,
been investigated.
Its
extraordinary power of multiplication is characteristic
of Sacch. apiculatus.
This and
its
competitive relations with
Sacch. cerevisicB have been mentioned on p. 230.
Hansen's investigations on the circulation of Sacch. apiculatus in
nature are described on
p.
246.
This fungus
generally distributed in nature on fruits and also in
Miiller-Thurgau found
cm.,
it
is
soil.
in the latter to a depth of 20 to 30
and Berlese to a depth of 36 cm.

it is found commonly
According to Will
in
bottom
mentation breweries, but only in small amount.
fer-
It is
generally present in those Belgian breweries where beer
is
prepared by spontaneous fermentation.

According to the investigations of Miiller-Thurgau and Wortmann it
manufacture of wine, as it has a retarding
influence on the fermentation, but not, however, if the liquid contains 3
It is most efiective during the first stages of fervol. per cent, of alcohol.
is
especially detrimental in the
mentation.
Sacch. apiculatus possesses in a higher degree than true wine
296
and absorbing organic acids. MullerThurgau's experiments show that this property also asserts itself when it
works simultaneously with true wine yeasts, as is the case in the progress of
ordinary wine fermentations.
Finally, by the formation of volatile acids
and other products it is injurious to the bouquet and flavour of the wine.
According to investigations by W. Seifert it formed the largest amount of
yeasts the power of decomposing
volatile acid (0-064 per cent.) and volatile ester of six pure yeasts in the
same grape must. The amount of ester expressed in cubic centimetres
of y'j normal alkali on 100 c.c. of wine corresponded to 10-8, while with the
other yeast species it varied between 1-32 and 4-4. When it ferments grape
must a cider-like taste and smell are exhibited. Although the increase of
Sacch. apiculaius occurring on fruit and grapes is not prevented by the
addition of pure yeasts to the must, yet, as experimental results show,
its
detrimental influence can be very considerably restrained by the addition

of
a quick-growing, vigorously-fermenting yeast (Miiller-Thurgau).
Accord-
ing to some French investigators Sacch. apiculatus yields a good cider with
a strong bouquet but, according to Miiller-Thurgau,

a harmful influence on the cider fermentation.
;
it
has, on the contrary,
IVlycoderma Species.
These fungi are the so-called true film fungi which

are
by the rapid formation on nutrient

on beer and wine, of a covering film
between the cells. The cells are usually short
distinguished
liquids, particularly
having air
and sausage-shaped.
Mycoderma
They
cerevisiae,
are strongly aerobic.
Desm.
{Sacch.
rnycoderma).
Several species are included under this name.
The
species
usually to be found in the Copenhagen breweries forms a

dull, gray,
wrinkled film on wort and beer.
contain from
nature.
no
1 to
The
cells
3 refractive granules, which are of a fatty
This fungus does not induce fermentation, contains
invertase,
and occurs
in practically all lager beer,
but
does not succeed in growing so long as the bottles are well

stoppered.
It is
known with
certainty that at least
some
of these
forms do no harm in breweries under ordinary conditions

this holds
good with regard to the species observed by
Hansen, A. Petersen, Gronlund, Jorgensen and Prior.
houbek and Kukla, on the
conti-ary,
Belo-
mention a species which
MYCODERMA
The same
causes turbidity in beer.
297
applies also to three
forms observed by Lasche in America, which are said to

cause a bad smell and taste in beer.
which forms
are said, more-
Lafar has described an
over, to produce alcohol in Avort.

allied species
They
acetic acid in beer.
These forms are easily obtained if beer or wine
is
allowed
to remain at a temperature of 10 C. with a free supply

of air.
Mycoderma
or
to,
Desm,
vini,
very nearly related
probably identical with, the above species
is
The fungus
it
This film can become over 1 cm.
forms the film of wines.

thick.
(Fig. 120), is
acts, like
the other species, as an oxidis-
ing agent on the alcohol in the wine, forming carbonic acid
Fig. 120.
Mj/coderma
and water.
vini,
About
(After
-if*-.
Wortmann.)
It can also attack other constituents of the
By decomposing
wine.
Desm.
a part of the free acid
it
favours
the growth of acetic acid bacteria, and consequently the
production of a
this
vinegar
taint.
Wortmann
states
that
fungus can also influence directly the flavour of a
wine.
Forti mentions a
Mycoderma
species
which has a detrimental
influ-
ence on yeast in wine.

W. Seifert has made complete experiments with two related forms
isolated from red wine, which he names Mycoderma vini I. and II.
Mycoderma vini I. are 3 to 10 /i long, and 2 to 4 /x broad.

smooth at first, later strongly wrinkled, coherent and
grayish-white. The temperature limits for growth in wine with 8 vol.
per cent, of added alcohol are Maximum, 30 C. optimum, 25 to 28 C.
The
The
cells of
films are
298
and minimum,
vol.
artificial
vol.
This species grows even in the presence of 12'2

and vigorously attacks malic acid. In an
5 to 6" C.
per cent, of alcohol,
culture liquid (Pasteur's solution) containing malic acid and 4'8
per cent, of alcohol,
weeks
at the
it
formed 0-152 per
same time the whole
ordinary Austrian white wine
it
of
cent, of glycerine in fourteen
the alcohol had disappeared.
increased the
amount of
cent, to 0-82 per cent.), formed acetic acid (0-904 per cent.),
amount
In
glycerine (0'68 per
and reduced the
of alcohol (7-8 to 3-8 vol. per cent.) in twenty-six days.
Mycoderma
II. differs from the above species in having tempergrowth in wine with 8 per cent, of alcohol as follows
Maximum, 28 to 30 C. optimum, 22 G. and minimum, 1 to 2 C. This
fungus attacks malic acid only to a small extent. In the culture solution
referred to above, it only formed 0-016 per cent, of glycerine after fourteen
weeks, and at the same time the amount of alcohol was only lowered from
ature limits for
vini
its
4'8 to 4-1 vol. per cent.
No
increase of glycerine was effected in white wine after twenty-six
<^'
Fig. 121.
Mmtilia
ca?(rfirfff
(Bonorden), Hauseu.
with refractive granules occur in some of the
SedimcDtary
cells.
-'-Oj-QO..
Vacuoles
yeast.
(After HanseD.)
days, only 0-064 per cent, of acetic acid was formed, and the alcohol
only reduced from 7-8 to 6-8

Tartaric acid
acid not at
all.
is
vol.
was
per cent.
and citric
formed are gradually used
practically not attacked by either species,
The glycerine and
acetic acid
up again.
Monilia Candida (Boiiorden), Hansen.
This fungus (Figs. 121, 122 and 123)

in nature
on fresh cow dung and on
investigations described are due to
generally found
is
The following
fruit.
Hansen
In nutrient liquids containing sugar this fungus quickly

develops a growth
vacuoles
with
of
Saccharomyces-like
cells,
in
which
one or two strongly refractive granules
frequently occur (Fig. 121).
When such a
culture
is
allowed
MONILIA CANDIDA
to remain
some time the
cells
299
become elongated, and there
results finally a complete mycelium, a mealy, white, tufted
growth
mould which forms chains
of
of yeast-cell conidia
or divides into members like Oidium (Fig. 123,
growth
When young and
vigorous
is
on
and vigorous fermentation
effected
e.g.,
When
beer wort,
like a top fermentation
even while the bubbles are rising a
their surface.
are
species
of this
cells
seeded in a fermentable nutrient solution,

a rapid
This
d).
on solid culture media.
also appears
film
forms
the frothing has finished, the film
gradually extends over the whole surface
during this pro-
^te
Qbl
\_j^
Fig. 122.
Cells derived from a
Moiiilia Candida (Bonorden), Hansen.
film growth.
here.)
j(
J-\--.
(The shining granules appearing

(After Hansen.)
in Fig. 121 are not
young
shown
cess the large film-covered air bubbles gradually burst,
often cause folding of the

fibn
forms before there
of fermentation.
formed
any perceptible macroscopic sign
is
In wort after sixteen days
1"1 vol. per cent, of alcohol; after nine
months, 6'5 vol. per cent.

6"7 vol. per cent.
reached and the

cent,
of alcohol in 15
were dead.
and a half
It
maximum had been

formed 55 vol. per
per cent, dextrose-yeast- water in
fourteen days at 25 C.
solution in
this species
and after twenty-six months,
After this time the

cells
and
If old cells are seeded the
film.
in
twenty days, 0'7
six months, 3 vol. per cent.
a
vol.
and
10 per cent, saccharose

per cent, of alcohol
in
in twenty-seven months.
Monilia
Fig. 123.
culture,
re.
aiiii/i'ln
Chain.? of
(Bonorden), Hansen.
more or
less
of oval-shaped yeast cells often occurs,
yeast
cells,
shaped
cells.
c,
b,
Lemon-shaped
cells.
at each
-L-Y-"--
rf,
O-idiwmAikQ
cells,
(After Hansen.)
old
node a whorl
The same form, but without
Typical mycelium with septa,

/,
Mould growth from an
thread-shaped cells
e,
oval
Pear-
CHALAEA MYCODERMA
4-9 vol. per cent.
The
cells
were then
30]
still alive.
fermentation proceeded very slowly in these
fungus withstands high

vigorously at 40 C.
temperatures,
e.g.,
cases.
The
The
ferments
it
In wort, the temperature limits are
42 to 43 C. and 6 to 4 C.
remarkable circumstance that in the fermentation
It is a
of the saccharose solution neither invertase nor invert sugar

could be detected.
Consequently the vigorous fermentation
generated in the saccharose solution by Monilia Candida must

be quite unique, because saccharose was only
fermented after previous inversion.
by
detectable
The
known
to be
invertase not being
existing chemical methods,
it
follows that
the fermentation must be regarded as a direct one.
Never-
Hansen indicated the possibility that the inversion

takes place inside the cells and that the invert sugar protheless,
duced
and
is
P.
fermented as soon as
Lindner by
it
is
formed.
grinding the
cells
discovered an invertase insoluble in water,
which
is
closely connected
E. Fischer
have recently
i.e.,
a ferment
with the plasma of the
celL
At
the same time they found that this species contains maltase.
According to Bau the fungus can ferment diastase dextrin.

Monilia Javanica, Went and Prinsen Geerligs, occurs in " Raggi,"
which is applied in the manufacture of arrack in Java (see p. 260). On
solutions containing sugar this species forms a film, which, however (and
this distinguishes it from the previous species), disappears as soon as
fermjintation begins. It further differs from M. Candida in that it inverts
saccharose in the usual way, and the latter is then fermented it also
ferments dextrose, levulose, maltose and raffinose. When 5 per cent, of
alcohol has been formed, growth and fermentation cease. The alcohol it
produces has an unpleasant smell and taste.
A Monilia species has been described by Forti which has a detrimental influence on the yeast in wine.
;
Chalara mycoderma, Cienkowski.
Like Monilia, this fungus (Figs. 124 and 125) forms a film
on
liquids
abstricts
it is
here
composed of a branched mycelium which
and there globular or
oval,
but seldom
302
pear-shaped conidia, 4 to 11
greatest diameter.
Fig. 124.
They
/i,
most generally 4
are formed
by
to 6
/i
in
abstrietion, in part
Chalara mycoderma, Cienkowski.
* JJ-.
The
if-"-.
(After Cienkowski.
figure to the left
Connected mycelium with conidia.

shows separated mycelium branches and conidia.
from sterigmata and partly from the surface of the limb.

This species thrives on wort and lager beer.
the free state on grapes and on cow dung.
It is
found in
OIDIUM LACTIS
Oidium
Oidium
colourless,
lactis,
lactis, Fresenius.
Fresenius (Figs. 126, 127 and 128), develops
branched hyphse, which form a white
conidia develop
rule,
303
by
a division of the threads,
as a
a rectangular longitudinal section, but other forms
are also
fco
be observed
their length
is
most
generallj^
The temperature
fi, and breadth 3 to 5 fi.
growth in wort are near 37| C. and below
10 to 30
for the
for the film formation 364 to 374 C.
An
Oidmm
Fig. 125.
and about
limits
4 C.
3 C.
inter-growth similar to that observed by P. Lindner
was found by the author and Schicinning

When, for example, a young,
in Botrytis cinerea
in
The
felt.
and have,
lactis
(Fig. 128).
Chalara mycodenna, Cienkow.ski.

conidia.
vigorous mycelium
more vigorous
neighbouring
cell
cell
This fungus
been standing.
-'--Y-.
Mycelium members,
abstricting
(After Hansen.)
seeded in a thin layer of water, a
is
here and there grows into its

and there forms conidia chains.
is
feebler
found in general on milk which has
According
to"
Hansen's investigations
it
generates a trace of alcohol in wort and dextrose yeast

water.
According
generate 1
vol.
to
Lang and Freudenreich
per cent,
of alcohol
in
it
can
milk-sugar and
dextrose solutions.
In breweries
it
is
to be found on malt, lager vessels,
304
casks,
piping,
pressed yeast.
Fig. 126.
Oidium
etc.
It
also
is
found
occasionally
Jorgensen states that he has found
lactis,
Fresenius.
A,
distributed horizontally in the liquid

air at the line x-x,
it
large
projecting obliquely into the
a branch divided by septa into a chain of
cylindrical
7;.
amount on top yeast when
in
branched mycelium thread m-m,
medium
B, Conidia chain at the commencement of the separation of

members from each other. About if-. (After De Bary.
conidia,
on
this is allowed to
at rest after fermentation has ceased.
its
remain
DEMATIUM PULLULANS
305
Oklitan conidium formations have been observed by Brefeld in many

fungi {AgaricinecB).
Some botanists are therefore of the
mushroom
opinion that Oidium lactis is really a stage of development

fungi. Proof of this has not yet been advanced.
Dematium
The fungus
this
131 and 132) designated by
a branched, colourless mycelium on the
surface of which, apparently without order,
produces yeast
mostly oval.
such
pullulans, de Bary.
(Figs. 129, 130,
name has
of
cells or eonidia
These yeast
by budding
cells
it
frequently
the latter are
are often situated on the
CZD>
Fig. 127.
Oidium
lactis,
Ranvier's chamber.
The germination
Fresenius.
1 at lOJ a.m. , 2 at
of a conidium in wort in a
2 p.m., 3 at ij p.m. 4 at 7J
,
P.
M.
--p
(After Hanson.)
threads on small humps which are sometimes discernible after
They can then

by budding or develop germ
the release of the yeast
cells.
either produce
threads, which
more yeast cells
grow into mycelia. Swollen parts are frequently found in the
mycelium, which, after a certain time, become thick-walled
and dark
coloured, usually greenish-brown
developed by budding
this way.
cognised
formed.
may
also
the single cells
undergo transformation in
Gemmae, which, among other ways, can be
by their contents
(large oil
and
re-
fat drops), are thus
Dematium pullulans does not induce fermentation.

20
306
Fig.
130 represents an inter-growth similar to that
Such
which has been described under Botrytis and Oidium.

endogenous
conidia.
endospores.
FiG.
'[2%.
have been regarded and described as
The author
Oidium
laciis,
has
Freseuius.
(After Klocker
junction
with
shown, however, in con-
Pheuomenon
iji.
of inter-gi'owtli.
and Schionning.
Schionning, that
in
these
cases
it
is
matter of inter-growth only.

about when a
The phenomenon is brought

vigorous mycelium cell in the immediate
neighbourhood of a feebler
cell
acts as a parasite
on the
three mem/, A chain of gemmae

heniatiun pulhdans, de Bary.
(, b, c) have produced mycelium tubes (;/i)on which conidia (at
Fig. 129.
bers of this
d) are developed.
//,
ohaiu of gemmiE developing conidia directly (at d).
conidium a, which has grown a mycelium thread m, on whicli

IV, Conidium
it has also formed conidia directly.
conidia are formed
I', Conidium divided into
divided into two cells under similar conditions.
VI, a-//, Continuous development of one and
two cells developing conidia.
the same gemma, in a very shallow water layer with free air supply, into a
Ill,
double-celled,
thick-walled,
brown gemma
rich
in fat.
VJI and VII],
Mycelia divided up into simple, short, swollen members, which have become
thick-walled gemmte, generally much browned and furnished with large oil
drops.
which
At VIII a some of the gemmae are seen still further divided by septa,
the same direction as the axis of the thread.
^\K (From
lie in
ZoDf s handbook.!
308
Fig.
Deviaiium jmUulans, de Bary.
130.
show intruded mycelinm
Phenomenon
Conidia are being abstricted on both sides,

e,
d,
with conidia.
/,
Cell with
is
A conidinm in the act of budding.
mycelium thread has grown through the septa
filled
a and
forming conidia.
r,
of inter-growth,
threads, in 6 the thread
of
two
cells into
a cell
four conidia strongly resembling endo-
One of the cells has developed conidia, a mycelinm thread has

from the other. The remaining figures show various examples of
endogenous conidium formation, e was observed at about 20 C. in a water
culture about one month old the remainder were observed in water cultures
(After KlScker and Schionning.)
two days old at 20 and 25 C.
apores.
grown
g,
in
^K
DEMATIUM PULLULANS
309
and forms chains of yeast conidia at the end next

and within the feebler cell or, less frequently, the
latter,
to,
vigorous
130
(Fig.
cell injects
a, h, e
and
interior of the cell
a longer or shorter mycelium thread

g).
may
The yeast conidia formed

further increase here
in the
by budding
V-
h^
fc,
64
^t
l>t
Dematium puUulans, de Bary. Two development series of endogenous oonidium formations directly observed in cover glass water cultures.
In the series a the development proceeded for five hours, and in b for twenty,
four hours at 20 C. i^2. (After Klocker and Schionnlng.)
Fig. 131.
(Fig.
130
d),
while the intrusive mycelium thread forms
conidia (Fig. 130
b).
When
weak
cell lies
between two
vigorous ones, both of these latter can grow into the feeble
one and form chains of yeast conidia (Fig. 131

cell
a^-a^),
or one
intrudes a mycelium thread, while the other simply
810
forms conidia (Fig. 130
e).
shows two develop-
Fig. 131
mental series of such endogenous conidia, and Fig. 132 the
As with Oidium, the phenomenon
germination of the same.
when young,
when placed
appears
water
vigorous mycelium
seeded in a
is
little
wort or on moist gypsum blocks,
in
the mycelium can also display inter-growth, but
it
does
not take place nearly so frequently.
This fungus forms a very strong layer on nutrient

Skerst observed the following limits of temper-
liquids,
v.
ature for
its
mum
C, and minimum 05
16
growth in wort: Maximum 31
to 32
C,
opti-
In a related form
to 2 C.
Hoffmann had found that the gelatinised cell walls were

by iodine. The author observed that this also
stained blue
occurs sometimes in the typical Bematixim puUulans.
The fungus
on
fruit.
to
Lindner
It
mann
tinous
It is
is
wort and makes
is
also transforms grape
substance,
which
is
cell
ropy.
must
into a thready gela-
derived from
membrane.
In wine, however, the fungus

killed,
by
According
according to Wort-
is
product of
This appearance
marked when cane sugar
especially
though not
in nature, especially
it
clouds white beer wort
it
the outer parts of the
liquid.
common
found in moist places in breweries.
decolorises
it
extremely
is
present in the
quickly suppressed,
the evolution of carbonic acid.
the presence of about 8 vol. per cent, of alcohol
not grow, but
is
also not killed.
The
so-called
it
In
does
cork or
stopper flavour of wine arises from the cork of the bottle
being overgrown with various fungi, Dematium and others.

According to Wortmann, Dematium pullulans is the cause of adisease of
wine grapes. The attacked grapes looli like those afiected with " schwarzen
Brenner," but the black spots are
soft,
not brittle, and depressed; further
they have a white, mealy spot in the middle.

of
At this place the mycelium
the fungus breaks through the epidermis of the grapes, and the yeast
buds appear.
The
Dematium
spots often reach half
pullulans
is
round the grapes.
one of the mould fungi which
CLADOSPORIUM HERBARUM
311
has been repeatedly regarded as the original form of the

saccharomycetes.
Communications
relative
to
this
have,
however, suffered the same fate as those in which the same

is maintained for Penicillium, Aspergillus, Mucor, etc.
as
;
soon as exact experiments were made, these ideas were
found to be quite incorrect.

it may also be mentioned that various Ascomycetes are found,
Sphierul'na intennixta, Berk, and Br., and Dothidea ribesia, Pers.,
Finally,
(-(/.,
which can produce Deinatium-Vike growths.
Fio. 132.
/Jeinatium /ndlidaiis,
De
If
Brefeld says that the
Germinatiou of endogenous conidia
Bary.
conidia (a-i) swiÛed up after twentygerm threads (a.,) the same has also taken place
with the uppermost two cells in the mycelium thread,
.
(After Klocker
and Schionning.
in
wort
in a cover glass preparation.
The
four hours and put forth
^--f
species in question produce
Dematium
pullulans, he
is
in error, because
these growths do not entirely correspond'with the species named.
More-
over, Brefeld has also been unable to produce Ascomycetes from a typical
Dematium pullulans.
of the
Under certain conditions it is also found that some

forms of Cladosporiuni herbarum produce Dematium-like growths.
Cladosporium herbarum, Link,

a collective name.
is,
as already mentioned,
In addition to the conidial form of
SphcBrella Tulasnei, referred to
on
p.
283, a
number
of other
name
known
fungi are found, which have been classed under the
none of
these,
however,
is
312
Some
to possess ascus fructification.
are similar to
tium pullulans, and some investigators

for
reason,
this
classed
Gladosp.
same development
pullulans in the
(e.g.,
Dema-
Laurent) have,
and Demat.
herharum
series,
Lopriore has
studied a parasitical Cladosporium herharum form occurring
on wheat, which forms
sclerotia.
He
states that
duces Hormodendron cladosporioides as well as Demat.
it
pro-
jjullu-
It is therewith stated that several species occur that
lans.
produce conidial forms, which can be identified with the

species referred to above.
Fission Fungi (Schizomycetes).
II.
General.
Structure and Form of Bacterial Cells.
1.
The
membrane.
contains a
show
cell
Like
mass
of
consists
cell
Contents.
Cell
yeast
cells,
the
bacterial
by
protoplasm surrounded
of
has been contended as to whether
It
nucleus or not.
it
Later investigations, however,
that in this respect also bacterial cells resemble other
cells.
As with yeast
is
is
vacuoles and granules occur in
In some bacteria a substance
the protoplasm.
which
cells,
stained blue
by
iodine.
observed, especially in the
cells of old
is
found
in
occasionally
many species, often
the colonies are highly coloured,
brown,
between the
The
it
fat
globules
cultures.
are
Colouring
in such quantity that
red, yellow, blue, violet,

is
found partly in the
and partly secreted as granules lying
cells.
Cell
cell
e.g.,
The colouring matter
etc.
interior of the cells,
If the
found
Sulphur granules or oxide of iron
are found in other forms
matter
is
probably granulose or an allied carbohydrate, as
is
Wall
and
its
Gelatinous
placed in a solution of
Formation,
common
salt,
the
STRrCTURE AND FORM OF BACTERIAL CELLS

plasma separates from the membrane so that the
becomes
molysis.
plainh'^ visible.
This phenomenon
The membrane does not
is
313
latter
called plas-
consist of cellulose but
of albuminoids, probably modifications of those forming the
The membrane often
protoplasm.
of
forming gelatine and swells up.
possesses the
property
The growths
are then
enveloped in mucilage and form gelatinous films or masses,

called ZooglcBse (Fig. 133).
is
by
stained blue
iodine, in others it gives the cellulose
reaction (blue stain
and sulphuric
Fx(i.
In some species the mucilage
by iodine
in zinc chloride or iodine
acid).
ISS. Bacteriii in Pasteurianv.m, Hansen. Gelatinous formation in an old

growth on beer.
The three cells to the left have fallen out. The cells are
prepared and stained by L'dfller's method, li'f''' (After Hansen.)
Flagella.
Many
bacteria
microscope to be in motion
physical causes and
which
this
mm. per
discovered in
can only be
is
the
often due to
then the so-called Brownian molecular
movement
second.
is
cilia.
motion attains, has, with some

?,
under
observed
motion
organs of motion, flagella or
value of about
were
are
this
Another kind of
movement.
special
is
efiected
species,
The
by
The rapidity
an average
flagella or cilia
1836 by Ehrenberg, and, as a
rule,
observed after a special preparation (see p. 91).
In some forms they are found at the poles of the

in others at the sides: occasionally several are
cells,
found to-
:314
jrether.
Fischer distini^niishes between bacteria
Alt'r.
with one flageUuui at the end (Fig. 135),

of flagella at the end. and
surface
(Fig.
(3)
with
Flagella
134).
(2)
flagella
furnish
FlG.
1-34.
c,
Vegetative motile cell;
.sponilatini;
b,
and
char-
for classih-
Butyric acid bacterium with
Cl"StruU)t,ti hi'ftjrintin, Prazmow.ski.
Hagella.
with a cluster
over the whole
important
acteristics for the determination of species,
(1)
motile
cell.
.'.-Y''-
(After Alfr, Fischer.)
cation
it
is
importance to determine
of
if
the
species
concerned occurs with ilagella or without.

Bacteria with flagella
motion
may
movement
be temporarily stopped by certain means,
by an increase
in the acid content of the nutrient
or by a deficiency of oxygen
Fl>;.
1S:i.l'fi:iiiJjcic/friiiHi
itrctl,
tlagellum.
is
then
known
as
neutralisation or
moved.
This
are called motile.
When,
the condition of the organism
Zeidler.
J-7".
Acetic acid bacterium witli one
(After Zeidler.)
flagella-stiffness
by aeration the
By
(Geisselstarre).
stiffness
can again be
therefore, bacteria in a culture do not
just at the instant,
e.g.,
medium
re-
move
one cannot be always certain that the
power of motion has altogether
left
them.
Various sub-
STRUCTURE AND FORM OF BACTERIAL CELLS

stances can attract or repel motile bacteria
is
known
Shape of the
Ceil,
^' ^'
Some
of the
""3
cell
shapes which
Spherical bacteria are
''"-/
.*
o^ ^/V
this property
as positive or negative Cheinotaxis.
occur most frequently are as follows
3L5
,--
B
.-r/
^.
Fig. 136.
m'
Form and
i!;^'
\.'J
size of various bacteria.
A,
1.
Micrncuixus of various
sizes.
Micwcuccus letragoiius.
5. Sarcina
B, 1, 2, 4. LoEg rods.
veiitrimdi, package form.
6. Staphylococci.
3.
Short rods. 5. Connected chains of long and short rods. 6. Long threads.
2.
Diplococci.
Afs.
(^,5.7-5-1.)
known
Streptococci.
4.
(After P. Baumgarten.)
as cocci (Fig. 136, A), rod-shaped ones,
which are at
bacilli
3.
least double as
i.e.,
those
long as they are broad, as
or long rods (Fig. 186, B,
i, 2,
4, s),
and those of which
the length and breadth are more nearly equal, as bacteria
816
(in the true
sense) or short rods (Fig. 136, B,
3,
more or
Bacillus displays (as the result of spore formation) a
decided club shape
less
if
curved
Spirillum.
it
called
is
called Clostridium (Fig. 137)
is
it
If a
s).
Vibrio,
and
if
it
screw shaped,
is
known as
common for
The very long thread-like bacteria are
Gladothrix, Streptothrix, Grenothrix, etc.
It
is
the same species to occur in different forms.
Most bacteria are very

tion
e.g.,
and 4
10
/J.
and a powerful magnifica-
The smallest
required in order to observe them.
is
not more than
cells are
are,
small,
/a
Bacillus oxalaticus,
forms
fj,
long
fj.
broad.
Methods of Reproduction.
As
Fission.
Cell
whose rods may be 30
and Bacteriiom megatherium, whose rods are
thick,
long and 2"5

2.
Especiallj'^ large
1 fi long.
above mentioned, the vegetative
by division or fission
one method of multiplication,
multiplication of bacteria proceeds
many
forms have only
this
whilst others can, in addition, form endospores.

teria
become elongated, and divide by means
the latter never occurs lengthways
not
alter
their
taken place.
Rod
bac-
septum
spherical bacteria
do
appearance before division has
original
In the
of a
division
latter,
can take place in
The new-formed cells often remain in

union with the mother cells, whereby long chains may be
several directions.
formed.
By
seeding on various nutrient gelatines the species
produce colonies, which have a more or
less varied
macro-
and condition
connection the method
scopical appearance, as regards both shape
of the surface (slimy, dry).
In this
by which the colonies were started is also of influence,

it was by stab or streak cultures or giant colonies
whether
from the seeding of a drop.

the colour of the colonies
It has already
may
vary.
been said that
METHODS OF REPRODUCTION OF BACTERIA
317
If a nutrient solution remains clear after seeding, the
species present
one with pronounced chain or thread
is
growth and without self-movement.

oxygen for their growth develop a
Forms which require

on the surface of
film
If the latter
the, as a rule, clear liquid.
is
uniformly turbid,
the forms are isolated, living and sometimes motile
in this
case also the oxygen-absorbing species form a film.
Spore Formation.
Endospores
are seen
refractive granules in the interior of the cells

cell
contains one spore, seldom two.
They
as
;
strongly
usually each
are formed
by
the contraction of the contents which become gradually
by an
thicker and more refractive and, finally, bounded
The
independent wall.
species
is
known
latter is usually
which a membrane of
in
with longitudinal ridges
With
(Fig. 137, B,/,
h).
only one
special structure
to be observed (Arth. Meyer).
is
assumes a special shape during
cei'tain species the cell
spore formation,
smooth
becoming,
example, spindle-shaped
for
The spore has a much greater power
of
resistance to external influences than the vegetative cell
up by
aniline dyes are taken
the remaining plasma

obstinately retained
wards employed.
it
much more
slowly than by
on the other hand these colours are
when
a decolorising reagent
Extreme degrees
of cold
and
after-
is
heat, besides
complete drying, are endured by the spores without death

ensuing.
On
the contrary, light appears, in the generality
of cases, to be the deadliest
enemy
of bacterial spores.
The
spores of several species resist a boiling temperature for
some hours their germinating power may even be increased

by this means.
Through the great resisting power of bacterial spores,
which is more marked in the dry than in the moist state,
;
sterilisation is
order to
if
found to be
sterilise
water
it
difiicult in
must be
this is not convenient,
raised
many
cases,
e.g.,
in
boiled under pressure, or,

to
boiling temperature
318
several times at intervals (see

after boiling
is
added
that this
is
the case can be seen
yeast water,
to
when
of
in
flasks of
wort after simple boiling
that the culture
medium
is
many
may
be due to the fact
not favourable for the germina-
Wort
tion of the spores present.
a sample
That no growth appears
makes
appearance.
if
frequently a growth
bacteria
its
Wort is often not sterile
p. 78).
indeed a liquid in which
is
species of bacteria, even in vigorous condition, are
not able to grow.
.%
I I
.1
II
to iO ' c <^
Fig. 137.
^t
Clostridiini butyricum, Prazmow.ski.
A, Vegetative cells. B, Sporewith the exception of a and b. In/ and h, the cells are swollen
to spindle shapes, the formation of the spore being here complete.
Two spores
have grown in g. C, Germinating spores. (After Prazmowski.
bearing
cells,
As soon
as the spore
conditions, to germinate.
is
ripe
it is
able,
under favourable
Before germination proceeds the
spore swells out and thereby loses
its brilliancy,
then bursting and a small knob protruding.
the skin
This protrusion
can take place either at the pole of the spore (Fig. 137, C), the
new
growth lengthwise from the spore, or

the spore skin bursts in the middle and the young cell grows
cell
continuing
its
VARIATION OF BACTERIA
319
in a direction perpendicular to the longer axis of the spore.
These are the two commonest types of germination of bacterial spores
and other ways are quite exceptional.
The causes
of the production of spore formation are to
be sought in unfavourable nutrimental conditions, accumulation
of
products of growth,
injurious
Like the
etc.
saccharomycetes, certain bacterial forms require free access

of air in order to
form
spores.
Variation.
3.
Hansen's experiments with the acetic acid bacteria

Bacterium
have shown that temperature

shape.
Bad. Pasteurianum and Bact. Eutzingianum,
aceti,
a factor for influencing
is
His conjecture that the results found by him have
more general application was contirmed by the author's

species.
Henneberg came to
the same conclusion from experiments with Bact. oxydans
The investigations of Hansen demonand Bact. acetosum.
a
experiments with four other
strated that the species

cell
forms,
144, 145
140),
viz.,
and
146),
three different
When
favourable culture
(Figs.
139
a,
sometimes as threads (Figs. 138, 139 and
and at other times
(Fig. 140).
named appear with
sometimes as short rods in chains
as pear-shaped or globular swellings
young, vigorous
medium
beer (Danish beer with
rich in extract,
little
seeded in a
e.g.,
"
double
"
and high extract) or

and 34 C, the chain
alcohol
wort, at a temperature between
form with the short rods appears

be inoculated in the culture
cells are
5
;
medium
if,
now, such a growth
of a
new
flask
and kept
at 40 to 40^ C, the cells are quickly changed into threads

(Figs. 138
fji,
and
while the
139).
cell
The
latter can attain a length of
seeded only measured 2
fi.
500
If the thread
form be now brought again to 34 C, the length continues

to increase
swellings
here and there globular, spindle, or pear-shaped
may
occur,
whereupon the threads begin
to divide
320
again and to assume the chain form (Fig. 140).
By Nageh
and others such swollen forms were regarded as not belonging

to normal development, but as an indication that the cells
are about to die.
Flo. 138.
It is
shown by Hansen's above-mentioned
Bacterium Pasteurianum., Hansen. The thread form after twenty-four

double " beer at 40 to 40^ C. i,<!-0
(After Hansen.
hours in
'
'
investigations that, under certain culture conditions, these

peculiar
forms appear normally and indicate a vigorous
growth.
Beyond the above-mentioned investigations on
acetic
acid bacteria, only
subject are as
321
few and incomplete contributions
to this
Macfadyen and Blaxall
yet to be found.
found that some thermophilic bacteria investigated by them

also
formed long threads
at high temperatures (approaching
70 C).
It
generally
is
known
that
among organisms
different
conditions can produce identical or nearly corresponding
'wQq
'cues
a'
."
'Oo=oO<i^
Fig. 139.
by
*Oooo<^
*'=ooc'^ ^*'='
^C::^
Bacterium Pastetirianuni, Hansen. Development of the thread form

on " double " beer agar-agar in a Bottoher's chamber at
cultivation
about 40^ C.
after 10
a,
A chain
a"', after
consisting of 8
20 hours.
6,
members
a',
five-membered chain
the same after 6

;
V, after 5
a",
b", after
9 hours, c, Development after W;d, after 21 hours. Tlie times are reckoned
from the beginning of the experiment. -'Y'S- (After Hansen.)
forms of development.
It is therefore not
remarkable that
various authors have stated that the change of shape referred to can also be obtained as the result of the influence
of factors other than temperature
H. Buchner has found,
in regard to Bacillus subtilis, that the chemical composition
of the culture
medium was
responsible for similar changes
of shape.
21
322
The abnormal and in general

by many bacteria when they have
inflated shapes
assumed
to exist for a long time
known
as in-
may take up the most varied
shapes.
in unfavourable
environments are usually
volution forms.
These
Bacterium Pasteurianum, Hansen.

Transformation of the thread
form into swellings and chains by cultivation in "double" beer at 34 C.
I. after four, II. after five, III. after seven hours from the beginning of the
Fig. 140.
experiment.
Some,
e.g.,
bacillus,
^^K
(After Hansen.
the acetic acid bacteria (Fig. 141), the diphtheria

tubercle
mycelium-like,
bacillus,
branched
etc.,
sometimes
appearance,
and
it
assume
has
been
attempted, without grounds, however, to
fact in
among
make
use of this
order to identify the parent forms of bacteria

the hyphomycetes.
only abnormal formations.
grown and developed
Fig. 141.
323
They
are,
however, as stated,
bacterium has never been
into a hyphomyces,
Bacteriuin aceti (Kutziug), Hanaeu.
days' cultivation in wort and
"double" beer
Unusual
and
cell
vice versa.
forms after several
at 39 to 41 C.
i-Y"^.
(After
Hansen.
All communications
which have hitherto appeared on the
development of bacteria from higher fungi are without

proof.
With many
species an enfeeblement takes place during
continued culture, so that, for instance, the fermentative
21*
324
activity becomes lost (as distinguished
from the saccharo-
mycetes, in which this has never been observed).
which cause diseases
in
man and
virulence (pathogenic properties) by
treatment.
Bacteria,
animals, can lose their
means
of
change of another kind has been likewise
observed in the splenitis bacillus, which, during
porary change of form, can

tion
of
this
special
lose the
change of form displays
its
tem-
power of spore formaitself in
the production
rudimentary spores.
With regard to variation among vinegar bacteria,

Hansen has further shown that the mucilage of his two
species, Bact. Pasteurianum and Bact. Kiltzingianimi, which
by iodine, loses this property under certain
Whether developed on the surface of a liquid
conditions.
or solid medium, a time arrives when they no longer give
In some beer cultures he found that
the blue reaction.
this point was reached in three to four months at the
is
stained blue
ordinary temperature of the room

trary,
it
was not even attained
These abnormal
normal
4.
cultures
in others,
on the con-
after seven to nine months.
usually return
quickly to the
state.
Disease Bacteria in the Alcoholic Fermentation

Industries.
The harmful influence of bacteria in the fermentation

is by no means small
there are, however, only
relatively few species which do an appreciable amount of
damage. The majority, in fact, do not thrive, or do so but
industries
indifferently, in acid liquids

acid,
and are thus protected
beer and wine contain free

to a great extent.
By
the
introduction of the pure culture system this injurious action

of bacteria has been very
much
lessened in bottom
and top
fermentation breweries, as well as in the manufacture of

wine.
DISEASE BACTERIA
325
The decomposition products of bacteria

They can develop acetic acid, lactic
kinds.
A large number
acid, various alcohols, etc.
jêlatine
are of manj'
acid, butyric
liquefy nutrient
through the possession of peptonising power.
Pasteur had discovered in the year 1861 that butyric

acid bacteria are anaiirobic, that
the absence of oxygen.
is,
they can only grow in
The majority
thrive best with free access of
air.
of species, however,
The
acetic acid bacteria
are typical examples of aerobic forms.
Pasteur (1876) was also the
first
to point out the injuriouK
part played by bacteria in the fermentation industry.

disease
phenomena brought about by
liquids are:
bacteria in fermented
mucilage formation, decolorisation, turbidity,
acid formation, disagreeable smell

Pastern- gives
in beer
The
and wine
and
taste.
some information on mucilage formation

he mentions a Micrococcus which makes
the liquids mentioned ropy.
Kramer
isolated
from thick
wines a Bacillus with which he could induce the same
From ropy
disease in sound white wine.
van Laer isolated

L.
it
bacilli
which were the cause
Belgian beers,
of the disease.
Vandam also found in English beer a Bacillus that makes

thick.
At the same time this species only attacks the
when
number at the beginning of the

Brown and Morris describe a Coccus,
The disease
likewise causing ropiness in English beer.
attacked the beer when it was six weeks to two months old.
beer
present in large
primary fermentation.
The source
of infection appeared to be a slaughterhouse for
pigs in the neighbourhood of the brewery.
was found by Lindner
The
disease
known
in
ropy
"
Weissbier
as the turning of
Pediococcus
".
wine
consists in red
wine assuming a brown colour, while white wine becomes

turbid and
colouration.
this
disease
discoloured
and frequently develops a dark

wine is changed by
The
tartar occurring in
into
potassium carbonate, which causes the
326
change of
Kramer
colour.
two Micrococcus
isolated
species
from turned wines.

bacteria of lactic acid, butyric acid and acetic acid
The
may
be mentioned as acid producers in fermented liquids.
In breweries the
are especially active in the mashing
first
According to van Laer a
stage.
species, Saccharobacillus
Pastorianus, causes the turning of beer

loses its brilliancy,
the beer thereby
becomes disagreeable in smell and taste
and forms a sediment the

;
latter consists partly of nitrogen-
ous products which separate as a result of lactic acid production,

lactici,
p.
and partly of the bacteria themselves.
according to Kramer, causes the
343) of wine.
"
Zickenwerden
" (see
Butyric acid bacteria are responsible for a
They are
very unpleasant smell and taste in beer.

injurious in the
Bacillus acidi
mashes
especially
in distilleries.
Acetic acid bacteria are
found particularly in wine
manufacture, where they occasion the tartness of the wine.
When
once the wine has been strongly attacked by them,
and
in consequence vinegar sour,
is
then no means of removing the

of
most danger
it is
evil.
in top fermentation breweries,
conditions for their development are

in
valueless
in practice with Bacterium aceti
and
there
where the
more favourable than
Hansen experimented
Bact. Pasteurianum in
the presence of Carlsberg bottom yeasts No. 2 and No.
He came
is
In brewing they are
1.
to the conclusion that the bacteria in question
were indeed present
in the finished lager beer if the infec-
tion took place at the beginning as well as at the end of
the primary fermentation, but that this infection appeared

neither in the fermentation cellar nor in the lager cellar.
The
beer
bacteria can propagate themselves only after the lagered

is
drawn
gar sour
bottles
if
off;
care
the beer, however, did not become vine-
was taken that the transport vessels and

filled.
The same holds good
were well closed and well
DISEASE BACTERIA
when
also
beer after
327
the bacteria are introduced into bottom fermented

has left the lager
it
ally occasioned
were observed.
no bad
effects
The
cellar.
when
infection gener-
named
the precautions
High temperature and
free air access are
necessary for the development of the bacteria.
Gayon and Dubourg found

a short non-motile rod
wine containing mannite,
in
which forms masses of
reduces invert sugar to mannite.
exposed to this disease in
zoogloea.
Wine, however,
warm
is
It
most
(As formerly
climates.
mentioned, Penicillium glaucum can cause the same pheno-
menon.)
According to the investigations of V. H. and L.
the disease of rum,
called " faultiness," is
a bacterium which these authors
This species
is
name
especially distinguished
Veley,
brought about by
Coleothrix meth^stes.
by
its
great power of
resisting alcohol, the bacteria retaining life in
alcohol content of 75 per cent,
J.
rum with an
by weight.
Pediococcus or Sarcina species frequently occur in brew-
However, only certain of these cause harm, and these
eries.
only under special conditions.

sen,
It has
been shown by Han-
Jorgensen and Ant. Petersen that when certain of the
species occur even in considerable
without appreciable influence on
amount
in beer, they are
its quality.
These forms
have been especially studied by P. Lindner (1888).
Accord-
ing to him and other authors, some species under certain conditions occasion " sarcina turbidity
".
In this connection
Reichard states that the disease appears when a strong afterfermentation takes place in beer inoculated with Sarcina,
while equally strongly infected beer did not succumb to the
disease
when
the primary fermentation was a strong one,
so that in place of the vigorous after-fermentation, only a
An
addition of hops to the
lager casks will, according to the
same author, prevent an
slight
maturing took place.
outbreak of the Sarcina disease.
328
Schonfeld has also investigated the infection of yeast by
and
Pediococcus
Sarcina.
His results are the following
Culture yeasts are susceptible to Sarcina in varying degree.
Wild yeasts are more
development of
prejudicial to the
Sarcina than culture yeasts
with favourable conditions a
Sarcina acclimatised to any yeast race can spoil the beer
prepared by means of this yeast
than aerobic
Sarcina
the virulence of Sarcina
is
medium temperatures, an absence
certain
motion
the
multiplication
of
more anaerobic
dependent upon
is
Sarcina
of air,
is
and on
restricted
by
strongly hopping the beer, by high alcohol content and by

aeration.
Kupfer seeks
for the causes of the poor "
head
"
and
slight stability of beer in Sarcina infection.

S.
V.
tartaric
Huth has recommended

acid
remedy
tartaric acid are
for
for six to twelve hours, after
duced
the
into
which 6 grams of
added in aqueous solution to every kilo of
After stirring, the whole
yeast.
the employment of a
Sarcina, in
fermenting
always be applied with
care,
is
put on one side to rest
which the mixture

vessel.
and
is
is
intro-
This method must
only to be recommended
when
the stock yeast contains practically no wild yeast.
For
this
if
is
not the case the latter develops at the expense
of the culture yeast,
and the
result proves correspondinglj'
unfavourable.
5.
Application of Bacteria
in
the Alcoholic Fer-
mentation Industries.
Only the
lactic
acid-forming bacteria are directly em-
ployed in the alcoholic fermentation industries, these beingapplied in distilleries in subduing butyric acid bacteria and
"
in the
"
to the
bottom of the dilute wort; such does not happen in
Weissbier
distilleries,
breweries.
In breweries the yeast
settles
where the thick mash does not allow of
this.
CLASSIFICATION OF BACTERIA
The
pitching
yeast
for
this
ment
of harmful
Avhich
are
effected
order
in
unable
thrive
to
of
culture of lactic acid bacteria.

starting from a trial
lies
at about 50
develop-
C, that
acid
acid
liquids.
therefore
is
young and vigorous pure

The latter is obtained by
which the souring is satis-
mash of
factory.
the
strongly
in
formation of
by the addition
in
of nutrient liquid
prevent
to
itself
such as butyric acid bacteria,
germs,
and suitable
sti'ong
amount
requisite
acidified
is
by
produced
therefore
is
The
special vessels.
329
for lactic acid bacteria
on the
of butyric acid bacteria,
other hand, at about 40 C.
The sweet yeast mash
is
therefore kept at about 50 C, in which case only a strong
growth
of lactic acid bacteria develops.
of acidity
killed
is sufficiently
by warming
the
When
the degree
high, the lactic acid bacteria are
mash up
to 70
C, following this it
and a pure culture of a
is quickly cooled to 17 to 20 C,
selected yeast race added.
part of the yeast produced
is
then taken out to be used in pitching a fresh portion of

sweet mash.
As formerly mentioned,
F.
Lafar was the
duce the systematic selection of a

this purpose.
lactic acid
Wehmer, however, has
first
to intro-
bacterium for
lately attempted to
suppress butyric acid bacteria with commercial lactic acid,
which, according to his investigations, has given good
Of the remaining
acid are employed, as
results.
bacteria referred to, those of acetic

is
well known, in the manufacture of
vinegar.
Systematic.
Ferd.
Cohn showed
that bacteria are plants, and set
system based on cell form.
We
have
seen,
up a
however, that
the same species can assume various forms of growth.
For the investigation of
different species, the
morpho-
330
and developmental
logical
characteristics are, in the tirsb
applied as far as possible,
place,
the presence of fiagella
the shape of the
e.g.,
mode
their arrangement, the
and
of germination of the endospores,
cell,
Further, the forms
etc.
which the growth assumes on various nutrient media,

on culture gelatine, in which case
what extent the
it is
e.g.,
also observed
species liquefies gelatine,
at
if
to
The
all.
products resulting from growth are also of importance.
The
are
cultures
cultures, the
by stabbing
latter
prepared
either
as
streak
or
stab
former on the surface of the gelatine, the

in a thick layer of gelatine (see
Drop seeding on the surface of the

ployed.
The behaviour of bacteria
gelatine
is
p.
98)_
also
em-
species with various
staining methods takes a large place, especially in medical

bacteriology.
The bacteria considered here can be divided into two

chief groups, viz., spherical bacteria (Coccacea) and rod
bacteria (Bacteriacece),
we
by which means, on practical grounds,

which shape was the
retain the old classification, in
criterion,
known.
and
The
also the
older
author, however,
is
at present very uncertain,
no
down.
little
fixed,
is,
on
The
nor against this arrangement.
since
names which are generally
and
this point, neither for
classification of bacteria
is still
subject to change,
systematic, distinctive lines
That the shape, moreover,
is
can be laid
in several respects of
value as a systematic distinguishing feature follows,
among
other things, from
what has been
said in the fore-
going on the polymorphism of bacteria.
I.
Family
Spherical Bacteria (Coccace^).
In the isolated state the

place along one,
cells
two or three
are spherical.
planes.
Fission takes
COCCACE^
Genus
1.
The
331
Micrococcus, Cohn.
joined together irregularly.
cells are
Included in
genus are several forms which, like Sarcina, divide
this
along three planes, but the
cells
quickly separate from one
another and do not remain united as in Sarcina.

species
ought rightly to be
Micrococcus viscosus
is,
Such
genus named.
classified in the
according to Pasteur, the cause
of the ropiness of beer.
Micrococcus saprogenes vini

obtained by
Kramer
I.
and
in pure cultures
II.,
Kramer, has been
from turned wines
from Styria and Croatia.

Diplococcus
I.
two
II.,
Genus
2.
The
and
cells are
Aderhold, makes wine ropy.
Pediococcus, Francke.
arranged in
flat colonies
they divide along
planes.
Pediococcus cerevisise, Francke, was

pure culture by P. Lindner.
diameter.
tetrads,
They occur
and are
to be
The
first
prepared as a
cells are 0'9 to 1'5
fj,
in
either as coccus or diplococcus or in
found
in large quantities in beers
with
the so-called sarcina turbidity.
Pediococcus viscosus, Lindner, has been isolated by P.
Lindner from thick
" Weissbier."
Pediococcus sarcinseformis, Reichard, causes disease in

beer, according to Reichard.
Pediococcus acidi
10
in diameter.
/i
tion in
growth
in
is
40
C,
5)
cells are
The
cells
are 0-6 to
lactic acid
fermenta-
whilst
hopped wort nor in
The
Lindner.
This species excites
malt mashes.
3.
A,
lactici,
dies at 62 C.
it
for its
It does not thrive
beer.
Genus
Sarcina, Goods.
arranged in packet-formed octads (Fig. 136,
they are divided by three
fission planes.
To
this
332
iênus belong species
which form white, yellow, red or brown
colonies.
Sarcina maxima, Lindner,

cells are 3 to 4
found in malt mashes.
is
Lindner, has been isolated from
Sarcina aurantiaca,
Bei-lin " Weissbier
This species, as also S. flava and S.
".
alba, is said to give rise to diseases in
The
The
in diameter.
American
beer.
two foregoing genera
species belonging to the
are
very common in nature, and occur, among others, also in

Jorgensen and Lindner, for example, found these
water.
Their occurrence in brew-
forms in their water analyses.

been mentioned above.
eries has
'
* e
Fig. 142.
II.
The
never
Family
cells
ta
Sarcina.
and
Rod Bacteria (Bacteriace^).
aftei-
all
wid 2
Bacterium and Bacillus.
sometimes
as
At present
it
long rods.
names on
The author, however,

practical grounds, the
being hence represented with their old names
that, for
im-
is
two genera completely from one

For the species appear sometimes as
retains both these generic
dium,
for example, called all short
long rods Bacilli.
possible to separate the
another by this means.
ui.,
previous elongation of the rod.
The older systematists, Cohn

rods Bacteria,
species
(After Hansen.)
Fission takes place only in one direction,
Genus
short,
are of various lengths, cylindrical and straight
spiral.
ti'ansversely,
"a
so
example, the generic names Granulobacter, Clostri-
etc.,
are retained in
what
follows.
For distinguishing-
purposes the spore formation and the flagella have been
BACTERIACE^
suggested
333
but these characteristics also have proved to be
insufEcient.
Acetic Acid Bacteria.
Kiitzing (1837)
whilst
first
described an acetic acid bacterium,
Hansen was the
show that various species

give at the same time
the outHnes of their life history (1879). The investigations
of A. J. Brown, Henneberg, Zeidler and Zopf show that
a few of these species have a swarming state under certain
conditions.
One of these acetic acid bacteria with flagella
is shown in Fig. 135.
In this case the flagella are of
of acetic bacteria
first to
and
exist,
to
With
particular importance in characterising the species.
some
species the mucilage
which
is
formed by the
cells
gives a blue reaction with iodine (Hansen), that of others

yields the cellulose reaction (a blue stain with iodine
sulphuric acid
Hansen
has
J.
and
Brown).
given
information
on the
vitality
of
these bacteria in different nutrient media and also in the
dry
state.
He
found that Bacterium
aceti lived in "
double
"
beer more than six years, in some cases, however, not five
years
further, that after nine years in lager beer
about two years in a saccharose solution
it
was
and after
still
alive
Bacterium Pasteurianum was alive after six years (in one

case
was dead after two and a quarter years) in " double "
after more than ten years (in one case death occurred
one to two years) in lager beer, and after one year
it
beer,
after
and a quarter
was dead
in a saccharose solution
after
but in the latter
one year and a half;
lastly.
Kiltzingianum was alive after about six years in

(in
some
cases
it
was dead
seven years in lager beer

only).
after five years),

(in
some cases
"
it
Bacterium
double
"
beer
and after about

after five years
In the dry state on small pieces of platinum wire
in Freudenreich flasks the life limit of the cells of the three
334
named was about five months. When the abovementioned dry pi-eparations were introduced into glass
species
tubes,
and these closed by fusing, and preserved at the
ordinary room temperature and at 2 C,

the lifetime at the former
it
five
months, and more than a year at the
the
cells
latter.
When
were introduced into the tubes in a moist state
they very soon
died.
The formation
is
appeared that
named temperature was about
of acetic acid induced
by
these organisms
brought about by the alcohol being converted into acetic
As Pasteur
acid in the presence of a plentiful air supply.
showed
(1864),
and A.
bustion can proceed
already formed
F.
aceti
is
J.
Brown confirmed
still
the com-
later,
further, so that the acetic acid
oxidised to carbonic acid and water.
Lafar studied the influence of temperature on Bad.
and
forming
Bact. Pasteitrianum
acetic acid.
with regard to their power of
He found
as low a temperature as 4 to 5
can set
up a strong
acetic
that the former species at
C, the
limit of its growth,
fermentation, but that Bact.
Pasteurianuni, on the other hand, formed no acetic acid at

4 to 4| C.
that the
With the last-named
maximum
after seven days,
W.
Seifert
acid formation
when 33
came likewise
species he found, further,

is
reached at 33 to 34 C.
per cent, by weight
produced.
is
to the conclusion that
morpho-
logically different species of acetic acid bacteria also exhibit
substantial difl'erences in regard to their chemical behaviour.
In his experiments he employed Bact. Pasteurianum and

Bact. Kiitzingianum.
He draws
"
the conclusion,
that the
fermentative power of acetic acid bacteria in the presence

of the
monatomic primary alcohols decreases as the propor-
tion of carbon in the latter increases
that, further, Bact.
Pasteurianum has the feeblest fermentative power in the

presence of the polyatomic alcohols and dextrose
berg carried out investigations similar to
''.
Seifert's,
Hennebut with
ACETIC ACID BACTERIA

a whole
series of species of
at the same general
335
which some are new
he arrived
result.
In the following table from Henneberg will be found
numerous substances which can be converted

the various species.
that
no
The
sign
+ indicates
acidification takes place.
into acids
by
that acidification,
336
proceeded slowly in barrels. As these barrels were employed for several

years without emptying and cleansing, large quantities of Vibrio aceti were
frequently developed, which caused considerable trouble.
The Pasteur
he thus endeavoured to provide the most favourable conditions for the development of
the film of acetic bacteria and by this means to bring about as quickly as
method consisted
in using shallow vessels instead of barrels;
possible the formation of vinegar so that the Vibrio aceti could not develop.
For various reasons the method did
not, however, obtain a foothold in
were so uncertain since iliere

a selected species or race. In the
his Untersuchungen aiis der Praxis der Ocirungsindustrie
practice, partly because the results obtained
was no question
edition of
first
Hansen had,
the application
of
of
in this connection, already directed attention to the
problem
applying the pure culture system to the manufacture of vinegar

but only of late has the way been paved for reform in this direction by the
researches of the experimental station in Berlin the goal has not yet been
reached, however. The question here concerns the most important method
of also
Fig. 143.
Baclerium
aceti (Kiitz.
),
Hansen.
beer at 34 0.
of
vinegar manufacture,
Schiitzenbach.
The
i.e.,
Young
film formation on " double
(After Hansen.
the so-called quick vinegar manufacture of
acetic bacteria occurring in this connection will be
described later.
Acetic bacteria naturally
fall into
groups according as
they possess flagella or not, and according as their mucilage

gives a blue reaction with iodine or not.
it is
expressly stated
if
In the following-
the species in question has flagella
or gives the blue iodine reaction.
The
first
three follo'iîng,
the descriptions of which are from Hansen, belong to species
without
flagella.
Bacterium Aceti
species forms a
(Kiitz.))
Hansen
smooth gelatinous
(Figs. 141, 143).
film
on
"
double
"
This
beer at

34 C. after twenty-four hours.
The
337
film cells are usually
hour-glass shaped rod bacteria arranged in chains (Fig.
148)
long rods and threads with or without swellings are
only exceptionally found.
40 to 40| C. long thin
At
The mucilage is not stained by a solution

by a solution of iodine in potassium iodide.
After four days at 25 C. it develops colonies on wort
gelatine which are grey, waxy, round, usually arched and
threads develop.
of iodine or
with unbroken edges, seldom star-shaped, and which consist

chiefly of free, small rod bacteria
The maximum temperature
here.
beer
the chain form
is
about 42 C, the
Fig. 144.
minimum
Bncterivm Pasteurianum, Hansen.

beer at 34 C.
The
species
is
J-^--.
of
growth
is
repressed
in "
double
"
4 to 5 C.
Young film formation on "double
(After Hansen.
found both in top fermentation and in
bottom fermentation beers; Hansen observed it frequently in

the dust of the air and Holm now and then in his analyses
of water.
Bacterium Pasteurianum, Hansen (Figs. 133, 138, 139,

140 and 144).
C. after
This bacterium forms on
"
double " beer at 34
twenty-four hours a dry film which very soon assumes
wrinkled and folded appearance, and
rises
but
little
from the surface of the liquid along the sides of the flask.
Like those
of the foregoing species, the cells are arranged in
long chains, but are altogether larger and especially thicker

22
338
The thread form (Figs. 138, 139 and 140) is

somewhat thicker at 40 to 40| C than with Bad. aceti.
(Fig. 144).
also
The mucilage
stained blue with a solution of iodine or
is
Plate culture colonies on
of iodine in potassium iodide.
wort gelatine
after four days at 25 C. are usually smaller
than those of Boat,
with
aceti,
They
this species.
otherwise they are the same as

consist chiefly of typical chains.
After about three weeks the surface of the colonies
On wort
folded.
gelatine
and without 10 per
C,
solid, dry,
and on lager beer
cent, of saccharose,
waxy and
ature for growth on
"
''
the growth
is,
is
with
at 25
The maximum temper-
yellowish.
double
gelatine,
beer
C, the minimum,
42
is
5 to 6 C.
Hansen found this species in the same places as the

previous one, but more frequently in top fermentation than
in bottom fermentation breweries.
W. Seifert found it in
vinegar-tainted wine.
Bacterium KUtzingianum, Hansen
formed on
differs
double
''
"
(Fig. 145).
The
film
beer after twenty-four hours at 34 C.
from that of Bact. Pasteurianum by growing high
above the liquid along the sides of the
It consists of
flask.
small rod bacteria which are, as a rule, free, or at most

joined in pairs
form
at
40
they form long chains rarely.
to
40i C.
contains relatively
threads than Bact. Pasteurianum.
The thread
more short
The microscopical appear-
ance differs distinctly from that of the latter
on the other
hand, both species are alike in that the mucilage

blue
by
is
stained
a solution of iodine or of iodine in potassium iodide.
In plate cultures on wort gelatine this species develops

colonies after four days at 25 C,
which consist almost
exclusively of small, free, rod bacteria
found only
ver^^ seldom.
three weeks
is
The surface
smooth, not folded.
long chains are
of the colonies after
The growths
at 25
C,
both on lager beer gelatine and wort gelatine, with and

without 10 per
cent, of saccharose, are shiny,
The maximum temperature

double " beer, the minimum,
bluish-grey.
about 42
Hansen found
bacterium in
this
"
slimy an
of growu'i
double
"
beer
i^
6 to 7
C. in "
it
probably diffused to the same extent as Bact. Pastearianum.
In addition to the above-cited differentiation betwasn

Pasteurianum and Bact. Kiltzingianum furnished by
Bact.
Hansen, Henneberg and Seifert have given an extensive

physiological
series of
characteristics,
example, are the following:
among which, for
Bact. Pasteurianum forms a
most half as much acetic acid as Bact. Kutzingianum in

lager beer with added alcohol.
Thus the former, in lager
trnm
Fig.
145.
Young film formation on

Kutzingianum, Hansen.
"double " beer at 34 C. i-V-- (After Hansen.)
Baderiuni
with 8 per cent of alcohol, produces 3-23 per
beer,
acetic acid in twelve days, while the latter

cent.
the formation of acid
by
cent, of
forms 656 per
Bact. Pasteurianum
is
like-
wise only half as great as that by Bact. Kutzingianum
when
the cultivation takes place in a 2 per cent, glycol
When
solution.
yeast water with 3 per cent, of dextrose
is
used as the nutrient solution, the growth of Bact. Pasteurianum

at
26
to
30 C. consists
Kiltzingianum,
on the
connected together.
of long chains, that of Bact.
contrary, of single
cells,
The same microscopical
or
pairs
differences
are thus found here as between the growths of both species
on beer and wort
Of the many
gelatine.
species of acetic acid bacteria
22*
which have
340
been described in the
among
others
last
few years we
will
mention here
Bacterium oxydans, Henneberg

species is distinguished
from
{Bact. aceti, Zopf).
This
Hansen described
Bact. aceti of
by having, as shown by Zopf and Henneberg, a

swarming state it was isolated from a bottom fermented
above,
beer from Halle.

Further, Beijerinck mentions a species which he calls Bact. aceti, and
It
states to be the active species in quick vinegar manufacture.
which he
is
distinguished by forming a film on artificial nutrient liquids
this
common with other species, is, however, not permanent

in this species, and may be lost after a previous cultivation on solid nutrient
property, held in
media.
it
All gradations of film
development are presented here. Further,

cane sugar, this being due, according to
liquefies beer gelatine containing
Beijerinck, to the inversion of the sugar.
To what extent Beijerinck's Bact.
aceti is the acetic
bacterium
of
Eothenbach
quick vinegar manufacture is, however, not established.
found that the active bacteria of the process named are not in a condition
to form a film.
The loss of this power of film formation rests, in his
opinion, on the circumstance that they are never allowed the opportunity
of film formation in practice, because the mash is in continual motion.
According to Bothenbach the quick vinegar bacteria are acclimatised
Some of them closely resemble Bact. aceti, Hansen, and can
furnish vinegar containing a high percentage of acetic acid from a mash
containing little nutrient substance and a high percentage of alcohol, in
forms.
the Schiitzenbach
acetifiers.
Of other acetic acid bacteria the following
Bacterium Xylinum, A.
and furnishes
cartilaginous,
sulphuric acid,
i.e.,
a blue
J.
Brown.
is
with iodine and

This species also occurs in the
Further, the following is described
colour.
Bacterium acetigenum, which was

quick vinegar manufactory.
also
from the vinegar of a

under certain
swarming; Bacterium
isolated
It yields a cellulose reaction
conditions, and has especially intense power of
industrium
be mentioned
gelatinous substance
the cellulose reaction
acetifiers of the quick vinegar process.
by Henneberg
may
The
has a swarming
state.
Several species have likewise been described under the

generic
name
Termobacterium, as for example
Termobacterium
aceti, Zeidler,
which
is
illustrated in
Fig. 135 and which possesses a long flagellum
further
Termobacterium lutescens, Lindner, which renders beer

turbid and gives rise to a celery odour in
it.
SLIME-FORMING BACTERIA
We
341
few more Bacterium species

Bacterium vermiforme, iVIarshali Ward, forms cells which
will
measure
mention
0"5 to 50
still
long and 0'5
fi
/x
The
broad.
gelatinisa-
tion of the cell wall with this species consists in the loosening
of the strongly swollen outer layer of the cell
whereby the
cells are
membrane
enclosed as in a capsule.
with the Saccharomyces pyriformis mentioned on
forms the so-called ginger beer

Bacterium termo, Cohn,
is
Together
261
p.
it
plant.
a collective expression for motile bacteria
occurring in decomposing substances.
According to Cohn's description
it
a feebly fluorescent, strongly motile, short, ovate rod but which of the
numerous putrefactive bacteria is thus indicated cannot be determined.
Wiudisch considers that the cause of the so-called cellar flavour, by
is
which is understood usually a raw, musty, damp taste in the beer, is

undoubtedly to be looked for in the contamination of the wort during
cooling, as the beer sometimes remains too long in the coolers during the
warm summer months, and is, in consequence, infected by one of the
bacteria belonging to the Bact. termo group.
They
thrive well in
hopped wort, but cannot
live associated
with
yeast.
Most of the species included

form endospores.
They can be
in the
genus
Bacillus,
classified in various
Cohn,
groups
according to their physiological activity.
Slime-forming Species.
Bacillus vlscosus
ropy beers.
and
I.
II.,
and a breadth of
/x
slimy, yellowish islands

rise, in
1"6 to 2*4
/j,
they are usually isolated, not infre-
quently, however, in pairs.
They give
van Laer, were isolated from
Both form rods with a length of
I.
forms on the surface of wort
which appear to branch downwards.
consequence, to a slimy covering contain-
ing bubbles, which result from the evolution of carbonic acid

II.
does not form a slimy covering.
production and viscosity are
less.
The carbonic
acid
The wort becomes dark-
brown and a peculiar odour is evolved. A

grams of saccharose and 1 gram of peptone
solution of 3
in 100
c.c.
of
342
water
rendered ropy and viscous by
is
I.,
while
causes turbidity and carbonic acid evolution.
amount
of sugar
prejudicial to the
which reason,
bacteria, for
is
is
development of these
33 C.
is
higher
therefore, hghtly fermented beer
comparatively seldom ropy.
for their development
only
II.
The
bacteria are frequently
found in water analyses.

Bacillus viscosus
2'0
/i
07
long and
L.
III.,
/x
broad
Vandam, forms rods which are

they usually occur isolated
sometimes they are joined in chains of two or three members.

This species was found in English beer
ditioned
by the presence
development of the
Bacillus
of sugar
0'6 to
membered
viscosus
08
vini,
broad
fj,
the ropiness
is
con-
air is necessary for the
species.
viscous in the absence of
and
Kramer, makes white wine
air.
It
forms rods 2 to 6
these are often joined in
/x
long
many-
chains.
Lactic Acid-forming Species.
Mention has been already made of the discovery of

acid bacteria
and
by
lactic
Pasteur, of their importance in distilleries,
of their recent introduction into the latter in the
form
of pure cultures.
The
first to isolate
a pure lactic acid bacterium for the
The species
said, F. Lafar.
and applied by him was Bacillus acidificans longisslmus, Lafar, the cells of which are 2-5 to 25 jx long and
above purpose was, as has been

isolated
about
1 fi
broad.
Bacillus acidi lactici, Hueppe, consists of immotile rods
which are 10 to 17
/a
long and
3 to
fi
broad; they
are frequently connected in pairs, rarely in four-membered

chains.
This bacillus
is
It loses its souring
aerobic and does not liquefy gelatine.
power
if it is
cultivated for a long time on
LACTIC AND BUTYRIC ACID BACTERIA

a sugar-free nutrient
species in wine,
As
medium.
Kramer produced
343
a result of seeding this

"
Zickenwerden "
(lactic
acid sharpness).
Saccharobacillus Pastorianus, van Laer,
is
responsible
known as the turning of beer. Pasteur

made the discovery that the disease is caused by bacteria.
The species thrives best on malt extract-agar with a little
alcohol added
this medium should be used when it is
"
'
for that disease
'
desired to obtain a pure culture.
when
It develops in beer only
the latter contains a small
According to van Laer
it
amount
of
hop
extract.
readily ferments saccharose (with-
out inversion), maltose and dextrose, but lactose only with

Lactic acid, ethyl alcohol and small amounts of
difficulty.
acetic
and formic acid are found
Fig. 146.
development in beer
is
in the fermentation.
Its
Lactic Acid Bacteria.
prevented by over 7 per cent, of
alcohol.
Butyric Acid-forming Species.

It has been stated that Pasteur discovered a but3iTic
acid bacterium
was shown
species,
which have already been alluded
much harm
to as the cause of
in breweries.
Clostridium
bacter,
which he named Vibrion hutyrique later it

fermentation is caused by many
that this
butyricum, Prazmowslci (Bacillus amylo-
van Tieghem), probably includes several species. The

(Figs. 134 and 137) consists of
form described by Prazmowski

rods 1
/A
broad, of which the
young individuals contain a
substance granulose which, however,
is
only formed
if
the
344
bacteria live in an entirely anaerobic state,
starch (amylum),
stained blue
is
by
and which,
like
(From
this
iodine.
the generic names Amylohacter and Granulobacter, sometimes
They
used, are derived.)
are provided with flagella (Fig.
When the spores develop,

134), and display active motion.
the rods swell out at one end and become club-shaped
(Clostridium form) (Fig. 137). According to Prazmowski,
the spores survive the temperature of boiling water for five
minutes
few of the strongest are

The species is
minutes.
after ten minutes only a
alive, whilst
in
die
all
fifteen
strongly anaerobic.
Bacillus
Hueppe,
butyricus,
differs
from the above
species in being aerobic.
saccharobutyricum,
Granulobacter
Beijerinck,
is
of
general occurrence on barley corns, and therefore also on
green malt, groats,
flour, etc.
species in distilleries.
maltose,
It is
one of the most injurious
decomposes dextrose as well as

acid, butyl alcohol, carbonic
and produces butyric
and hydrogen
acid
It
gelatine
is
not liquefied by this species.
Bacillus lupuliperda, Behrens, is classified here inasmuch as it

orms butyric acid when it is cultivated in a nutrient liquid containing
sugar.
Behrens found it in hops which had become warm. The cells
It liquefies
are motile; they are 0'7 fi to 2'5 |U long and 0'7 ^ broad.
gelatine, and can be cultivated in an extract of hops.
This bacillus
is partly the cause of
the spontaneous heating of hops, and forms
trimethylamine and ammonia.
Finally, two bacilli have yet to be mentioned which cannot be
classified in the above groups.
One is
Bacillus piluliformans, Miiller-Thurgau. The rods are 3^tol05/i
long, most frequently i /ito d fi, and are 0-75 /i broad. It causes in wine
a remarkable disease described by Miiller-Thurgau. In a red wine it had
formed rounded corn-like grains, which clung to the sides of the bottle, but
mostly
to
the bottom
these were in greater part detached by gently
which as many as a hundred were found

much in size, some being hardly discernible, whilst
had a diameter of as much as 45 mm. The wine was of a darker
moving the
The
bottle.
grains, of
in a bottle, varied very
others
colour than usual

clear, but not of
its
flavour and smell were but slightly altered
it
was
good quality.
Bacillus subtilis, Ehrenberg.
Hay
bacillus.
It
is
found frequently
BUTYRIC ACID BACTERIA
345
on hay.
The spores are very resistant towards high temperatures

according to Brefeld, they survive heating to 100 C. for three hours, to
105 C. for a quarter of an hour, and to 110 C. for five minutes. This species
can therefore be obtained by pouring water over hay and boiling the liquor
drawn
The rods
off.
flagella.
During
formed
its
cane sugar, and then oxidises
it
to the last trace.
action on dextrose a strongly reducing Isevo-rotatory body
(A. J.
is
met with in physiological fermentation

unhopped wort it does not thrive in acid
frequently
It develops easily in
and
is
Brown).
This species
work.
are strongly motile, and are furnished with several
It first inverts
on that account without significance in the brewery.

According to Esaulow, it occurs in kefir it here participates in the
formation of kefir grains, the film it forms on the milk serving as a collecting place for the lactic acid bacteria and yeast.
liquids,
is
LITEEATUEE EEVIEW.
Section
Spallanzani, Lazzaro
I.
(Pages
I.
15).
Saggio di osservazioni microscopiche,
Needham
relative al sistema della generazione di signori
II.
ScHEELE, Carl Wilhelm

Tom.
lingar.
Appert
Anmarkningar om
Le
Stockholm, 1782,
iii.
livre
de tous
IV.
î^me 4d.
ScHULZE, Franz
experimentellen
V.
les
I'art
de conserver
substances animales et
Paris, 18.31.
Vorlaufige Mittheilung der Resultate einer
Beobachtung
(PoggendorfFs Annal.
No.
nya Hand-
p. 120.)
manages ou
les
pendant plusieurs ann6es, toutes

v6g6tales.
sattet att conser-
(Kongl. Vetenskaps Academiens
vera attika.
III.
Modena, 1765.
BufFon.
d.
iiber
generatio
Phys. u. Chem.
sequivoca.
Bd. xxxix., 1836.
11, p. 487.)
Cagniard Latoue, Charles
(1) His communication appeared in the Parisian " L'Institut " on 23rd November,
:
1836.
(2)
Memoire sur
la
fermentation vineuse.
(Laid before
the Acad^mie des Sciences on 12th June, 1837; published
Compt. rend, de I'Acad. des Sc, Tom. iv., 1837, p. 905,

and in Annal. de Chim. et Phys., Tom. Ixviii., 1838, p. 206.)
in
Cagniard Latour states in the above that yeast is organised matter

it is probable that the formation of carbonic acid and of
alcohol is caused in some way by the growth of the yeast.
and that
348
Schwann, Thiodor.
VI.
In September, 1836, Schwann demonstrated before the Naturforsoher-Versammlung in Jena that the yeast fungus described by
him
is the cause of alcoholic fermentation.

In the publication which appeared in 1837 under the following
title
Versuche
Vorlaiifige Mittheilung, betreffend
(1)
Weingarung und Faulnis (PoggendorfF'i3 Annal.

Chem. Bd. xli., 1837, Nr. 5)
iiber die
Phys.
d.
u.
he also communicates the discovery that putrefaction is caused by

living corpuscles.
His words are
Then putrefaction must be thus explained, that these
germs, while developing and while feeding at the expense of the
organic substance, cause in it a decomposition of such a nature that
;
it
phenomena
gives rise to the
of putrefaction.
"
Mikroskopische Untersuchungen iiber die Uberein-
(2)
stimmung in der Struktur und dem Wachsthum der Thiere

und Pflanzeu. Berlin, 1839.
In the above research, Schwann again establishes that yeast
and that
fungus,
causes fermentation.
it
and
his discovery of yeast spores
He
of antiseptics.
"
fungi.
of
is
further communicates
lays the foundation of the science
expresses himself thus
All conceivable proofs point to the ferment particles being
Their form
is
that of the fungi, their structure
many
the fungi, for they consist of cells
young
He
cells
they grow like fungi by producing
is like
that
which again contain
of
new
cells at their
ends, they propagate themselves like fungi, partly by separation of

single cells, partly by production of
by the bursting
of these
mother
cause of fermentation follows
fermentation
cesses
VII.
KuTziNG,
iiber die
V.
Friedbich
stopped by
is
all
pro-
boiling heat, potassium
Mikroscopische
vegetabilischen
LiEBiG, Justus:
Anweudung
Theil.
e.g.,
"
Cheniie, Jahrg. 1837, Bd.
because they constantly occur in
the fungi,
kill
and
Untersuchungen
Hefe und Essigmutter, nebst mehreren anderen dazu
gehorigen
VIII.
first,
cells in the existing ones,
Now, that these fungi are the
second, because fermentation
which evidently
arsenate, etc.
new
cells.
(1)
Gebilden.
ii.,
p.
(Journ.
f.
prakt.
385.)
Die organische Chemie in ihrer
auf Agrikultur und Physiologie.
1.
Aufl., 2.
Braunschweig, 1840.
Licbig had as early as 1839 expressed the leading principles of his
LITERATURE REVIEW
theory in a treatise
nis
349
" Uber die Erscheinungen der Gahrung, Faulihre Ursaotten ".

(Poggendorff's Annal.
und Verwesung und
Chem., 1839, pp. 106-150.) He expresses the following

"The form and condition of the insoluble precipitates in fermentation have led to a very strange conception of fermentation on the part of many physiologists. When beer and wine
yeasts are distributed in water and looked at under a good magnifying glass, transparent, flat, compressed corpuscles are seen, which
sometimes, attached to one another in chains, assume the form of
growths in the eyes of others they are similar to many infusoria.
It would certainly be an extremely noteworthy phenomenon if plant
substance and albumen, which are precipitated in a changed condition in the fermentation of beer and of plant juices, were to
assume a geometrical form when deposited, since these bodies have
never been observed in the crystallised condition. Now this is not
the case they precipitate, like all substances which do not possess
crystalline properties, in the form of spherical corpuscles, which either
swim about free or are connected with others. These investigators were
misled by this form to regard the ferment as consisting of animated
organic beings, whether plants or animals, which in their development assimilate the constituents of sugar and excrete them again in
the form of carbonic acid and alcohol
they explain the decomposition of the sugar and the increase of the mass of the added
ferment in beer fermentation. This view confutes itself in pure
sugar water the so-called seeds disappear with the plants on fermentation
fermentation takes place, the decomposition of the sugar
ensues with that of the ferment, without any development or reproduction of seeds, plants or animals being observed, which is regarded
by these investigators as the cause of the chemical process.''
Phys.
u.
views on
p.
d.
142:
LiBBiG, Justus
(2)
Ueber die Gahrung und

der Konigl.
die Quelle der
bayer. Akad.
Muskelkraft.
(Sitzungsber.
Wissensch.
1869, pp. 323 and 393, and Annal. d. Chemie
u.
ii.,
Pharmacie, Bd.
In the above
tation theory
cliii.,
1870, pp.
and 137.)
treatise, Liebig's last opinion
is
expressed.
It
may
d.
on the Pasteur fermen-
be inferred from the following
two quotations
Page 2 " From the chemical standpoint, which I should not like
to give up, it is an 'act of life,' 'a condition of motion,' and taken
:
in this sense Pasteur's view
prove mine
Page 6
is
not opposed to mine nor does
it dis-
".
"It might be that the physiological process stands in
no-
other relation to the process of fermentation than to produce the

substance in the living cell, which, by an action peculiar to itself
similar to that of emulsin on salicin
and amygdalin, causes the
breaking up of the sugar and other organic atoms
the physiological
350
process would be necessary in this case to produce the above substance, but with the- fermentation as such
would have no further
it
connection "
Hoppa-Seyler in
" Physiologische
his
same view
expresses substantially the
Chemie,"
Berlin,
1877,
that of Liebig's given
as
According to Hoppeabove and that given formerly by Traube.

Seyler, fermentation is caused by a purely chemical ferment conOn page 115 he says " Liebig has shown
tained by the yeast.
:
very clearly the untenable and harmful nature of Pasteur's views,

yet his deductions received
little
IX.MiTSCHERLiCH, EiLHARD
"
attention
(1) Uber die chemische Zersetzung und Verbindung vermittelst Contaotsubstanzen, given
:
a lecture in the Kgl. Akad. d.
as
reference
machung geeigneten Verhandlungen

d. Wissensch. zu Berlin, Dec, 1841.
On
390 he writes as follows
p.
sugar changes,
when
yeast
appears to be different to grape sugar

to crystallise
it,
and
its
d.
into
which cane
a solution of the latter,
to
Kgl. preuss. Akad.
The sugar
the author has not been able
much
polarises light
it
quantity of grape sugar
"
added
is
a
Bekannt-
Wissensch. Berlin
to be found in Bericht iiber die zur
is
formation
is
less
than the same
very remarkable
it is
in
mixed with the yeast spherules, and can be removed

by water extraction, and a clear solution of it causes the transformafact a substance
".
tion of cane sugar into this kind of sugar
(2)
Another communication
is
to be found ibid., Feb.,
184.3.
He
exhibited on this occasion
among
some engravings which represented

The figure given on
other things the increase of top yeast.
p. 192 of my book is a reproduction of one of these engravings,

which, however, so far as can be found, were never published
Some of the figures reproduced are to be found in

together.
Mitscherlich's " Lehrbuch der Chemie," i. Ausg., Bd. i., 1844 and in
Begnault, " Cours ^l^mentaire de chimie," 2ine ^d., torn, iv., p.
all
179
1.
X.
further iu Jos. Bersch,
" Giihrungs-Chemie
ScHROEDER, H., und VON
DuscH, Th.
Praktiker,"
Ueber Filtration der
Luft in Beziehung auf Fiiulnis und Gahrung.
Chem. und Pharm.

XI.
fiir
Teil, Berlin, 1879, p. 60, etc.
Pasteur, Louis
lactiquc.
1857, p.
(1)
(Compt.
91.3.)
Bd. Ixxxix., Heft
M6moire sur
rend,
la
2,
(Annal. der
1854, p. 232.)
fermentation appel6e
de I'Aead. des Sc, Tom. xlv.,
LITERATURE REVIEW
The above memoir
year he published his
(Ibid.,
He
first
research on this subject, and
In the same
paper on alcoholic fermentation, viz.
M6moire sur
(2)
first
shows, of lactic acid fermentation.
treats, as the title
Pastbue, Louis
Pasteur's
is
351
la
fermentation alcoolique.
1032.)
p.
same
arrives at the
results here as his predecessors,
Schwann,
that yeast splits up the sugar in consequence of its vitality.

then publishes a series of smaller papers in the " Compt. rend."
etc., viz.,
He
all
matter referring to alcoholic fermentation
paper, viz.
(3)
Memoire sur
Chim. et de Phys.
The publications
la fermentation
(4)
De
I'origine
des ferments.
Fermentation
(Ann. de
1860, p. 323.)
and among
Nouvelles experiences
(Compt. rend.
dites spontan6es.
1860, p. 849.)
1.,
which he controverts the doctrine

(5)
alcoolique.
xlviii.,
aux g^ndrations
de I'Acad. des Sc, Tom.

in
collected in a larger
in the " Compt. rend." are continued,
these might be mentioned
relatives
Tom.
3 Sen,
is
of generatio aequivoca,
butyrique.
Animalcules
and
infusoires
vivant sans oxygene libre et determinant des fermentations.
(Compt. rend, de I'Acad. des Sc, Tom.

in which, for the
first
Hi.,
1861.)
time, the doctrine of anaerobiosis
is
put
forward.
For some years he continues

generatio aequivoca
to fight against
the doctrine of
on this point are

especially Chamberland and
his particular opponents
Pouohet and Joly after him his

Koux, take up the matter.
;
pupils,
Pasteur at the same time upholds his theory of fermentation till

the beginning of the seventies his writings in this connection are
to be found in the " Oompt. rend." His opponents are chiefly Liebig,
;
Traube, Brefeld, Berthelot and

(6)
Etudes sur
le vin.
(7)
Etudes sur
le
(8)
Etudes sur
la bifere.
01.
Bernard.
Paris, 1866.
vinaigre.
Paris, 1868.
Paris, 1876.
In the three researches named, which with (4) must be looked

upon as the chief works of Pasteur in the science of fermentation, is
to be found what he has accomplished for these branches of
industry; in (8), especially, there is a collation of his researches on
fermentation and the organisms of fermentation as a whole, and
the book practically ends his researches on this subject.
He
352
publishes here for the
first
time his methods for purifying yeast

which were kept secret in his first
(the tartaric acid method, etc.),
smaller communications.
While the works of Pasteur on generatio aequivoca and on hisfermentation theory caused prolonged contests, his attempts to
introduce reform into technical fermentation very soon came to an
His methods and suggestions were tested, but without giving
end.
most important thing, the pure yeast, was
In Denmark, the Messrs. Jacobsen began experiments
successful results, as the
wanting.
Old and New Carlsberg breweries shortly after Pasteur's

work had appeared in 1876, using the methods there recommended
in the
for the purification of the yeast;
but the results were not satisfac-
This was the case also in France and other countries. His
" Etudes sur la bi^re " are mentioned in the technical writings of
tory.
that time with respect, but gradually become treated as something
do with practice thiey did not come into vital

The only upholder of Pasteur's work on
technical fermentation is Velten, his co-worker on the subject of
brewing.
In the " Eevue universelle de la brasserie et malterie,"
1888, No. 742 and No. 743, he points out that it is the mixture of
several different pure species oE yeast which gives the beer the
required flavour and bouquet. This is obtained, he asserts, according to Pasteur, by cultivation of the yeast in a cane sugar solution
to which a little tartaric acid has been added, or in wort to which
carbolic acid and alcohol have been added. As early as 1878 he
had given, in the lectures which he delivered to the Congress at the
International Exhibition in Paris, an exact description of this
method, as Pasteur and he considered that the brewers should use it
Velteu published these lectures
for the purification of their yeast.
later under the title, " De la fabrication de la biere par le precede
Pasteur" (Revue universelle de la brasserie, 1881, No. 372).
They
are also to be found in the publications of the Congress.
Duclaux also recommends Pasteur's above named methods in his
" Chimie biologique," 1883, p. 301. As is mentioned in the introductory chapter, no one understood at that time the importance of the
disease yeasts and their competition with the culture yeast
consequently also, it was unknown that Pasteur's method was specially
favourable to the development of yeast diseases in beer. Thus in
the above work Duclaux, in agreement with Pasteur, mentions
which had
little to
contact with the latter.
(p.
618) only bacteria as the disease
did not prevent
germs
him and other members
of
of beer.
This, however,
the French school from
attempting later on to give quite a difierent interpretation to

" Etudes sur la bi^re "
It became a regular practice to find in this
work the new results produced by science after 1876 on the biology
" Etudes sur la bidre "
of yeast fungi and their use in technique.
abounds in contradictory opinions where the yeast fungus is treated.
LITERATURE REVIEW
353
without being decisive as to which is the right one. This is, the
case, e.g., in the question of the parent forms of yeast species (pages
155, 165, 177) and on top yeast and bottom yeast.
(On p. 189 of the
work cited, the view is expressed that a conversion of the one into
the other does not take place, but on p. 833 the contrary is stated,
viz., that the conversion of bottom yeast into top yeast can take
,
method even being described to prevent this.)

means much confusion in the literature, and now and then
little disputes, have been caused.
The discussions of possibilities
which occur in so many places in Pasteur's work enabled any one
to find nearly always what he desired.
"Etudes sur la bi^re."wa3
place in breweries, a
By
this
not used as a scientific work, but rather as a Bible.
On
the dispute which Hansen's work caused see
Teaube, Moeitz
XII.
p. 355.
Theorie der Fermentwirkungen.
Bertrn,
1858.
XIII.
DB
rend.,
XIV.
Seynes, Jules
Tom.
Reess,
Max
Brefeld,
Pilze.
Oscar:
(1)
XVII.
Lister, Joseph
1877.
also in
iiber
Schimmelpilze.
On the Lactic Fermentation and
its bearing
(Trans, of the Path. Soc. of London, Vol.
Georg
Zymotechnische Riickblicke auf das
(Zeitschr.
XVIII.
XIX.
Hansen, Emil Chr.
V.
1874
1875, p. 160.)
1878, p. 445.)
HoLZNER,
.Jahr
iv.,
1874 and 1881.
4,
on Pathology.
xxix.,
Methoden zur Untersuchung der
Untersuchungen
(2) Botanische
(Ber. d. med.-phys. Ges. zu Wiirzburg,
Heft 2 und
(Compt.
vini.
Leipzig, 1870.
Landwirtsch. Jahrb., Bd.
XVI.
Mycoderma
le
Botanische Untersuchungen iiber die Alko-
holgahrungspilze.
XV.
Sur
1868, p. 105.)
Ixvii.,
Nageli, Carl
f,
d. ges.
Brauw., 1878, p. 142.)
Theorie der Ganing.

:
(1)
Miinchen, 1879.
Contributions a la connaissance
des organismes qui peuvent se trouver dans la hihre et le

mofit
de hihte
et
laborat. de Carlsberg,
vivre.
Tom.
(Compt. rend, des trav. du

i.,
livr.
ii.,
1879,
p".
49.)
In the above work Hansen communicates the preliminary
in-
vestigations for his reforming researches published in the following

years.
23
354
Hansen, Emil Chr.
Recherches sur
(2)
difKrentes 6poques
de I'annê,
les organistnes qui,
trouvent dans Fair, a
se
Carlsberg et aux alentours, et qui peuvent se d^velopper
dans
mout de
le
Tom.
(Ibid.,
biere.
livr.
i.,
1882.)
iv.,
206 the chief features of Hansen's spore analysis

pure culture method is described on p. 212, and
on p. 217 are described the first experiments on diseases in beer
caused by fungi of alcoholic fermentation.
In the note on
His
are given.
Maladies provoquês dans
(3)
alcooliques.
Les
(4)
et
livr. iv.,
(6)
torn,
chez
ascospores
(Ibid.,
Tom.
la biere
livr.
ii.,
le
livr.
ii.,
ii.,
par des ferments
1883, p. 52.)
Saccharomyces.
genre
ii.,
1883,
28.)
p.
M6thodes pour obtenir des cultures pures de Saccharo-
(5)
Tom.
(Ibid.,
M6thodes.
myces
p.
first
de microorganismes analogues.
Qu'est-ce que la levfire pure de
iii.,
(Ibid.,
Tom.
ii.,
1886, p. 92.)
livr. i.,
M. Pasteur?
[Ibid.,
1891, p. 24.)
The most important researches
of
Hansen
are contained in the
four works mentioned below.

(7)
Recherches
sur
les
organismes
6poques de I'annê, se trouvent dans
aux alentours,
de
biere.
et qui
qui,
I'air,
diflF6rentes
a Carlsberg et
peuvent se d6velopper dans
le
moiit
(Compt. rend, des trav. du laborat. de Carlsberg,
1879, 1882.)
(8)
Recherches sur
ferments alcooliques.
la physiologic et la
{Ibid.,
morphologic des
1881, 1883, 1886, 1888, 1891,
1898, 1900, 1902.)

(9)
Recherches sur
rend, des trav.

(10)
du
les bact^ries ac6tifiantes.
(Oompt.
laborat. de Carlsberg, 1879, 1894, 1900.)
Untersuchungen
aua
der
Praxis
der Garungsin-
dustrie.
The first edition of this work is given in Danish, with a, risumi

French in the "Compt rend, des trav. du laborat. de Carlsberg,"
Of the more complete German edition (Oldenbourg,
1888, 1892.
in
LITERATURE REVIEW
Munich and Leipzig) there appeared Part I.,
:
1890; edition
2,
1895; Part
3,
11., 1892.
355
edition
The
1,
1888
edition
collected English
(Spon, London and New York.)

The first to advocate Hansen's pure culture system in the literature was Carl Lintner, sen., who was then professor and director of
the Royal Bavarian Agricultural Oentralschule in Weihenstephan
(Zeitschr. f. d. ges. Brauw., 1884, p. 245).
Aubry at the same time
edition appeared in 1896.
introduced Hansen's innovations into the scientific brewing station

at
Munich.
journal), Will
In his report for the year 1897 (p. 591 in the above
mentions that "the epoch-making ideas of Hansen
have found a home and fostering ground in this station for more
" Our station," he adds, " was the only one for a
than a decade "
long time which disseminated them among brewing circles on the
Continent and gave them due recognition. The stormy period and
the vigorous struggle is probably still in the memory of each of us,
which was necessary to overcome the many prejudices which opposed
the introduction of the pure cultivated yeast into practice, not only
on the part of practical men, but also of eminent theorists, and to
allay the misunderstandings which were always cropping up."
Later on the acceptance became general.
In Denmark, Ghr. Gronlund and Alfr. Jorgensen in particular
advocated Hansen's reforming works, and the latter particularly has
striven to obtain their general acceptance.
(See his text-book " Die
Microorganismen der Garungsindustrie," 1-4 editions, 1886-1898
also " Centralbl.
f.
Bakt., Parasitenk. u. Infektionskr.," 2 Abt., 1897,
section: "Die Theorie der Garung," in

Thausing's " Malzbereitung und Bierfabrikation," 1898, p. 651,
besides his numerous articles in the brewing journals.) In this conp.
665,
and
finally the
may
be
named
two jubilee reports which the Old

published in 1896 and 1897 respectively
likewise the articles published by Kjeldahl in the
" Nationaltidende," 15th May, 1887, and 10th November, 1897, by
W. Johannsen in his textbook and E. Rostrup in the " Biographisk
Lexikon ".
Particulars of the dispute which the pure culture system aroused
are given in the " Wochensohr. f. Brauerei " and the " Zeitschr. f.
Velten and Duclaux
ges. Brauw.," in the years following 1883.
have been mentioned on p. 352. The attacks were not always made
in the most considerate manner, and they have been continued
down to the present time. (On this point see J. Chr. Holm,
"Hansen's Eeinzuchtsystem in Prankreich, Centralbl. f. Bakt.,
Parasitenk. u. Infektionskr.," 2 Abt., 1899, p. 641, where still further
references are given
they supplement the final chapter in Jornection
and
New
Carlsberg
also the
breweries
gensen's text-book, 1898.)
In the introduction to his " Mikroskopische BetriebskontroUe in

1 Aufl., 1895; 2 Aufl., 1898, P. Lindner on
den Garungsgewerben,"
23*
356
p.
10 writes appreciatively
becomes the reformer

in that of bacteria ".
"
Hansen by
the use of the pure culture
in the science of yeast organisms, as
The object
of his book,
Koch was
however, consists in
substituting in several places other methods in place of those intro-
duced by Hansen methods, viz., which more resemble those of

Koch. In it, he attacks several points in Hansen's work in that
direction which have been defended by Alfr. Jorgensen and Will.
;
The
latter
says (Zeitschr.
f.
d.
ges.
Brauw., 1899,
612);
p.
"The
pioneer researehes of Hansen have brought about a complete revolution in this direction.
The extension of Pasteur's study of the
organisms which produce diseases in beer, by Hansen,
who showed
that diseases are also produced by yeast species, and the building up of this theory by the latter investigator, his pupils and the
zymo-technical laboratories, have advanced to such a, point, that

we can with comparative quickness and certainty, by characteristic appearances, recognise all those organisms, in particular
to-day
those yeasts, which are foreign to a normal yeast or fermentation

and consequently also to a normal beer." Will and Jorgensen
themselves have contributed.
Among
others.
Prior's
laboratory
up Hansen's analytical methods. The latter depend in

general on the study of important and characteristic physiological
functions, but morphological characteristics were also included
where this was possible. The same analysis can, of course, in many
cases be performed in different ways. There is room enough for
other methods besides those given by Hansen.
For illustrating the progress of the development it is instructive
to go through various successive editions of textbooks and handbooks of the fermentation industries, e.g., such works as that of
Thausing mentioned above. Even at the beginning of the eighties
no mention is made of a reform with regard to the yeast question
but in the following years Hansen's ideas make themselves inalso took
creasingly
felt.
As regards the pure culture system in wine manufacture, see

Wortmann's book, "Die Auwendung und Wirkung reiner Hefen in
der Weinbereitung," Berlin, 1895. On the pure culture system in
spirit and pressed yeast manufacture, see Delbriick and P. Lindner
in the " Zeitschr. f, Spiritusindustrie," and " Wochenschr. f.
Brauerei," 1892 and 1895.
Although a large part of Hansen's investigations deals with the
study of species, and although they depart from new points of view
and have given rise to important progress, systematic botany has
hitherto paid little or no attention to them. Descriptive botanists
repeated substantially the methods of description of Reess, and proceeded from the old fallacy that a microscopical investigation is
sufficient to distinguish the systematic units from one another.
It
is still more unfortunate that such a doctrine is taught in more than
LITERATURE REVIEW
357
one of the Teohnisohe Hochsohulen. The theoretical aspects of

Hansen's research received special recognition in mycological textbooks
(e.g.,
Zopf s Die
fermentation
and in those of the chemistry of

1889; Langer, 1890; Ad. Mayer,
It has introduced a new chapter into this
Pilze, 1890),
Bourquelot,
(e.g.,
1895; Prior, 1896).

literature.
XX.
LiNTNER,
Carl
und
Haltbarkeit
Brauw., 1880,
XXI.
Koch, Egbert
1881,
Einfluss auf die
(Zeitschr.
f.
d.
ges.
Zur Untersuchung von pathogenen
(1)
(Mitt, aus d. Kaiserl. Gesundheitsainte, Bd!

1.)
According to Hueppe, the important plate culture
(2)
was
p.
imd dessen
des Bieres.
Giite
p. 384.)
Organismen.
i.,
Tiber Malz
demonstrated on the occasion of the Hygiene Exhi-
first
by Koch during the XI deutschen
bition in a lecture given
Arztetage, 1883,
were
cultures
first
Special
Berlin.
at
directions for these
published by Biedert in the Deutsche
Medicinal-Zeitung, 1884.
XXII.
Thausing, Julius
Einfluss
gahrung, Hefe und Bier.
der Hefegabe auf HauptZeitschr.
(Allg.
Bierbrauerei,
f.
1884, p. 872.)
XXIII.
ihre
Fischer, Emil: (1) Die Chemie der Kohleuhydrate und

Bedeutung fiir die Physiologic. Berlin, 1894.
(2) Einfluss
der Konfiguration
Enzyme.
(Ber. d. deutsch. chem.
and xxviii., 1895.)
XXIV.
Bd.
XXV.
Delbruck,
xii.,
Max
BucHNBB,
(Wochenschr.
f.
Brauerei,
Eduaed
Fortschritte
in
der
Chemie der
Tubingen, 1897.
Section
De
Lecture.
Wirkung der
Ges., Bd. xxvii., 1894,.
1895, No. 30, pp. 732-733.)
Garung.
auf die
II.
(Pages 16 to 169).
Text-Books, Handbooks and Journals.
Bary, a., and Woronin, M.

Physiologie der Pilze.
-2.
Beitrage zur Morphologie und
Reiho,
Frankfurt, 1866.
358
Bbhrbns, W.
DippEL,
L.
zum Gebrauch
Tabellen
tnikroskoipschen
bei
Braunschweig, 1898.
Arbeiten.
Daa Mikroskop und
seine
Anwendung.
Braun-
schweig, 1882.
Friedlandeb
Mikroskopische Technik.
Bearb.
Eberth.
v.
6.
Berlin, 1900.
Aufl.
HuEPPE, F.
Die Methoden der Bakterienforschung.
5.
Aufl.
Wiesbaden, 1891.
JoBGENSBN, A.
Lafae, F.
Die Mikroorganismen der Garungsindustrie.
4-
Berlin, 1898.
Aufl.
Teohnische Mykologie.
I,
Schizomyoeten-Garungen.
Jena, 1897.
Lindner, P.
Mikroskopische BetriebskontroUe in den Garungs-
gewerben.
LiNTNEB, C.
J.
Berlin.
Ausg., 1895; 2 Ausg., 1898.
Handbuch derlandwirtschaftl. Gewerbe.
Berlin,
1893.
Considers the application of the pure culture system not only in
breweries, but also in distilleries
MoRiTZ and Morris
and
Text-book
in pressed yeast
manufacture.
the Science of Brewing.
of
London, 1891.
Strasburger, E.
Stkes,
W.
J.
Das botanische Practicum.
The
Jena, 1887.
Principles and Practice of Brewing.
London,
1897.
References to the pure yeast system in English top fermentation
breweries.
Centralblatt
fiir
Bakteriologie, Parasitenkunde
krankheiten.
2.
Abt.
Emil Chr. Hansen.

Jahresbericht iiber die
Garungsorganismen.
und
lufektions-
Herausg. von Oscar Uhlworm and
Jena, 1895
f.
Fortschritte
in
der
Lehre
Herausg. von Alfr. Koch.
von
den
Braun-
schweig, 1891.
Zeitschrift
J.
fiir
wissenschaftliche Mikroskopie.
Behrens.
Braunschweig, 1884
f.
Herausg. von
W.
LITERATURE REVIEW
Brefbld, 0.
Works.
Special
2.
859
Methoden zur Untersuchung der

zii Wiirzburg, 1874; also
med.-phys. Ges.
Jahrb.
Bd.
iv.,
Pilze.
in
(Ber. d.
Landwirtsch.
1875.)
Botanische Untersuchungen iiber Schimmelpilze.

2
and
of
The above researches are specially important

studying morphology and life history.
Greg, P. H.
Cane.
Fermentation
in
Rum
as regards
Distilleries.
Emil
Chb.
Contributions
bifere et
vivre.
de Carlsberg, Tom.
Sur
le
la nature.
i.,
livr.
ii.,
Here
206)
{Ibid.,
Tom.
les
livr. iv.,
i.,
also are
and
livr.
On
livr.
iii.,
ii.,
1881, p. 159.)
organismes qui, a difF6rentes 6poques

I'air,
a Carlsberg et aux alentours,

le inoftt
de bi6re.
{Ibid.,
1882, p. 197.)
found the principles of Hansen's spore analysis
his first pure culture
Les ascopores chez

ii.,
1879, p. 49.)
peuvent se d6velopper dans

i.,
mofit
Saccharomyces apiculatus et sa circulation dans
Recherches sur
et qui
bifere et le
(Compt. rend, des travaux du laborat.
de I'annê, se trouvent dans
(p.
(The Sugar-
eonnaissance des
la
organismes qui peuvent se trouver dans la
Tom.
methods
Vol. xxv., 1893, No. 292.)
Hansen,
de
Parts
1874 and 1881.
4,
le
method
(p. 212).
genre Saccharomyces.
{Ibid.,
Tom.
1883, p. 13.)
and following there is a somewhat more detailed descripHansen's pure culture methods and of his spore culture
p. 23
tion of
method.
liber Wiesner's neue
Priifungsmethode der Presshefe.
(Dingler's polytechn. Journal, 1884.)
M^thodes pour obtenir des cultures pures de Saccharomyces et de microorganismes analogues. (Compt. rend, des
travaux du laborat. de Carlsberg, Tom. ii., livr. iv., 1886,
92.)
p.
detailed description of the manipulations in his pure culture
methods.
360
Hansen, Emil Chr
Tom.
(Ibid.,
Les voiles chez
livr. iv.,
ii.,
le
1886, p. 106.)
Untersuchungen aus der Praxis der Garungsindustrie,

Heft
1.
1,
1888;
Ausg.,
1895, Heft
Ausg.,
3.
2,
1892.
Miinohen und Leipzig.

The contents of Part 1 are as follows 1. The pure culture of
methodically selected yeasts in the service of the industry. 2. Studies
of yeast species.
3. On the practical testing of beer in lager casks,
with respect to its stability. Part 2 contains 1. The technical
:
and water. 2. What is

Investigations on diseases caused in beer
analysis of the micro-organisms of air
Pasteur's pure yeast?
3.
by alcoholic fermentation fungi.

pure yeast culture system.
4.
On
the present-day use of
my
The majority of the above works are to be found in Danish, with

risumis in French, in the journal of the Carlsberg laboratory. Also
the English edition mentioned above, London, 1896.
Sur
dans
livr.
la vitality des
iii.,
ferments aloooliques et leur variation
milieux nutritifs et a I'etat sec.
les
{Ibid.,
Tom.
iv.,
1898, p. 93.)
Methods are given here
for preserving
pure not only yeast species,
but also alcoholic fermentation fungi in general.
Neue Untersuchungen
Saccharomyceten.
Bd.
v.,
La
iiber die
(Centralbl.
1899, No.
f.
Sporenbildung bei den
Bakt., Par. u. Inf.
variation
des
(Compt.
Saccharomyces.
travaux du laborat. de Carlsberg, Tom.
Holm,
J.
Chr.
Sur
les
v., livr.
m^thodes de culture pure
sur la culture sur plaques de M.
Abt.,
Koch
et,
i.,
rend,
des
1900.)
sp6cialement,
et la limite des erreurs
(Compt. rend, des travaux du laborat. de
de cette m^thode.
Carlsberg,
Tom.
Zeitschr.
d. ges.
f.
2.
1.)
livr.
iii.,
i.,
1891,
p.
1.
In German
in
Brauw., 1891, S. 458.)
Analyses biologiques et zymotechniques de I'eau destin6e
aux
brasseries.
Carlsberg,
Holm,
J.
Tom.
(Compt. rend, des travaux du laborat. de

iii.,;;iivr.
2,
1892, p. 107.)
Chr., et Poulsen, S. V.
Jusqu'i quelle limite peut-
m6thode de M. Hansen, constater une infection de

levdre sauvage " dans une masse de levdre basse de Sac-
on, par la
"
charomyces
cerevisiee
LITERATURE REVIEW
de Carlsberg, Tom.
livr. v.,
1886,
iv.,
p.
88 et Tom.
ii.,
1888, p. 137.)
JoRGENSEN, Alfr.
mit
livr.
ii.,
361
Vorlaufiger Bericht iiber Versuche
absolut
dargestellt.
nach
Oberhefe,
reiner
(Allg. Zeitschr.
Hansen's
im grossen
Methode
Bierbr. u, Malzfabp.,. 1885,
f.
No. 36.)
This has a special interest as
it is
the
first
communication on the
application of pure yeast species in top fermentation.
Anwendung der nach Hansen's Methode
Die
reingeziich-
teten obergarigen Hefe in der Praxis.
(Brauer
Kalender
Stuttgart, 1889-90.)
f.
Deutschland
u. Osterreich.
Referat von Will hi Zeitschr.
shown here that
It is
f.
d. ges.
Malzer-
u.
Brauw., 1890.
in certain cases a special aeration of the wort
can hasten the clearing.
Analyse der obergarigen Hefe in Brauereien und
Ziir
Brennereien nach Hansen's Methode.
(Zeitschr.
d. ges.
f.
Brauw., 1891.)
Uber
die
Anwendung von Hansen's System
garungsbrauereien.
On Hansen's System
Top Fermentation.
vii.,
Ober-
in
{Ibid., 1893.)
of
Pure Yeast Culture in English
(Trans, of the Inst, of Brewing.
Vol.
1894.)
In the above two communications the same subject is treated but

from different standpoints. A special interest attaches to the communication of 1893.
JoRGENSEN, Alfr.,
pour
et
HoLM,
Chr.
J.
Le proc6de dc M. EfFront
la purification et la conservation de la levure a I'acide
fluorhydrique
et
des
4 S6r.
Quesneville.
(Monit.
fluorures.
Tom.
vii.,
1893.
scient.
Zeitschr.
f.
du Dr.
d. ges.
Brauw., 1893.)
KooH,
R.
(Mitt,
Untersuchung von
Zur
aus
dem
Kaiserl.
pathogenen
Organismen.
Gesundheitsamte, Bd.
i.,
Berlin,
1881.)
Fundamental
investigations for the technique as regards the use
of nutrient gelatine for cultivating micro-organisms.
Miquel: Dixi6me m^moire sur

et des eaux.
les poussi6res organisês
(Annuaire de Montsouris.
Of importance as regards the methods
of
de
I'air
Paris, 1888.)
examining
air
and water.
362
Muller-Thuroau,
H.
Neue
Gebiete der Weingariing

Praxis.
(Ber.
iiber
d.
Forschungsresultate
Verh.
d.
deutsch.
xi.
deia
auf
und deren Bedeutung
die
fiir
Weinbau-
Kongresses in Trier, 1889.)
dem
Ergebnisse neuer Untersuchungen auf

(Ber. iiber d. Verh. d.
Weinbereitung.
Gebiete der
Wein-
deutsch.
xii.
bau-Kongresses, 1891.)
Anwendung
IJber neuere Erfahrungen bei

in der Weinbereitung.
Pasteur, L.
der Reinhefen
Mainz, 1896.
Paris, 1876.
jfetudes sur la bi6re.
Of the extremely rich and varied contents the following are

specially important for this section
flasks (pp. 27, 29, 88, 328
(pp. 29
and
and
332),
and
Descriptions of various culture
331), sterilisation of nutrient solutions
composition of
nutrient liquids (pp.
artificial
89-
319), etc.
ScHioNNiNG,
H.
Matras pour cultures sur blocs de
platre.
(Compt. rend, des travaux du laborat. de Carlsberg,

iv., livr. ii.,
W.
Sbifbrt,
1896,
Verwendung von Reinzuchthefen

(Osterr. landwirtsch.
schaft.
WicHMANN, H.
Neuere
osterr. Vers. -Stat.
Tom.
p. 89.)
f.
in der Kellerwirt-
Wochenbl., 1897.)
Hefereinzucht-Apparate.
Brauerei
u.
(Mitt.
Malzerei in Wien, Heft
d.
6,.
1894.)
Will, H.
ges.
Wie wird
reine
Hefe geziichtet
(Zeitschrift
f.
d.
Brauw., 1885.)
Notiz betr.
den Nachweis von
wildeu
Brauereihefen und Jungbieren, sowie das

Sacch. apiculatus in denselben.
1.
in
[Ibid., 1893.)
Zur Untersuchung hefentriiber

iiber Lebensmittel.
Hefearten
Vorkommen von
Biere.
(Forschungsber.
Jahrg., Heft 10, 1894.)
Die Methoden, welche bei der Reinziichtung von Heffr
und ahnlichen Organismen durch Einzellkultnr auf festem

Nahrboden zur Feststellung der Lage der ausgewahlten
Zellen in den Kulturen zur
tralbl.
f.
Anwendung kommen.
Bakt. u. Par., 2 Abt., Bd.
ii.,
1896.)
(Cen-
LITERATURE REVIEW
Will, H.
363
Einige Bemerkungen iiber die Lebensdauer getrockneter
Hefe.
(Zeitschr.
found
ges. Brauw.,
d.
f.
Addenda are
1896.
1897, 1898 and 1899.)
ibid.,
Einiges aus der Praxis des physiologischen Laboratoriunis.

(Zeitschr.
WoRTMANN,
d. gea.
f.
J.
Brauw., 1899.)
Untersuchungen
wirtsch. Jahrb. 1892
iiber reine
Hefen
i., ii.
(Land-
and 1894.)
Uber die Verwendung von reinen AVeinhefen bei der

(Weinbau und Weinhandel, 1893.)
Apfelweinbereitung.
Uber
Anwendung von
die
Schaumweinbereitung.
rein geziichteten
Mitteilung iiber die Verwendung von
Most
fiir
Pilzkulturen.
Hefen bei der
[Ibid.)
(Botan. Zeit., Bd.
konzentriertem
li.,
1893.)
Die Verwendung und Bedeutung reiner Hefen bei der

Weinbereitung.
bau vereins
(Ber.
iiber die
Verb.
deutsch.
d.
Wein-
Neuenahr, 1893.)
in
Anwendung und Wirkung
reiner
Hefen in der Wein-
Berlin, 1895.
bereitung.
Comprehensive review and guide
for practical
men.
Uber Fehler, welcbe bei Anwendung von Reinhefen

(Ber. iiber d. Verhandl. des xvii.
gemacht wurden.
deutschen Weinbau-Kongresses in Trier, 1898.)
Untersuchungen iiber das Umschlagen
Section
1.
Db Bary,
a.
III.
Text-Books, Handbooks
and Journals.
Vergleichende Morphologie und Biologie der Pilze,
Chimie biologique,
JoRGENSEN, Alph.
4. Aufl.
Weine.
(Pages 170 to 345).
Mycetozoen und Bacterien.
DucLAUx, E.
der
Leipzig, 1884.
Paris, 1883.
Die Mikroorganismen der Garungsindustrie.
Berlin, 1898.
364
Lindner, P.
Mikroskopische Betriebskontrolle in den Garungs-
gewerben.
Mayer,
Adolf
Garungschemie.
Die
Berlin, 1898.
Aufl.
2.
4.
Aufl.
Heidelberg,
1895.
Prior, E.
zig,
ZoPF,
Leip-
Chetnie u. Physiologie des Maizes u. des Bieres.
1896.
W.
Die Pilze in morphologischer, physiologischer, bio-
logischer
Centralblatt
fiir
Bd.
iv.)
und Infektipns-
Herausg. von Oscar Uhlworm und
2 Abt.
Emil Chr. Hansen.
der
Breslau, 1890.
Bakteriologie, Parasitenkunde
krankheiten.
(Handb.
Beziehung.
systematischer
iind
Botanik, herausg. von Schenk.
Jena, 1895
f.
Jahresbericht iiber die Fortschritte in der Lehre von den Garungs-
Herausg. von Alfr. Koch.
organismen.
2.
Aderhold, R.
Braunschweig, 1891.
Works on True Fungi.
Special
Uber die Morphologie deutscher Weinheferassen.

f. Obst-, Wein- u. Gartenbau
(Ber. der Konigl. Lehranstalt
zu Geisenheim
a.
Rh.
f.
1893-94.)
Die Morphologie der deutschen

soideus-Rassen.
Amthor, C.
Studien iiber reine Hefen.
Chemie.
Bd.
Uber
den
xii..
Heft
1 u. 2,
Saccharomyces
physiolog. Chemie.
Bainier, G.
Saccharomyces
(Landwirtsch. Jahrb., Heft
Bd.
xii.,
4,
(Zeitschrift
ellip-
1894.)
fiir
physiolog.
1888.)
apiculatus.
Heft
6,
(Zeitschr.
f.
1888.)
Observations sur les Mucorin6es et sur les zygo-
spores des Mucorinees.
(Ann.
d. Sc. nat. Bot., 6. Ser.
Tom.
XV., 1883.)
Nouvelles observations sur

{Ibid.
Tom.
DB Bary, a.
Pilze, III.
xix.,
les
zygospores des Mucorinees.
1884.)
Beitrage zur Morphologie
und Physiologie det
Frankfurt, 1870.
DE Baby und Woronin
Zur Kenntnis der Mucorineen.
1866.
LITERATURE REVIEW
Bau, a.
Der Sammelbegrifi' Sacoharomyces
chenschr.
Bbhrens,
J.
Die Infektionskrankheiten des Weiiies.
(Wo-
cerevisiae.
Brauerei, 1894, No. 43.)
f.
Bakt. u. Par.,
f.
365
2.
Abt., Bd.
ii.,
1896, Nos.
Beitrage zur Kenntnis der Obstfaulnis.
(Centralbl.
6, 7.)
Bd.
{Ibid.
iv.
,.
1898.)
W.
Befjerinck, M.
Die Lactase, ein neues Enzjm.
(Centralbl.
Bakt. u. Par,, 1889.)
f.
Schizosaccharomyces octosporus, einejachtsporige Alkohol{Ibid., 2. Abt., 1894.)
hefe.
Weitere Beobachtungen iiber die Octosporushefe.
{Ibid.^
1897.)
Bbnecke, W.
Die zur Ernahrung der Schimmelpilze uotwendigen
Metalle.
(Jabrb.
f.
wissensch. Botanik, Bd. xxviii., Heft 3,
1895.)
Die Bedeutuug des Kaliums und des Magniums Mr

Entwicklung und Wachstum des Aspergillus niger, v. Th.,
sowie einiger anderer Pilzformen. (Botan. Ztg., Heft 6, 1896.)
Brefbld, 0.
I.,
1872
Botanische Untersuchungen iiber Schimmelpilze.
II.,
1874.
Of special interest in part
and
in part
2,
the
life
tfber Giirung.
Brown, A.
J.
Yeast Cells.
No.
are the researches on
Mucor
Mxtcedo,
history of Penicillium.
(Landw. Jabrb., 1874,
187-5, 1876.)
Some Experiments on the Numerical
Increase of
(Trans, of the Laboratory Club, Vol.
iii.,
1890,
4.)
The
Yeast
Specific Character of the Fermentative Functions of
Cells.
(Trans, of the
Fermentative Power.
Duclaux.
(Centralbl.
1897, Nos.
2, 3.)
An Answer
to Criticism
Bakt. u. Par.,
2.
by M. E.
Abt., Bd.
Oxygen on Alcoholic Fermentation.

Chem. Soc, 1892, Nr. 107.)
Influence of
of the
f.
Chem. Soc, 1894.)
iii.,
(Trans.
366
BucHNER, E.
Fortschritte in der Chetnie der Garung.
Tiibingen^
1897.
Besides the above communication, Buchner has published, sometimes by himself, sometimes in conjunction with Kapp, a series of
special researches in " Ber. d. deutsoh. chem. Ges.," 1897, 1898 and
1899.
Calmette: La levure
(Ann. de
chinoise.
I'Inst. Past.,
Tom.
vi.,
1892.)
Casagrandi, 0.
Uber
(Centralblatt
f.
die
Morphologie
der
Blastomyceten.
Bakt., Par. u. Inf., 2. Abt.', Bd.'iii., 1897, Nos.
21,22.)
Chamberland, Ch.
Recherches sur I'origine et
des organismes microscopiques.
CiENKOWSKi
Die Pilze der Kahmbaut.
le
developpement
Paris, 1879.
(Bull, de I'Acad. imp6r.
des Sc. de St. P6tersbourg, Vol. xvii., 1873.)
EiJKMAN
Mikrobiologisches iiber die Arrakfabrikation in Batavia.
(Centralbl.
Engbl
f.
Bakt. u. Par., Bd. xvi., 1894, No. 3.)

Paris, 1872.
Les ferments alcooliques.
Leo
EkiRERA,
Sur I'existence du glycogene dans
la
levdre de
(Compt. rend, de I'Acad. des Sc, 1885.)
bifere.
In connection with the above, Laurent (Annal. de I'Inst. Past.,

and Clautriau (Mem. publ. par I'Acad. royale de Belgique,
1895) have published later communications on glycogen in yeast.
One of P. Lindner's papers mentioned below is also on this subject.
1889)
Fischer, Emil
tung
fiir
Die Chemie der Kohlenhydrate und ihre Bedeu-
die Physiologie.
Berlin, 1894.
Einfluss der Konfiguration auf die

II.
(Ber. d. deutsch.
chem. Ges., Bd.
Wirkung der Enzyme

xxvii., 1894.)
Fischer, besides the above, has published several papers in con-
junction with P. Lindner and H. Thierfelder in "Ber. d. deutsch.
chem. Ges.," 1894 and 1895, on the enzymes contained by saccharomycetes, and on the behaviour
GiLTAY, E.
the latter to the difierent sugars.
of
Pasteur und die alkoholische Garung.
wissensch. Bot., Bd. xxx., Heft
GiLTAY,
E.,
und Aberson,
stoffzutrittes auf
J.
H.
1,
(Jahrb.
f.
1895.)
Uber den
Einfluss des Sauer-
Alkohol und Kohlensaurebildung bei der
LITERATURE REVIEW
alkoholischen Garung.
(Jahrb.
f.
367
wissenscli. Bot., Bd. xxvi.,
1894.)
Hansen,
Emil
Chr.
Contributions
connaissance
la
organismes qui peuvent se trouver dans la bi6re et
de bi6re et y vivre.
de Carlsberg, Tom.
Describes a large
des
mout
le
i.,
livr.
number
1879.)
ii.,
micro-organisms which, belong to the

various divisions oi the system gives systematic descriptions and
morphological and physiological investigations. (Communications
on the vinegar bacteria are to be found in the literature review of
of
bacteria.)
Recherches sur
les
organismes qui, a diff6reutes 6poques
de I'annê, se trouvent dans

tours, et qui
I'air,
a Carlsberg et
peuvent se developper dans
le
aux
modt de
alen-
hikre.
(Ibid.)
Sur
dans
le
que Tintroduction de
I'influence
mout
HypothSse de Horvath.
Sur
{Ibid.,
le
I'air
atmosph6rique
qui fermente exerce sur la fermentation.
(Ibid.)
(Ibid.)
Sacch. apiculatus et sa circulation dans la nature.
Tom.
i.,
livr.
1881.)
iii.,
The first communication on this subject was published by Hansen

in " Hedwigia " in 1880. The researches on the circulation of true
saccharomycetes in nature are begun here and in the above paper
on the organisms
of the air.
Recherches sur
les
organismes qui a differentes 6poques
de I'annee se trouvent dans

et qui
peuvent
Memoire.
I'air,
se d6velopper
{Ibid.,
Tom.
i.,
a Carlsberg et aux alentours,
dans
livr. iv.,
le
mofit de bi6re.
2i^nie
1882.)
Contains not only researches on the circulation of various microorganisms in nature, but also gives in the concluding section special
on several species {Dematium pullulans and the allied

lactis, Chalara mycoderma, and various Saccharomyces,
among them one species which gives a bitter taste to beer).
investigations
forms, O'idium
Les ascospores chez
le
Tom. ii., livr. ii., 1883.)

On p. 13 are given the essentials
of what was known at that time

formation in the saccharomycetes, also a full bibliography.
23 and following, Hansen's spore cultivation method and his
of spore
On
p.
{Ibid.,
368
pure culture method for mass cultures of saoeharomycetes are described.

On p. 31 experiments are described, and with them are
given spore curves for the six species Sacch. cerevisice I., S. Pastori'
:
anus
I., II.
and
III., S. ellipsoideus I.
and
tion of temperature to spore formation
Hansen, Emil Chr.
Sur
In addition, the rela-
II.
treated in general.
Torulas de M. Pasteur.
les
Neue Untersuchungen
is
(Ibid.)
iiber Alkoholgarungspilze.
(Ber.
der deutsch. botan. Ges., 1884.)

Researches on Monilia Candida.
Vorlaufige
Mitteilungen iiber
Garungspilze.
(Botan.
Centralbl., Bd. xxi., 1885, No. 6.)
The
gelatinous network, coalescence of spore walls, septum forma-
tion, the
behaviour of Sacch. apiculatus under the action of sunlight.
Les voiles chez

des travaux
dii
le
laborat.
(Compt. rend.
de Carlsberg, Tom.
livr.
ii.,
iv.,
1886.)
In addition to the investigations indicated by the
observations on the cell nucleus of saccharomycetes
new
researches on the gelatinous network
title
there are
125),
(p.
and
(p. 126).
Noch eiu Wort iiber den Einfluss der Kohlensaure ûf

Garung und Hefebildung.
(Zcitschr. f. d. ges. Brauw.,
1887, No. 13.)
Neue Bemerkungeu zu Foth's Abhandlung

der Kohlensaure auf Garung und Hefebildung "
schr.
f.
" Einfluss
(Wochen-
Brauerei, 1887.)
Action des ferments alcooliques sur les
di verses esp6ces
(Compt. rend, des travaux du laborat. de CarlsI
de Sucre.
berg,
Tom.
ii.,
livr. v.,
Also descriptions of
new
1888.)
peculiar species.
Untersuchungen aus der Praxis der Garungsindustrie.

(Beitriige zur Lebensgeschichte der
Heft,
1.
Aufl.,
1888
3. Aufl.,
As regards the contents, see

tjber die
1895
Mikroorganismen.)
;
2.
1.
Heft, 1892.
p. 360.
im Schleimflusse lebender Baume beobachteten
Mikroorganismen.
(Centralbl.
f.
Bakt.
u.
Par.,
Bd.
v.,
1889.)
First investigations on asporogenous varieties of Saccharomyces are
also given here.
LITERATURE REVIEW
869
Hansen, Emil Chr. Nouvelles recherches sur la circulation du

Saccharomyces apiciilatus datis la nature. (Ann. d. sc. nat.
Bot., 7 Ser., Tom. xi., No. 3, 1890.
Ann. de Micrographie,
:
1890.)
Contains also researches on the circulation
Production des varietes chez
de Micrographie, Tom.
Sur
la
ii.,
oi true
saccharomycetes.
(Ann.
Saccharomyces.
les
1890, No.
5.)
germination des spores chez
Saccharomyces.
les
(Compt. rend, des travaiix du laborat. de Carlsberg, Tom.

livr.
iii.,
i.,
1891.)
Kritische Untersuchungen iiber einige von
Ludwig und
beschriebene Oidium- und Hefenformen.
Brefeld
(Botan.
Zeituug, 1892, No. 19.)
Uber
die
streichen.
No.
Bakt. u.
f.
Par.,
Bd.
1893,
xiii.,
1.)
Uber
f.
neucn Versuche, das Genus Saccharomyces zu
(Centralbl.
d. ges.
kiinstliche
und
natiirliche Hefereinzucht. (Zeitschr.
Brauw., 1895, No. 14.)
Experimental Studies on the Variation of Yeast

(Annals of Botany, Vol.
ix.,
Cells.
189.5.)
Anlasslich Juhler's Mitteihaig iiber einen saccharomyces-
bildenden Aspergillus.
Abt., Bd.
2.
i.,
(Centralbl.
fiir
Bakt., Par.
u.
Inf.,
189.5.)
Biologische Untersuchuugen iiber Mist bewohnende Pilze.
(Botan. Ztg., Heft 7, 1897.)

Besides biological researches on Coprini, the
opsis stercoraria, explanation of its life limits
life
history of Anixi-
and contributions
to
the general biology of the reproductive organs.
Sur
dans
la vitality
les
des ferments alcooliques et leur variation
milieux nutritifs et a I'^tat
sec.
travaux du laborat. dc Carlsberg, Tom.
Neue Untersuchungen
Saccharomyceten
Bd.
v.,
1899, No.
iiber die
(Centralbl.
1.)
24
f.
(Compt. rend, des

iv., livr.
iii.,
1898.)
Sporenbilduug bei den
Bakt., Par. u. Inf., 2. Abt.,
370
Hansen, Emil Chr.
La variation des Saccharomyces. (Compt.

du laborat. de Carlsberg, Tom. v., livr.
rend, des travaux
1900.)
i.,
Contains a review of the experiments described in the above preliminary communication, and new contributions are added. This
paper
is
it
contains a description of the
for the first
time that variations such as
especially important because
methods, and
it is
shown
those described arise from

generally supposed, from
o,
transformation, and not, as had been
a,
Further, the factors active in
selection.
producing the transformation are determined.
La spore de Saccharomyces devemie sporange.

Tom. v., livr. ii., 1902.)
[Ibid.,
de
la crois-
Recherches comparatives sur

sance vegetative et
le
moisissures de la fermentation
duction des levures et des

alcoolique.
Holm,
Chr.
J.
Tom.
[Ibid.,
les conditions
d^veloppement des organes de reprov., livr.
1902.)
ii.,
uber Oberhefe
Altere und neuere Ansichten
(Deutscher Brauer- und Malzer-Kalender,
and Unterhefe.
1886-87.)
Bemerkung zu Foth's Abhandlung
den Einfluss der
iiber
Kohlensaure auf die Garthiitigkeit der Hefe.
Bemerkungen
Einige
f.
(Zeitschr.
biiden.
f.
d. ges.
Mitteilung
der
anlasslich
Wachstum der Hefen
Lindner's iiber das
HiJCKBL
(Zeitschr.
d.
Brauw., 1889, No. 15.)
ges.
Brauw., Bd.
xvi.,
1893.)
Znr Kenntnis der Biologie des Mucor corymbifer.
P.
auf fasten Niihr-
Jena,
1885.
Janczewski, E.
ses
Recherches sur
compagnons habituels sur
Jaxssens, Fr. a.
zelle.
f.
Bakt.
u. Par.,
Janssbns, Fr. A., et Leblanc, A.

cellule de leviire.
studerede.
Nr.
2.)
O.
et
Krakowie, 1894.
les cer6ales.
Beitrage zu der Frage iiber den Kern der Hefe-
(Centralbl.
Johan-Olsen,
le
Bd.
1893, No. 20.)
Recherches cytologiques sur
(La Cellule, Tom.
Norske
xiii.,
xiv., fasc.
aspergillusarter
i.,
la
1898.)
udviklingshistorisk
(Christiania Videnskabs-Selskabs
Forh.,
1886,
LITERATURE REVIEW
JoEGENSEN, Alfr.
371
Vorliiufiger Bericht iiber Versuche im Grossen

mit absolut reiner Oberhefe, nach Hansen's Methode darge:
Zeitschr.
(Allg.
stellt.
und
Bierbr.
f.
Malzfabr.,
1885,
Nr. 36.)
Jdrgensen has continued his researches on cultivated top-yeast
and their use in
species
The papers
practice.
relating to this are
cited on p. 361.
Der Ursprung der Weinhefen.

Abt, 1895.)
(Central bl.
f.
Bakt., Par.
u. Inf., 2.
tlber den
Ursprung der Alkoholhefen.
(Berichte
garungsphys. Laboratoriums von Alfr. Jorgensen.
des
Kopen-
I.
hagen, 1895.)
Uber Pilze, welche tlbergangsfornien zwischen Schimmel

und Saccharomyceshefe bilden und die in der Brauereiwiirze
(Zymotek.
auftreten.
Centralbl.
f.
Kjobenhavn,
Tidsskrift.
1896.
Bakt., Par. u. Inf., 2. Abt., 1896.)
Die Hefenfrage.
(Centralbl.
f.
Bakt., Par. u. Inf.,
2.
Abt.,
1898.)
Jorgensen, Ai.fr.
pour
et
Holm,
J.
Chr.
Le precede de M. Effront
la purification et la conservation
de la levure a
I'acide fluorhydrique et des fluorures.
Quesneville.
4. S6r.
Tom.
1893.
vii.,
I'aide
de
(Monit. scient.
du Dr.
d. ges.
Zeitschr.
f.
Brauw., 1893.)
JuHLER,
J.
Umbildung
(Centralbl.
ceten.
eines Aspergillus in einen

f.
Bakt.,
Par.
u.
Inf.,
2.
SaccharomyAbt.,
1895,
S. 16.)
second communication on the same subject,
Klbbs, Georg
Die Bedingungen der Fortpflanzung bei eiuigeu
Algen und
Pilzen.
Jena, 1896.
Only the section on fungi,
Klocker, Alb.
ibid., p. 326.
p. 446, is of interest here.
Recherches sur
les
Saccharomyces Marxianus,
Sacch. apiculatus et Sacch. anomalus.
(Compt. rend, des
travaux du laborat. de Carlsberg, Tom.
iv.,
La formation d'euzymes dans

peut-elle
livr.
i.,
servir
caracteriser
1900.)
24*
les
livr.
i.,
1895.)
ferments alcooliques
I'espece
{Jbid.,
Tom.
v.,
372
Klucker, Alb.
1st die
Enzymbildung
bei
zen ein verwerthbares Artmerkmal

u. Inf., 2. Abt.,
Bd.
1900, No.
vi.,
Eine neue Saccharomyoesart

eigentiimliohen Sporen.
den Alkoholgarungspil-
(Centralbl.
Saturnus, mihi) mit
(Saccli.
Bd.
[Ibid.,
Bakt., Par.
f.
8.)
viii.,
1902, No. 5.)
Experimentelle UnterKlocker, Alb., and Schionning, H.

suchungen uber die vermeintliche Umbildung des Aspergillus
;
oryzse in einen Saccharomyceten.

n. Inf., 2. Abt., Bd.
i.,
Experimentelle
Umbildung
meintliche
Saccharomyceten.
(Centralbl.
Bakt., Par.
f.
1895, Nos. 22, 23.)
Untersuchungen
{Ibid.,
Bd.
ii.,
Que savons-nous de
die
iiber
Schimmelpilze
verschiedener
1896, Nos.
I'origine des
verin
6, 7.)
Saccharomyces
(Compt. rend, des truvaux du laborat. de Carlsberg, Tom.

iv., livr. ii.,
1896.)
Noch einmal Saccharomyces und Schimmelpilze.

(Centralbl.
f.
Bakt., Par
u.
Inf., 2.
Abt., Bd.
iv.,
1898, Nr.
11.)
Uber Durchwachsungen und abnorme Konidien-
bildungen bei Dematium pullulans de Bary und bei anderen

Pilzen.
{Ibid.,
Bd.
v.,
1899, Nr. 14.)
Phenomenes d'accroissement perforant et de forle Dematium pullulans, de
mation auomale des conidies chez
Kny,
Bary, et autres champignons.
(Compt. rend, des travaux
du
v.,
L.
;
KoRFF,
livr.
i.,
1900.)
Die Beziehungen des Lichtes zur Zellteilung bei Sac-
charomyces
Heft
Tom.
3,
cerevisioe.
(Ber. d. deutsch. bot. Ges., Bd.
ii..
1884.)
Einfluss des SauerstofTs auf Garung, Garungsenergie
und Vermehrungsvermogen verschiedener Heferassen

verschiedenen
Erniihrungsbedingungen.
(Bayr.
uiiter
Brauer-
Journ., 1899, Nr. 36 u. fF. Inaug.-Dissert., Jena, 1899.)
KiJSTER, E.
Bd.
xviii.,
Zur Kenntnis der Bierhefe.

1898, No. 9.)
(Biolog.
Centralbl.,
LITERATURE REVIEW
Van Laer
Notice sur une levure a attenuation-
tres-61evee.
Tom.
4. S6r.,
Lafar, F.
Denamur
et
limite
(Moiiit.
soientif.
Quesneville,
Dr.
d.
xlv., livr. dcxliii., 1895.)
Uber einen
bildet.
373
(Centralbl.
Sprosspilz,
f.
welcher
Essigsaure
kriiftig
Bakt. u. Par., Bd.
1893.)
xiii.,
Studien iiber den Einflxiss organischer Sauren auf Eintritt
mid Verlauf der Alkoholgaruug.

(Landw. Jahrb.,
Essigsaure.
LiXDNEB, P.
Die Weinhefeu and die
I.
189.5.)
Uber Durchwachsungen an
deutsch. bot. Ges., Bd.
Uber
rot
v.,
Pilzmycelien.
(Ber. d.
1887.)
und schwarz gefarbte
(Wochensclir.
Sprosspilze.
f.
Das Langwerden der Wiirze durch Dematium pullulans.

1888, Nr.
(Ibid.,
1.5.)
Die Ursache des langen Weissbieres.
(//;irf.,
Schizosaccharomyces Pombe
ein neuer
erreger.
n.
sp.,
1889, Nr. 9.)
Garungs-
{Ibid., 1893).
Sacoharomyces farinosus
u. Bailii.
(Ibid., 1894.)
Fruchtatherbildung durch Hefen in Griinmalz und in

Wiirzen.
{Ibid., 1896.)
Beobachtungen
iiber die Sporen-
und Glykogenbildung
Die Blaufarbuug der

Hefen auf Wiirzegelatine.
Sporen von Schizosaccharomyces octosporus durch Jodlosung.
einiger
(Centralbl.
LiNTNEK, C.
tralbl.
LoEW, E.
f.
J.
f.
2.
Abt., Bd.
ijber
Dematium
wissensch. Bot., Bd.
LoHMANN, W.
1896.)
Studien iiber die Selbstgarung der Hefe.
Bakt., Par. u. Inf., 2. Abt., Bd.
f.
ii.,
pullulans.
v.,
(Cen-
1899, Nr. 23.)
(Pringsheinis Jahrbiicher
vi.)
iJber den Einfluss des intensiven Lichtes auf die
Zellteilung bei Sacch. cerevisice und anderen Hefen.
Ros-
tock, 1896.
LoPRioRE, G.
Bd.
xxiii.,
Die Schwarze des Getreides.
(Landw. Jahrb.,
1894.)
Cladosporium herbarum and Dematium pullulans.
374
LoTT
Experiments on the Apparent Effect of Mould on Malt, as

shown in the Composition of the Extract and Beer brewed
from Mouldy Malt. (.lonrn. of the Feder. Instit. of Brewing,
:
Vol.
1899, Nr.
v.,
Meissner,
R.
1.)
Studien
das
iiber
Zahewerdeu von Most und
(Landw. Jahrb., 1898.)
Wein.
Torula species.
MnLLBR-THURGAU,
H.
Die
Edelfanle der
(Landw.
Trauben.
Jahrb., 1888.)
Botrytis cinerea,
Weitere Untersuchungen iiber die Physiologie der Hefe
und
Bedeutung
die
Heferassen
fiir
ausgewahlter
Weingarung.
die
und
reingeziichteter
(Schweiz.
Zeitschr.
f.
Obst- und Weinbau, 1894.)

Other papers by Miiller-Thurgau are mentioned on p. 362.
Nielsen,
Chb.
J.
Sur
developpement des spores du Sacch.
le
membrantefaciens, du Sacch. Ludwigii et du Sacch. anomalus.
(Compt. rend, des travaux du luborat. de Carlsberg, Tom.

livr.
iii.,
Pasteur, L.
1894.)
iii.,
M^moire sur
chim. et phys., Tom.
la
fermentation alcoolique.
Iviii.,
(Ann. de
1860.)
Etudes sur
le vin.
Note sur
fermentation des fruits et sur la diffusion des
germes des
Sc, Tom.
la
leviires alcooliques.
Ixxxiii.,
Etudes sur
Examen
Paris, 1866.
la bifere.
Paris, 1876.
critique d'un ecrit posthurne de Claude
sur la fermentation alcoolique.
Pedbrsen, R.
(Compt. rend, de lAcad. des
1876.)
Recherches sur quelques facteurs qui
I'influence sur la propagation de la levftre basse
Tom.
Sur
dans
le
i.,
livr. i.,
I'influence
(Zeitschr.
de
1878.)
que rintroductiou de
modt qui fermente
Petersen, A.
ont
du Sacch.
(Compt. rend, des travaux du laborat. de Carls-
cerevisise.
berg,
Bernard
Paris, 1878.
I'air
atmosph^rique
exerce sur la fermentation.
{Ibid.)
Einige Bemerkungeu iiber das Liiften der Wiirze.

f.
d. ges.
Brauw., 1888.)
LITERATURE REVIEW
Prior,
E.
Physikalisch-chemische
soheiniingen.
375
Erkliirung der Giirungsev-
(Bayr. Brauer-Journ., Jahrg.
Erklarung der Ganingserscheinungen.
1895.)
v.,
{Ibid.)
Siud die Hefen Frohberg und Saaz der Berliner Brauerei-
versiichsstation Hefetypen
Uber
im physiologischen Sinne?
Menge und Natur
die
{Ibid.)
der bei der Vergarung von
Bierwiirzen vermittelst veschiedener Heferassen gebildeten

Siiiiren.
{Ibid.)
dem Leben
Die Beziehuiigen des osmotischen Druckes zu

der Hefe und den Garungserscheinangen.
{Ibid.,
Centralbl.
ii.,
Rapp, R.
Bakt., Far. u. Inf.,
f.
2.
Einfliiss des Sauerstoft'es auf
Abt., Bd.
1896.
1896.)
garende Hefe.
(Ber. der
deutsch. chem. Ges., Bd. xxix., 1896.)
Raymann and Kruis
Chemisch-biologische Studieu.
(Mitt. d. Versuchsstat.
f.
1.
u.
II.
Spiritusind. in Prag, 1892 u. 1895.)
On
fermentation products evolved by saccliaromycetes, on the

tlie alcohol formed by films of the saccharomycetes, and
on the formation of amyl alcohol by saccharomycetes.
oxidation of
Reess, M.
Botanische Untersuchungeu iiber die AlkoholgarungsLeipzig, 1870.
pilze.
RoTHBNBACH, F.
niyces
Die Dextrin vergarende Hefe, Schizosaocharo-
Pombe und
(Zeitschr.
f.
ihre eventuelle Einfiihrung in die Praxis.
Spiritusind., 1896.)
Salamon, a. Gordon Cantor Lectures on Yeast

and Culture. London, 1888.
:
ScHiEWBK, 0.
its
Morphology
Uber Sak6, das Nationalgetrank der Japaner, und

(Beilage zum
Bereitung wirksamen Pilze.
die bei seiner
Jahresberioht
der
evangelischen
Realschule
I.)
Breslau,
1897.
Uber neue Erfahrungen auf dem Gebiet der Sakebereitung, 1898.
ScHioNNiNG, H.
leviire.
Tom.
Nouvelle et singulifere formation d'ascus dans une
(Compt. rend, des travaux du laborat. de Carlsberg,
iv., livr. i.,
See also Klocker.
1895.)
H76
ScHMiTZ, F.
d.
Uber
die Zellkerue der Thallophyten.
Niederrhein. Ges., 1879,
W.
Seifert,
"
Weinlaube
.Seiter, 0.
Klosterneuburg,
Schizosaccharomyces octosporus.
iiber
Erlangen, 1896.
(Abstract in
Nr. 13, and in:
Centralbl.
1896, Nos.
ii.,
de Seynek,
J.
Sur
des So., Tom.
V.
1868.
(Wochenschr.
Bary.
2.
Abt.,
11.)
mycodernia
Ixvii.,
Bayer. Brauer-Journ., 1896,

(Compt. rend, de I'Acad.
viui.
Ann.
d.
nat. Bot., 10. S6r.,
so.
Dematium
Beitrage zur Kenntnis des
.Skebst
und
le
9, 10,
f.
1869.)
v.,
SoLDAN, G.
(Also in
Studien liber die Abstainmung der Saccharomyceten
Tom.
1897.
".)
und Untersiichungen
Bd.
Geschiclitliche Dar-
tjber den Urspruug der Hefe.
stellung der Hefefrage.
(Sitzvmgsber.
Aug.)
4.
pullulans, de
f.
Die (xesamtarbeitsleiatung der Hefen Saaz, Frohberg
Logos
Saccharose-,
in
und Maltoselosung
und Ernahrungabedingungen.
Dextrose-
luiter verscbiedenen Versuchs-
(Bayer. Brauer-Journ., 1898, Nr. 47
VAX Tibghem
ff.)
Nouvelles reoherches sur les Mucorinees.
d. so. nat., G. Ser.,
Tom.
!.,
Troisieme m^moire sur
(Ann.
1875.)
les
Mucorinees.
[Ibid.,
Tom.
iv.,
1878.)
VAX TiEGHEM ct Le Moxniek

{Ihid.,
Ser.,
.5.
VuYLSTBKE,
J.
Mischsaaten
Tom.
xvii.,
Recherches sur
Ein
Beitrag
von
Saccharomyceten.
zur
Vol.
The Nucleus
xii.,
of the
Mucorinees.
Entwicklungsgeschichte der
Brauw., 1888, Nr. 24; 1889, Nr.
Wager, H.
les
1873.)
(Zeitschr.
f.
d.
ges.
1.)
Yeast Plant.
(Annals of Botany,
1898, No. 48.)
Ward, H. Marshall
Composing
it.
The Ginger-beer
(Pliil.
I'lant
and the Organisms
Trans, of the Royal Soc. of London,
Vol. cl-xxxiii., 1892.)
Wehmeb,
C.
Beitrage zur Keuntuis einheimischer Pilze, Heft
Jena, 1895.)
Penicillhim,
Mucor and
Botri/tis.
2,
LITERATURE REVIEW
AVehmer,
C.
Uber
(Chem.
377
die Verfliissiguug der Gelatine durch
Pilze.
1895, No. 91.)
Ztg.,
Aspergillus Oryzfc, der Pilz der japanisclien Sak^brauerei.

(Centralbl.
f.
Abt., Bd.
2.
i.,
189.5.)
Sak^brauerei und Pilzverzuckerung.
{Ibid.)
Uber
(Botan. Centralbl.,
einige neue Aspergillus- Arten.
1899, No. 51.)
Weleminsky,
F.
de Bary.
Nr.
Went,
Uber Sporenbildung
(Centralbl.
f.
bei
Dematium
2.
pullulans,
Abt., 1899,
9.)
C, and Prinsbn Gebrligs, H.
F. A. F.
C.
Beobachtungen
uber die Hefearteu und zuckerbildenden Pilze der Arrak-
Amsterdam, 1895.
fabrikation.
Akad.
Wetensch.
V.
WiLHELM, K.
te
Beitriige zur
(Also in Verh. d. koninkl.
Amsterdam,
2. Ser.,
Tom.
iv.,
Nr.
2.)
Kenntnis der Pilzgattung Aspergillus.
Berlin, 1877.
Will, H.
Uber Sporen- und Kahmhautbildung
(Zeitsohrift
f.
Uber das
{Ibid.,
eiiie
Geschmack
Vorkommen von Sporenbildung
iu
No. 17.)
wilde Hefe, welche
giebt.
bei Unterhefe.
Brauw., 1887, No. 16.)
natiirliche
Brauereien.
iJber
d. ges.
dam
Biere einen bittereu
(Ber. d. wissensch. Stat. Miinchen, 1889.)
Zwei Hefearten, welche abnorme Veranderungeu im Bier

veranlassen.
tjber die
(Zeitschr.
Wirkung
f.
d. ges.
Brauw., 1891, Nr.
7, 8.)
einiger Desinfektionsmittel auf Hefe.
{Ibid., 1893, 1894.)
Vergleichende
Arten von Bierhefe.
Untersuchungen
an
vier
untergarigen
{Ibid., 1895, 1898, 1899.)
Uber einen ungeformten Eiweisskorper, welcher der unterist, und dessen Beziehung zu
dam sogenannten gelatinosan Natzwerk, welches baim
garigen Bierhefe beigemengt
Eintrocknen der Bierhefe entstaht, uebst einigan Beobachtungen iibar Natzbildung in der Kahrahaut. {Ibid., 1897,
Nr. 35.)
378
WoRTMANN,
J.
Uutersuchungen
Hefen
iiber reine
und
I.
II.
(Landwirt. Jahrb., 1892 und 1894.)

iJber einige seltenere, aber in diesem
Sommer
stark auftretende Erkrankungen der Weintrauben.

und Weinhandel, Bd. xvi., 1898.)
teilweise
(Weinbau
Treats of the occurrence of Deinatium,
Vorkommen und Wirkung
lebender Organismen in ferti-
gen Weinen und ihre Bedeutung
fiir
Further references in the literature review
of
Section
Umschlagen
Untersuchuugen iiber das

ZiMMERMANN, 0. E. R.
iiber
Weine.
cells.
Chemnitz, 1871.
Das Genus Mucor.
Zukal: Vorlaufige Mitteilung
II.
der
Turbidity brought about by the solution of dead yeast
Entwicklungsgeschichte
die
crustaceum, Link, und einiger Ascobolus-
des Penicillium
(Sitzungsber. d. Wiener Akad., Bd. xcvi.,
Arten.
Wein-
die Praxis der
Berlin, 1898.
bereitung.
Abt.,
1.
Nov.-Heft, 1887.)
3.
Sjyecial
Works on Bacteria.
Textbooks and Handbooks.
DE Bary, a.
Vorlesungen
Fischer, Alpr.
FlUgge
Vorlesungen
Jena, 1897.
iiber Bakterien.
Die Mikroorganismeu.
Fraenkel, C.
Leipzig, 1885.
iiber Bakterien.
3.
Anfl.
Leipzig, 1896.
Grundriss der Bakterienkunde.
3. Aufl.
Berlin,
1890.
Lafar, F.
Technische Mykologie.
I.
Schizoniyceten-Garungen.
Jena, 1897.
Lbh.mann und Neu.mann
Atlas und (ilrundriss der Bakteriologie.
Miinchen, 1896.
ZoPF,
W.
Die Spaltpilze.
3.
Ausg.
Breslau, 1885.
Papers.
Bbhrens,
J.
Der Ursprung des Trimethylamins im Hopfen und
die Selbsterhitzung desselben.
(Arbeiten d. bakt. Inst. d.
techn. Hochschule in Karlsruhe, Heft
2,
1894.)
LITERATURE REVIEW
W.
Bbijerinck, M.
ferment.
379
tJber die Butylalkoholgarung
(Verb.
van
Akad.
koninkl.
d.
und das ButylWetensch.
te
Amsterdam, 1893.)
liber die Arten der Essigbakterien.
Par.
II.
Brown, A.
Inf., 2. Abt.,
J.
terium
News,
(Central bl.
The Chemical Action of Pure Cultivations of Bac(Journ. of the Chem. Soc, 1886. Chemical
aceti.
vol.
1886.)
liii.,
On an
the
Bakt.,
f.
1898.)
Acetic Ferment which forms

Chem. Soc, 1886.)
Cellulose.
(Journ. of
Further Notes on the Chemical Action of Bact.

(Journ. of the
aceti.
Chem. Soc, 1887.)
Note on the Cellulose formed by Bact. xylinum.
Note on Bacillus
{Ibid.)
(Journ. of the Fed. Inst, of
subtilis.
Brewing, 1895.)
BiJTSCHLi,
0.
Bau der Bakterien und verwandter
tJber den
Organismen.
Leipzig, 1890.
Weitere Ausfiihrungen
und Bakterien.
CoHN, F.
LTntersuchungen
Biologic der Pflanzen.
1875.
DowNES,
A.,
Bd.
iiber
den Bau der Cyanophyceen
Leipzig, 1896.
Heft
ii..
2,
iiber
Bd.
i.,
Bakterien.
Heft
1876, und Heft
and Blunt, Th.
P.
(Beitrage
1872, und Heft
3,
zur
3,
1877.)
Researches on the Effect of Light
ufion Bacteria and other Organisms.
Fischer, Alfr.
2,
(1877.)
Die Plasmolyse der Bakterien.
(Ber.
d.
k.
sachs. Gesellsch. d. Wissensch. Math.-phys. Kl., 1891.)
Untersuchungen
Bot., Bd. xxvii.,
Heft
Untersuchungen
iiber Bakterien.
1,
(Jahrb.
f.
wissensch.
1894.)
iiber
den Bau der Cyanophyceen und
Bakterien, Jena, 1897.
Hansen, Emil Chr.
Mycoderma
Pasteurianum, nov.
sp.
Tom.
aceti (Kiitz.),
(Compt.
i.,
livr.
rend,
ii.,
Pasteur et Myc.
des travaux
1879.)
du
380
Hansen, Emil Che.
saurebakterien.
Botanische Untersuchungen liber die Elssig(Ber. d. deutsch. bot. Ges., 1893.)
Recherches sur
des travaux du
1894, and torn,
Henneberg, W.
de Carlsberg, Tom.
v., livr.
i.,
Weitere Untersuchungen
Bakt., Par.
u. Inf., 2.
iii.,
(Zwei
Bacterium industrium und B. ascendens.)
deutsohe Essigindustrie, Jahrg.
f.
livr.
iii.,
1900.)
Zur Untersoheidung der Essigbakterieii.
neue Arten
(Compt. rend.
les bact6ries ac6tifiantes.
laborat.
ii.,
(Die
1898, Nr. 14 und 15.)

(Centralbl.
liber Essigbakterien.
Abt., 1898, Nr. 1-4.)
Beitrage zur Kenntnis der Essigbakterien.
[Ibid.,
1897,
Nr. 9-10.)
Bacterium industrium und B. ascendens und Erganziingen

zu den
Untersuchungen
bisherigen
liber
Essigbakterien.
(Die deutsche Essigindustrie, 1898, Nr. 19-23.)
JoRGBKSEN, Alfe.
(Allg. Brauer- u. Hopfenztg., 1890,
Sarciua.
Nr. 115.)
VAN Labr, H.
Note sur
les
(M^moires
fermentations yisqueuses.
de I'Acad. royale de Belgique, Tom.
xliii.,
1889.)
Contributions a I'histoire des ferments des hydrates de
carbone (Bacille des bieres tournês).
[Ibid.,
Lafar, F.
Tom.
xlvii.,
1892.)
Physiologische Studien liber Essiggiirung und Schnell-
essig-Fabrikation,
Bd.
2. Abt.,
i.,
2.
Abt.
(Centralbl.
f.
1895, Nr. 4-5.)
Die kiinstliche Siiuerung des Hefegutes der Brennereien.

{Ibid.,
Bd.
Lindner, P.
ii.,
Uber
1896.)
ein neues, in Malzmaischen
Milchsiiure bildendes Ferment.
vorkommendes,
(Wochensohr.
f.
Brauerei,
1887, Nr. 23.)
Die Sarcina-Organismen der Garungsgewerbe.
Berlin,
1888.
Ruft Sarcina im untergarigen Biere Krankheitserscheinungen hervor oder nicht? (Wochenschr. f. Brauerei, 1890.)
Bemerkungen zu Jorgensen's Aufsatz liber Sarcina.

No. 41.)
{Ibid.,
LITERATURE REVIEW
Meyer, Arthur
Pasteur,
L.
Meinoire
Kerne und
tJber Geisseln, ReservestofFe,
bildung der Bakterieu.
381
Heft
(Flora, Bd. Ixxxvi.,
sur
fermentation
la
(Conipt. rend, de I'Acad. des Sc,
Fermentation butyrique.
Tom.
5,
appel6e
Animalcules infusoires vivant
Memoire sur
la
fermentation ac6tique.
TEcole norm, sup., Tom.
Etudes sur
.
(Ann. scicntif. de
1864.)
i.,
Paris, 1868.
le vinaigre.
Sarcina im Biere ohne irgend eine Krankheits-
erscheinung.
Prazmowskt
(Ibid.,
1861.)
Hi.,
Petersen, A.
lactique.
xlv., 1857.)
sans oxyg^ne libre et determinant des fermentations,
Tom.
Sporeii-
1899.)
(Zeitschr.
f.
d. ges.
Brauw., 1890, Nr.
1.)
Untersuchungen iiber die Entwicklungsgeschichte

und Fermentwirkung einiger Bakterienarten. Leipzig, 1880.
Reichard,
Alb.
Studien iiber
(Zeitschr.
Biere.
f.
d. ges.
Reichard, Alb., und Riehl, Alb.

der Sarcinakrankheit.
Sarcina-Organismus
eiuea
im
Brauw., 1894.)
:
Zur Kenntnis und Bekiimpfung
(Zeitschr.
f.
d.
ges.
Brauw., 1895,
Nr. 8-10.)
Seifert,
W.
Beitrage
Essigsaurebakterien.
Abt., Bd.
Zeidler, a.
iii.,
zur PhysioJogie
(Centralbl.
f.
und Morphologic der

Beitrage zur Kenntnis einiger in Wiirze und Bier
vorkommender Bakterieu.
(Wochenschr.
f.
Brauerei, 1890.)
Tiber eine Essigsaure bildende Termobakterie.

f.
2.
1897.)
2.
Abt., Bd.
ii.,
1896.)
(Centralbl.
INDEX TO SUBJECTS AND AUTHORS.

Ascospore formation in aspergillus,
125.
Abbe's illuminating apparatus, 25.

Aberration, spherical and chromatic,
23.
Aberson, 216
Acetic acid bacteria, 333, 117, 319,
326.
Achromatic
saocharomyces, 121, 203.

Ascus formation in schizosaccharomyces, 211.
Aspergilleae, 170, 274.
Aspergillus, 274, 116, 125, 186.
fumigatus, 277.
glaucus, 274, 116, 273.
niger, 277.
oryzae, 277.
repens, 116, 272, 274.
Asporogenous varieties of saccharomycetes, 136, 236.
Aubry, 355.
Aujeszky's
method of staining
spores, 9
objectives, 24.
Aderhold, 203, 258.

Aeration of beer wort, 6.
Aerobic organisms, 5, 296, 342.
Agar-agar, 81.
Agaricineae, 305.
Air analyses, 143, 150.
Hansen's, 150.
Albumen, 82.
Alcanna tincture,
penieillium, 125.
92.
Autoclave, 52.
Auto-fermentation, 219.
Auxiliary apparatus for microscopy,
Algae fungi, 171.

Amici, 26.
Amthor, 295.
Amylobacter, 344.
85.
Amylomyces Rouxii,
181.
Anaerobic organisms, 5, 98, 325, 344.
Analysis, the biological, of soil, 143,
B.
155
of air, 143, 150.
of water, 143, 145.
of yeast, 133, 14.
Anixiopsis stercoraria, 272.
Antiseptics, action
of,
Bacilli, 332, 316, 324, 325.
Bacillus acidificans longissimus, 342.

lactici,
on yeast, 223.
Aperture, angular, 26.

numerical, 26.
Apparatus, 17.
for counting, 32.
illumination
for
artificial
microscopy, 34.
for spore cultivation, 64.
for sterilisation, 51.
Appert, 4.
Apple syrup, 79.
Arrack manufacture, 185, 260, 301.

Artificial illumination, 34.
Ascogonium, 279.
Ascomycetes, 186.
acidi
342, 267, 326.
amylobacter, 343.
butyricus, 344.
caucasicus, 267.
lupuliperda, 344.
oxalatieus, 316.
piluliformans, 344.
344, 100, 267.

viscosus
and
341, 342.
in
subtilis,
I., II.
III.,
vini, 342.
Bacteria, 332, 312, 315.

application of, in the alcoholic
383
fermentation industries, 328.

acetic acid forming, 6, 136, 333.
butyric acid forming, 343, 826,
328, 329.
cells, structure and shape of, 312.
INDEX TO SUBJECTS AND AUTHORS
384
Bacteria, lactic acid forming, 342,

10, 168, 326, 328, 329.
mode of increase of, 316.
preservation of, 117.
slime forming, 341.
staining, 90.
Bottles
5,
for
reagents
storing
apd
immersion
liquids, 35.
Botrytis cinerea, 286.
vulgaris, 286.
Bottom fermentation, 220.
yeast, 185, 220.
suppression in yeast growths,

difference between,
yeast, 220.
systematics 329.
Boullanger, 211.
variation 319.
Bourquelot, 281.
which cause diseases in fermen- Bread, 86, 280.
of,
and top
99.
of,
of,
tation industries, 324.

BacteriaceEe, 332, 3-30.
Bacterium aceti, 336, 150, 223, 319,
326, 333, 334.
Beijerinck, 340.
acetigenum, 340.
aoetosum, 319.
industrium, 340.
Kiitzingianum, 338, 319, 324, 333,
334.
megatherium, 316.
oxydans, 340, 319.
Pasteurianum, 337, 150,319, 324,
333, .384.
termo, 341.
vermiforme, 341, 261.
xylinum, 840.
Bail, 249.
Bainier, 124, 172.
de Bary, 274, 275.
Basidia, 274.
Brefeld, 10, 99, 100, 278, 279, 280,

305, 345.
Brewery yeast, preservation of, 118.
purification of, according to
Pasteur,
313.
Buchner, E.,
aeration of, 77.

with addition of tartaric acid,
j
72.
in Pasteur flasks, 72.

Behrens, 185, 275, 281, 288, 289.
Beijerinck, 214, 236, 264, 270, 340.
Belohoubek, 296.
Bench surfaces, preparation
Bendixen, 163.
Bergh, 163.
of, 18.
2,
347.
Cane sugar.
of, 80.
sterilisation of, 71.
sterilisation of, in Carlsberg

vessels, 73.
in Freudenreieh flask.'!,
cells, 191.
Oagniard Latour,
j
'
78.^
213, 217.
Butyric acid bacteria, 343, 325, 326.
Beer, sterilisation
Beer wort,
7,
H., 98, 321.
Budding
Ban, 220, 301.

de Bavay, 229.
Beck, 226.
6.
Brown, A. J., 216, 388, 334, 345.

H. T., 163, 325.
Brownian molecular movement, 188,
See Saccharose.
Carbol-fuchsine, 92, 88.
Carlsberg vessel, 56, 73.
bottom
yeast. No. 1, 252, 201,

217, 220, 221, 2.30, 234, 240,
244, 245, 326,
No. 2, 252, 215, 217,
221, 227, 230, 235,
326.
Casagrandi, 188, 189, 211.
Cell contents, 187.
215,
241,
220,
242,
chemical constituents of, 211.

nucleus, 89,
staining of the, of yeast, 89.
shape, 190, 312.
wall, 189.
Cells, resting, 201.
Chalara mycoderma, 301.

Chambers, moist, 68.
stand for, 70.
investigations
Chamberland
in, 92.
flasks, 59.
Chamberland's autoclave,
Berkefeld's filter, 81.

Berlese, 295.
Biemacki, 223, 243.
Chamotte
Blaxall, 321.
Chaptal,
Blunt, 99.
Chemical
52.
filter, 81.
blocks, 123.
5.
factors, influence
yeast, 222.
Bokorny, 224.
Bottcher's moist chamber, 69,92, 107. Chemotaxis, 315.
of,
on

Chlamydospores, 172.
385
Dubourg, 183, 214, 327.

Duclaux, 213, 275, 352.
Dusoh, 4.
See Flagella.
Circulation of yeast in nature, 246.
Cilia.
Cladosporium, 116, 283.
herbarum,
283, 311.
Cladothrix, 316.
Clostridium, 316, 332.
butyricum, 343.
Cocoacese, 330.
Cocci, 267, 315, 325.
Cohn, 277, 289, 329, 341.
Coleothrix methystes, 827.
Colonies, formation of yeast, on solid
culture media, 202.
Colour for object marker, 109.
Earthenware cubes,
Ehrenberg,
2, 99, 313.
Elion, 123, 163.
Emmerling,
176, 267.
decipiens, 263.
See Spores.
Energy of multiplication, 193, 231.
Engel, 122.
Endomyces
Bndospores.
Columella, 174.
Competitive relations, 229.

Condenser, 25.
Erlenmeyer's
Conidia, 17.
Coremium,
123.
Edelfiiule, 288.
Effront, 223, 243.
flask, 62.
Errera, 211.
Esaulow, 345.
278.
Counting chamber, 32, 127.

methods, 126.
Cover glasses, 29.
gauge for, 34.
removal of grease from,
Eumycetes, 170, 171.

Eurotium, 274.
87.
F.
squared, 30, 110.

Crenothrix, 316.
Crimean wine, 265.
Culture agar-agar, 81.
gelatine,
media,
of anaerobic organisms, 98.
vessels, 54.
yeast, distinction from
"Paultiness" of rum, 327.

Fermentation, influence of
temperature on, 214.
81.
71.
and
of,
solid, 81.
of,
air
bouquet, 218.
energy 217.
organisms, systematics
phenomena, 212.
products, 218.
theory of E. Fischer, 213.
of,
wild
173.
of Liebig, 2.
yeast, 205.
Cup fungi, 286.
Cupboard, Hansen's
of Nageli, 7.
of Pasteur, 7.
sterile, 21.
of Traube, 7.
Fermenting
D.
Delbriick, 15, 232, 243, 357.
Dematium, 116, 186, 305.
puUulans, 305.
Development, investigation
Dextrose solutions, 80, 265.
Dilution methods, 101.
Diplococous I. and II., 331.
Discomycetes, 170, 285.
Disease bacteria, 324.
yeasts.
See Wild yeast.
Distillery yeast, 253.
Dothidea ribesia, 311.
of,
cultures of yeast, 125.

Filter, Berkef
Chamberland's,
hot water,
314.
Fischer,
E., 213, 221, 301.
of, 92.
eld's, 81.
81.
83.
Alfr.,
7,
99.
culture, Lindner's, 138.

Drying, influence of, on yeast, 224,
227.
controlling contents
Ferments of the yeast cell, 212.

Film formation, 5, 115, 125, 195.
Drop
vessels,
137.
Downes,
cellar journal, 139.
power, 217.
Fission fungi, 312.

Fitz, 101.
Fixation of bacteria
90, 88.
Flagella, 313.
staining of, 91,
Flasks, 62.
25
and yeast
cells,
386
Forti, 297, 301.

Fractionated culture, 111.
Frankel, 98.
Freudenreich, 59, 267, 30.3.
flasks, 59.
Frew, 229.
Frohberg yeast, 215, 220, 231.
285.
Fungi imperfecti,
289, 170.
G.
Gauge
for cover glasses, 34.
Gayon, 183, 327.

Gelatine, 81.
Gelatinous formation of cell wall, 189,
312.
Gemmse,
172.
Generatio aequivooa,
Gerard, 281, 288.
Germination
3, 4.
saccharomyces
of
spores, 203.
Giesenhagen,
beer, 261, 341.
slips, 29.
mycelium-formation
of
90.
of
202.
juice
from
spores
yeast,
between the
culture yeast
wild yeasts, 205.
germination of the spores
and
Gronlund, 259, 260, 296.

Gymnoasceae, 170, 186.
of
203.
difference
raisins, 80.
concentrated, 80.
sugar.
See dextrose.
Greg, 271.
formation
spore
Gypsum
the
140.
formation of yeast colonies

on solid culture media,
staining bacteria,
Granules, 188.
Granulobacter, 332, 344.
saceharobutyricum, 344.
Grape
134.
yeast, 200.
hollow, 68.
Gram's method
119.
analysis for brewery purposes, 145.

Air analyses, 150.
Results of his air analyses, 154.
The pure culture system, 9, 156.
bottom
in
fermentation
breweries, 156.
in top fermentation breweries, 163.
in the manufacture of wine,
164.
in
the
manufacture of
spirits and pressed yeast,
168.
On the mucor species, 172, 174, 175.
yeast cell nucleus, 187.
net-work formation of yeast,
189.
shape of the yeast cell, 91.
multiplication of yeast, 193.
film-formation of yeast, 195.
Glass slip cultures after Brefeld, 100.
of
85.
Giltay, 216.
Ginger
Continued.
transmitting yeast,
of spore cultivation, 121.
fortheanalysisof yeast,
Tartaric acid method for
analysis of yeast, 135.
Stability test of beer in cask,
Method
Water
Fruit syrups, 79.

syrup gelatine, 84.
Fumago,
Hansen, E. Chr.
blocks, 64.
of
of
of yeast, 206.
temperature curves
of spore
formation, 209.
action of yeasts on sugars,
212.
action of oxygen on yeast,
Haas, 219.
Hsematimeter, 32, 130.
Hansen, E. Chr.
Investigations in general, 6, 9, 353.
First pure culture method, 10, 101.
Second pure culture method, 12,
106.
transformation
experiments
with top yeast and bottom
yeast, 221.
the influence of temperature

on yeast cells, 224.
vitality of yeast, 227.
Saccharose method for the preservation of

various
microorganisms, 114.
Method
215.
yeast types, 220.
on cotton
paper, 117.
of preservation
wool and on
filter
disease yeasts, 228.

competitive relations
yeasts
between
mixed fermentations,
229.
the variation
of yeast, 283.
387
Investigation of growth, methods of,

Contimied.
92.
the circulation of yeast in
Involution forms, 322.
naturOi 246.
Iodine-potassium iodide solution, 92.
Description of yeast species, 249.
Iodine tincture, 92.
On Anixiopsis stercoraria, 272.
Description of Torula species, 290. Irmisch, 246.
On Saccharomyces apioulatus, 295.
Hansen, E. Ghr.
On
Mycoderma, 296.
Monilia Candida, 298.
Oidium
303.
the acetic acid bacteria, 319,
826, 333.
Sarcina, 327.
61.
Hansen
Kuhlepurecultureapparatus,158.
laotis,
flasks,
Hansen's sterile cupboard,

Hantsch's solution, 87, 92.
21.
Hay bacillus,
100, 267, 344.

Hayduck, 193, 223, 242.
Hayduck's asparagine solution, 215.
Hayem and
Janczewski, 283.
Jansseus, 89, 188.
Jensen, 164.
Johannisberg II., 258, 237, 238.
Jorgenseu, Alfr., 30, 120, 135, 150,
163, 164, 168, 223, 236, 243, 246,
253, 264, 296, 304, 327.
flasks, 61, 120.
Journal, fermenting cellar, 139.
lager cellar, 144.
Nachet's haematimeter,
K.
32, 130.
Heating apparatus for the microscope, 45.
Katz, 281.
Kayser, 168, 211.
Heating, effect of, on yeast, 224.

Kefir, 266.
Heidenhain's method of staining
Kissling, 288.
Klebs, 272.
fractionated culture. 111.
Klein's method of staining spores, 91.
Klonne and MiUler's object marker,
cell nucleus, 89.
Heinzelmann, 223, 243.

Henneberg, 333, 334, 335,
Heron, 119.
339, 340.
Hesse, 98.
Higher fungi, 170, 186.

Hoffmann, 310.
Holder for sterilised water, 67.
Holm, J. Chr., 105, 109, 134, 147,
33, 108.
Kny, 225.
Koch, B.,
149,
223, 264, 271, 855.
Holm's water analyses,

Holzner,
149, 837.
cladosporioides, 285,
312.
Hot-water
Hueppe,
Huth,
84.
328.
Hydrofluoric acid method, 223.
Hyphomycetes,
11,
Koji, 277.
Kokosinaki, 164.
Korff, 215.
Kramer, 325, 326, 343.
Kruis, 200, 218.
Kruse, 106.
Kiihle, 158, 221.
Kukla, 243, 296.
Kupfer, 828.
Kuster, 188.
Kiitzing, 2, 5, 192, 333.
filter, 83.
Hotter, 164, 258.

V.
plate culture, 103.

streak inoculation method, 103.
Koehler, 264.
9.
Hormodendron
11, 98, 103.
Koch's burner, 50.
289.
I.
Immersion,
25.
liquids, bottles for, 35.

Imperfect fungi, 289.
Inoculation methods, 93.
method, streak, after Koch, 103.
Intergrowth, 287, 803.
Invertase, 801.
Invertin, 2, 301.
Laboratory, situation of, 17.

Laborde, 289.
Lactase, 213.
Lactic acid, application of, in
manufacture, 168, 828.
25*
bacteria, 342, 326.
spirit
388
van Laer, 325,
326, 343.
Lafar, 168, 297, 329, 334, 342.
Lager cellar journal, 144.
Lang,
303.
Lasche, 297.
Latour, 2, 347.
Laurent, 812.
Leblanc, 89, 188.
Leeuwenhoek,
table, 20.
testing
of, 28.
Mitscherlich,
191.
228, 267.
2, 10, 99,
Mixed fermentation,
Miyoshi, 281.
Moeller's method of staining
nucleus, 89.
1.
Lewkowitsch, 281.
Liebig, 2, 7, 348.
Liebig's theory of fermentation, 2.
Light, influence of, on beer, 226.
yeast, 225.
Lindner, 110," 138, 150, 163, 168, 201
236, 246, 264, 265, 270, 287, 301,
310, 327, 355.
Lindner's drop culture, 138.
droplet culture, 110.
Link, 283.
Lintner, 9, 219, 855.
Liquid nutrient media, 71.
Lister, 10, 101.
gelatine, 85.
Loffler's method of staining flagella,
Litmus
91.
Logos yeast, 215,
heater, 45.
Miquel, 105, 151.

Miquel's flask, 151.
Lesage, 281.
Microscope, 22.
220.
Lohmann,
225.
Lopriore, 812,
Lott, 273.
cell
Moist chambers, 68.
development
in, 92.
Monilia, 116, 298.

Candida, 298.
javanica, 260, 301.
Morris, 163, 325.
Moto, 277.
Mould
film fungi, 296.
fungi, preservation 114.

Mucor, 116, 124, 172, 174, 186.
alpinus, 172.
alternans, 183.
corymbifer, 183.
erectus, 175, 180.
Mucedo, 177.
neglectus, 172.
oryzse, 181, 260.
racemosus, 124, 179.
Eouxii, 181.
spinosus, 183.
stolonifer, 184.
of,
Mucoracese, 170, 171.
M.
Muencke's form
of
Lothar Meyer's
regulator, 48.
Macfadyen, 321.
Miiller, 2.
Mach,
Miiller-Thurgau, 1C4, 247, 258, 281,
164, 255, 289.

casse, 289.
Maltase, 213.
Maladie de la
Manipulation
of
nutrient liquids, 93.
Manure, 86.
Marx, 163, 164, 258.
Mass cultures, pure,
Mazun, 267, 264.
100.
Meat
extract, 79.
peptone gelatine, 84.
Meissl, 217.
Meissner, 82, 292.
Melibiase, 213.
Membrane of yeast cell, 189.
Meyen,
288, 289, 295.
Multiplication, power of, 231.

Miiutz, 281.
Mycelium formation in yeast, 200.
Mycoderma, 2, 116, 149, 224, 296.
cerevisiae, 149, 224, 296.
pulverulenta, 264.
vini, 264, 297.
L and n., 297, 298.

Myoomycetes, 170, 186.
N.
2.
Meyer, Arthur, 36, 317.

Lothar, 48.
Micrococcus, 289, 325, 326, 331.
saprogenes vini I. and II., 331.
viscosus, 331.
Micrometer, 31.
scale, standard, 31.
Niigeh,
101, 216, 320.

168.
7,
Nathan,
Needham,
3.
Net eye-piece, 32.

Network, the gelatinous,
Ney, 226.
Nielsen, 263, 264, 269.
189.

Nitric acid, 92.
Petersen, Anton, 44, 216, 296, 327.

Petersen's thermostat, 44.
Petri dishes, 64.
Pezizaceae, 170, 286.
Nucleus, 89.
Nutrient agar-agar, 81.
389
gelatine, 81.
substrata, 71.
Pfefier, 281.
solid, 81, 86.
Pfeiffer's
microscope heating appa-
ratus, 45.
Phycomycetes, 170, 171.

Physical factors, influence
o.
Objectives,
achromatic
and
apo-
chromatic, 24.
with correction,
changing, 28.
immersion, 25.
Object marker,
stage, 28.
Oidium, 299.
303, 267.
24.
Platinum brush,
Pombe, 269.
33.
lactis,
Oxygen, influence
of,
on yeast, 214.
of,
on
yeast, 224.
Pichi, 265.
Pink yeast, 294.
Pipettes, 70.
Plasmolysis, 313.
Plate cultures (after Koch), 11, 103.
71.
Portele, 164, 255.

Potatoes, 86, 103.
Poulsen, 134.
Prazmowski, 318, 344.
Preparation making, 86.

Preservation methods, 114.
Pressure vessel, 52.
Panum's thermostat,
Pasteur,
39.
112, 192,
215, 247, 281, 289, 325, 334, 335,
343, 349.
flasks, 54.
3,
5,
6, 7, 15, 54,
working with, 93.

Pasteur's theory of fermentation,
7,
3,
232.
215.
physiological
method, 112.
Prior, E., 59, 217, 220, 224, 278, 296.

Prior's vessel, 59, 77.
Promyoelium, 207.
Propagation, modes of, of yeast, 191.
Proteolysis, 214.
Protoplasm of the yeast cell, 188.
Pure culture, natural, of Delbriick,
pure culture
purification of brewery yeast, 6.

tartaric acid method, 6, 112.
Pasteurisation, 6.
Pedersen, E., 193, 216.
Pediooocous, 331, 325, 327.
acidi laotici, 331.
cerevisiae, 331.
sarcinseformis, 331.
viscosus, 331.
Penicillium, 116, 125, 150, 186, 273,
apparatus
controlling
278.
the
contents
106.
Lindner's, 110.
Lister's, 10, 101.
Pasteur's, 6, 112.
Sohoufeld's, 111.
methods,
99.
for bacteria, 113.
for mould fungi, 113.
system, Hansen's.
sen.
Pyrenomycetes, 273.
and
Klebs's, 111.
saochari, 278.
Perisporacese, 170, 271.

Perithecium, 271.
formation by aspergillus, 275.
penicillium, 280.
Permanent preparations, 87.
varieties, 236.
Permeability of cell membrane, 217.
Perosmic acid, 92.
Persoon, 2, 249.
Hansen
of the, 137.
method, Brefeld's, 10, 99.
Hansen's, 9, 11, 12, 101,
crustaoeum, 278.
glauoum, 125, 150, 278.
luteum, 278.
of
Kilhle, 158.
R.
Race
I.,
168.
168, 225, 253.

IX., 254.
II.,
Eaggi, 185, 260, 301.
See
Han-
390
Saccharomyces Ludwigii, 267, 200,
Eauvier's moist chamber, 69, 93.
Rapp, 217.
207, 212, 227, 235, 236, 287,

250.
mali Duclauxi, 265, 250.
Marxianus, 261, 194, 200, 205,
228, 236, 250.
membranaefaciens, 264, 265,
213, 228, 236, 238, 250.
II. and III., 265.
var. californicus, 265.
tauricus, 265.
mycoderma, 296.
n. sp., 250.
Pastorianus, 8, 102.
I., 254, 194, 195, 198, 207,
217, 218, 221, 226, 228,
235, 237, 238.
II., 255, 194, 195, 198, 202,
217, 218, 234, 238.
III., 256, 194, 195, 198,
207, 217, 218, 221, 229,
238.
pyriformis, 261, 225.
Saturnus, 250.
Vordermanni, 260.
Saccharomycetes, 170, 186.
budding of, 191.
cell wall of, 189.
Rauscher, 273.
Ray, 278.
Raymann,
Reagents, 92.
V.
bottles for, 35.
Recklinghausen, 100.
Reess, M.,
8, 203, 294.
Reiohard, 827.
Reichert's regulator, 46.
improved regulator,
Rice, 86.
beer, 277.
Rhizopus, 184.
nigricans, 150, 184.

oryzae, 185, 260.
Rohrbeck's thermostat,
Room,
36.
sterile, 20.
Ropiness, 292, 310, 325, 331, 341.
Rothenbach, 340.
Roux's regulator, 49.
Rubber tubes
for flasks, 71.
s.
Saare, 269.
Saaz yeast, 215, 220.

Sac Fungi, 170, 186.
Saccharobaoillus
Pastorianus, 326,
343.
Saccharomyces,
135,
228,
2, 8, 102, 121,
149, 246, 249, 267.
anomalus, 195, 205, 207, 218,
238, 262.
var. belgicus, 250, 264.
apiculatus, 102, 143, 193, 230,
294.
Aquifolii, 260.
Bailii, 265.
cartilaginosus, 266.
cerevisi*, 102, 225, 230.
I., 194, 195, 198, 205, 207,
221, 224, 231, 240, 251.
ellipsoideus I., 257, 194, 195,
202, 207, 217, 218, 221, 238,
II., 258, 195, 198, 207, 217,
221, 224, 229, 238, 250.
exiguus, 262, 212, 226, 250.
farinosus, 250, 265.
fragilis, 266, 214, 250.
Hansenii, 218.
hyaloaporus, 265, 205, 250.
Iliois, 259.
212,
195,
48.
Reinke, 118.
Besting cells, 201.
Revolving nosepiece, 28, 109.
200, 218.
238,
246,
chemical constituents
of
the
210,
231,
207,
203,
231,
cell,
211.
circulation of, in nature, 246.
colony formation of, on solid
culture media, 202.
effect of drying, 224, 227.
heat on the, 224.
light on the, 225.
oxygen on the, 214.
ferments contained by the, 212.
film formation of the, 125.

germination of the spores of, 203.
independent fungi, 186.
influence of chemical factors on
the, 222.
physical factors on the, 224.
-^ modes of propagation of, 191.

mycelium formation of, 200.
209,
resting
201.
spore formation 203, 121.
sporeless varieties 136, 236.
structure and shape of
187.
variation 233.
vitality 227.
Saccharose solutions,
method of preservation, 114.
cells of,
of,
198,
250.
218,
of,
cell,
of,
of,
80.
Sake, 277.
Sand bath, 52.
Sarcina, 331, 150, 154, 267, 327, 328.
alba, 332.

Saroina aurantiaoa, 332.
Spore cultures
332.
maxima, 332.
flava,
Sohionning, 65, 123, 211, 270, 271,

303, 306.
Schizomycetes, 170, 312.

Sohizosaccharomyces, 191, 211, 269.
mellaoei, 271.
octosporus, 270, 211.
Pombe, 269.
Schmitz, 187.
Schonfeld, 111, 163, 253, 328.
of yeast, 121.
from bacteria, 123.

fungi, 124.
method, Hansen's, for yeast
analysis, 134.
walls, coalescence of, 206.
cultivation, apparatus for, 64.
Spores, staining of bacteria, 90.
yeast, 88.
Stab cultures, 98.
Stability test of beer in cask, 140.
Stage, microscope, 28.
Staining of bacteria, 90.
free
mould
Sohribaux's thermostat, 42.

Sohroeder, i.
yeast cells, 88.

dead yeast cells, 88.
Sohroter, 103.
Schukow, 219, 220.
Sohultze, W., 226.
Sohulz, 223, 243.
Sohulze, Fr., 4.
cell nuclei, 89.
Stand for moist chambers, 70.

Standard micrometer scale, 31.
Steam
steriliser, 62.
Schiitzenbaoh's vinegar makers, 336.
Stephenson, 26.
Sterigmata, 274.
Schwackhofer, 149, 219.
Sterigmatocystis, 186, 274, 278.
Schwann,
Sterile cupboard, Hansen's, 21.
2, 4, 7, 8, 203, 348.
Sclerotinia Puckeliana, 286.

Sclerotium formation in aspergillus,
273.
botrytis, 286.
cells,
126.
W., 164, 168, 219, 258, 263,

265, 296, 297, 334, 338, 339.
Salter, 271.
Septum formation, 172, 206, 207.
Seyffert, 242.
de Seynes, 8, 203.
Seifert,
Sterilisation of glass ware, 51.
apparatus, 51.
by
filtration, 81.
81.
Sterilising oven, 51.

Streak culture, 97.
inoculation method of Koch, 103.

Streptothrix, 316.
Sulphuric acid, 4, 92.

Surface plate cultures, 106.
Surfaces, preparation of bench,
Symbiosis, 267.
18.
Syrê, 135, 231.
Siebel, 224.
Simple sac fungi,

V. Skerst,
room, the, 20.
discontinuous,
penicillium, 125, 280.
Seeding of a definite number of
391
186,
310.
Systematics of fermentation organisms, 173.
Slips, glass, 29.

hollow, 68.
Soda
solution, 87.
Solid culture media, 81.
Solutions for washing bench, 19.
Soxhlet's regulator, 49.
Spallanzani,
Tartaric acid, 211, 265, 328.
method, Hansen's,
Sphseriaoese, 170, 282.

Sphaeriese, 170, 282.
Sphaerulina intermixta, 311.

Spherical yeast, 172.
Spirillum, 316.
Spontaneous generation, 3, 4.
Sporangium, 171.
Spore formation in bacteria, 317.
in yeast, 203, 8.
cultures of bacteria, 124.
for yeast
analysis, 135.
Pasteur's, 6, 112.
3.
Spermatia, 288.
Sphserella Tulasnei, 283, 311.
T.
Temperature curves for film formation in yeast, 198.

spore formation in yeast,
209.
Termobaoterium
aceti, 340.
lutescens, 340.
Thausing,
9, 163.
Thermometers,
50.
Thermoregulatora, 46.
Thermostats, 36.
Thiele, 280.
392
Thoma's chamber,
van Tieghem, 274,
Water holder, 67.

Wehmer, 88, 168,
32.
278, 281.
Top fermentation, 220.
yeast, 135, 253, 270, 304.
difference between,
bottom yeast, 220.
and
Torula, 289.
Transmission
Traube, 7.
flasks, 119.
2, 6, 7 and 93, 252.

True fungi, 170, 171.
Tube, microscope, 24.
Turbidity in beer and wine,
Tribe
"
214, 278,
123, 148, 163.
Wild yeast, 228, 6, 10, 255,
difference
between
yeast and, 205.
Will, 30, 118, 125, 188, 198,
214, 219, 228, 243, 249,
295.
Wilson, 164.
Windisoh, 341.
Wine, diseases of, 326, 343,
282, 329.
Wichmann,
229, 327.
259.
culture
201, 210,
259, 264,
344.
See Beer wort.
Wort.
Wort gelatine, 84.

Wortmann, 80, 164,
Turpin, 249, 289.
165, 219, 229,

247, 258, 278, 282, 290, 295, 297,
310, 356.
u.
Ustilago, 187.
V.
Yabe, 224.
Vacuoles, 188.
Yeast, analysis
Vandam,
325.
Variation of bacteria, 319.
yeast, 233.
Varieties, temporary, 233.
permanent, 236.
in brewery practice, 241.
Veley, 327.
Velten, 6, 352.
Vernier, 28.
Vessels, control of the contents
of,
137.
Vibrio, 316.
Vitality of yeast, 227.
Vuylsteke, 231.
of, for bacteria, 134,

136.
biological analysis of, 133.
cells, fixation and staining of, 88.
counting and seeding, 126.
shape and structure of, 187.
glucase, 213.
maltase, 213.
poisons, 223, 281, 289.
preservation of, 118.
ring formation, 115, 198, 201.
spores, 88.
transmission of, 119.
types, 219.
vitality of, 227.
water, 79.
gelatine, 84.
W.
Wager, 188.
Warm
chamber,
43.
Washing bench,
19.
Water,
z.
\.
Walporzheim yeast, 258.

Ward, Marshall, 225, 261.
sterile, 78.
analysis, hygienic, 145.

technical, 143.
for brewery purposes
Hansen, 145.
by Holm, 149.
after
Zeidler, 264, 333.

Zickenwerden, 326.
Ziehl's carbol-fuchsine, 88.
Zoogloea, 313, 327.
Zopf, 170, 218, 285, 333, 340.
Zukal, 278.
Zygomycetes, 171.
Zygospore formation, 172, 176.
Zymase,
213.
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Cornell University

the Cornell University Library.

There are no known copyright

the United States on the use of the

lilil (nil illl'l

mil liiii mil liiii iiM lIL'i

3 1924 073 864 781

Cornell University Library produced this volume to

Scaimed as part of the A. R. Mann Library project to

ASSISTANT IN THE CARLSBERG LABORATORY, COPENHAGEN-

TRANSLATED FROM THE GERMAN BY

FORMERLY LECTURER IN THE BRITISI1

AND REVISED BY THE AUTHOR

LONGMANS, GREEN, AND

PATERNOSTER ROW, LONDON

HONOUR AND GRATITUDE

PROF. EMIL CHR. HANSEN,

THE AUTHOE'S PEEFACE

had the honour to act as assistant to Prof. Emil

frequently expressed by students to have at their

handbook which would contain an adedescription of the fittings, apparatus and

of the biology of the organisms of fermentation

by means of which the usual experi-

of such a laboratory can be carried out.

similar request reaching

of Stuttgart, I could but con-

clude that a real necessity existed for such a book.

work are divided

into three sections.

organisms of fermentation has gradually

given of the most important steps which have

marked the progress

section describes the fitting

the laboratory and

necessary for con-

being given to the

important micro-organisms of the alcoholic

The book thus deals with

For each section there is a bibliography which

January, 1900, could not be included.

sults not hitherto published.

of the fermentation industries with

book is chiefly concerned is that of

science of the organisms of fermentation as

set forth in this

however, not only

PREFACE TO THE ENGLISH TRANSLATION

A VERY considerable and rapidly increasing amount

being given in this country

fermentation industries, and

a growing demand for sound textbooks on the

study of the " fermentation organisms

are taking up this special

forced on the notice of the writer, whose

quately satisfied at present.

too well supplied with textbooks on Bacteriology

the subject of "fermentation organisms"

works describing the more modern developments

devoted to the treatment of laboratoiy methods

study of "fermentation organ-

students of Technical Microbiology, for

placed in their hands a book which cannot

to be of great assistance to them.