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Part I. THE DISSOCIATION OF
SACCHAROMYCES ACERIS-SACCHARI
FABIAN AND HALL AND PICHIA
ALCOHOLOPHILA KLOCKER

Part 2. STUDIES ON THE PRODUCTS
OF FERMENTATION OF THE
8 AND R FORMS OF YEASTS

Thesis for the Degree of M. S.
Lynferd _] Wickerham
1935

 

 

 

 

 

 

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EICHIA ALJOEOLOPHILA "L CHI-IR

Part 2. STUDIE ON TEE 1RD DU CTS U‘E FLRLZI‘J' 41.101;

13‘ THE S AND R 30311.13 03‘ YASTS

Submitted to the Faculty of the Michigan
State College in partial fulfillment
of the requirements for the degree

of

Master of Science

by

_ «’9’; ’2
Lynferd J3 fickerhem

June, 1935

TH 5515

Part 1.

Part 2.

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Titles
Table of Contents

‘

Dissociation of Saccharomgces aceris~sacchari Faoian and Hall

 

and Pichia alcoholophila Klbcxer

 

ntroduction

Dissociation of Saccharomyces aceris-sacchari Figures 1-13

 

Dissociation of Pichia alcohologhila.Figures 14-18

 

Discussion
Summary
Conclusion
Literature Cited
Studies on the Products of Fermentation of the S and R Forms
of Yeasts
Introduction
Description of Cultures Used
Description of hedia Used
Inoculation of media
Chemical and Yeast Analysis
Results - Tables 1-21, Figures 1-5
Discussion
Summary'and Conclusion
Literature Cited

Acknowledgement

Dissociation of Saccharomyces aceris-sacchari Fabian
and Hall and Dichia alcOholophila Klbcker

Introduction

Dissociation in yeasts has been.studied extensively by
Fabian andlucCullough (l). Eenrici (2) has also reported
variations in colonies of'a species of Torula. As morphological
changes of the dissociating yeasts have not been studied in
detail, an inveSLigation to do this was undertaken. The
large size of the yeast cell as carpared to the size of the
bacterial cell makes the changes in internal structure as
well as the changes in shape more clearly apparent. The
novel forms which the cells assumed as they passed frcm.one
stage in their dissociation cycle to another made their study
interesting. Of the six yeasts with which we worked, only
two responded to the dissociating agent used. They were

Saccharomyces aceris-sacchari Fabian and Hall, and Pichia

 

alcoholophila Klbcker.

 

Dissociation of Sacchar mvces aceris-sacchari

L

Saccharomyces aceris-sacchari in the normal or smooth

 

form produces glistening colonies on agar plates (Figure 1).
No scum is produced in young broth cultures, but after a

few days growth a thin pellicle appears. The cells are round
and show little, if any, internal.structure. When rapidly
growing in liquid media the budding cells tend to remain
attached, thus forming small clumps, or "colonies", a

ccmmon condition among S yeasts. The cells range from 5.5

to 7:microns in diameter (Figure 2).

A

-z-

The dissociating media used contained lithium chloride
as the dissociating agent. Yeast extract broth with two
different concentrations of lithium chloride; i.e., 0.10
per cent and 0.25 per cent were used. Yeast extract broth
is in itself a mild dissociation agent and promotes the
growth of’yeasts. This medium consisted of 0.1 per cent
of digested yeast cells, 0.5 per cent tryptophane broth and
0.5 per cent glucose. Plain broth containing 0.25 per cent
lithium chloride was also used because Fabian and.McCullough
(1) used it to induce dissociation in many species of yeasts.

A rapidly growing culture of the 3 strain of g. aceris-
sacchari was inoculated into culture tubes of the 0.10 per
cent lithium chloride yeast extract broth and serially

transferred daily. Platings and microscopic examinations
were made at each.transfer in order tc>note any changes
which.might occur. The media used fa? plating were malt
extract agar and dextrose agar. The first transfer produced
a variation.from.the S form, for many of the cells instead
of producing round buds produced elongated buds having
large central vacuoles. There were scme clumps of cells
in which the mother, or primary, cell was round and in which
each succeeding generation became more elongated. These
elongated buds had large central vacuoles. They were common
by the sixth transfer, by which timeJnost of the cells,
whether elongated or round, contained vacuoles and small,
highly refractive bodies which gave the characteristics of
fat when.stained by Sudan III or osmic acid. These re-
fractive globules, or oil drops, appear in the photographs
either as very light or very dark spd;s, depending upon the

focus when the pictures were taken (Figure 5).

 

Figure 1 Figure 2

 

Figure 4

Figure l. S colonies of g. aceris—sacchari

Figure 2. Cells of the S colony, unstained. Photographed
from a hanging drop preparation.

Figure 5. Elongated and round cells showing vacuoles and
small oil drOps.

Figure 4. Twenty-four hours later. The oil drops are much
larger and more numerous. The dark centers are vacuoles.

In all photographs of colonies the magnification is 8
thnes. In all photographs of cells the magnification is
460 times.

-3...

The seventh transfer in the 0.10 per cent lithium
chloride yeast extract broth when examined microscopically
appeared to he cmmposed of more or less rounded cells of
various sizes. The central vacuole was very large and
constituted most of the interior oiftme cell. The oil drops
had become larger and.were usually two in number. They
were located around the periphery of the vacuole into which
they sometimes escaped and showed Brownian movement (Figure 4).

When plated these cells gave rise to colonies which had
an outer band that was dull and wrinkled and a center which
was smooth and glistening. The next, or eighth, transfer
produced colonies which when plated were completely rough.
These R colonies were two or three times as large as the
smooth colonies; they were snow-white, and consisted of
many radial ridges extending from the center to the edge of
the colony. Between the ridges was a lacy'network. The
cells forming these colonies produced a heavy growth at the
surface of liquid media and moderate growth at the bottan.
MicroscOpically the cells varied greatly in size and morphology.
Some were elongated and some were spherical. The oil drops were
large and vacuoles were prominent. When the cells of a young
rough colony were suspended in a drop of water and examined
microscopically the preparation showed a.great many oil
drops which had escaped from the cells and were moving
about by Brownian movanent. ‘Many such oil drops were present
when the Figure 5 was photographed; however, only those that
were adhering to the surface of the yeast cells show in the

photograph since they were not moving. The cells do not

-4-
form clumps as the S cells do, but appear as single individuals
(Figure 6). No further change occumed on ccntinued transfer
in the 0.1 per cent lithium chloride.

For further study this R form was inoculated into malt
extract broth std also into a medium containing 0.25 per cent
lithium chloride in yeast extract broth. The former is a
very favorable medium.for yeasts and aids in the reversion
of the R form.into the S form. It was prepared by adding
5.0 per cent Trommer's malt extract and 1.0 per cent glucose
to nutrient broth. The latter was used to learn whether any
further change would occur in the R form.

After one to three daily transfers of the rough in malt
extract broth the cells became rounded, and when plated on
malt extract agar produced spreading colonies which reached
70 to 80 millimeters in diameter after a few days growth.
These colonies were rugose and the pariphery was soneWhat
irregular. This type was designated the spreader form
(Figures 7 and 8).

A single transfer of the spreader in yeast extract
broth caused its return to the R type. when the spreader
colony was allowed to age a few days on the malt extract
agar plate and was then directly plated on the same medium,
the resulting colonies were of the R type. However, after
continued daily serial transfers of the spreader type in
malt extract broth, it was cunverted into the S form after
approximately four transfers. The growth was almost canpletely
at the bottom of the culture tube. The colonies were smoth

and glistening, and the cells were round and homogeneous

 

Figure 9

Figure 5. Rugose R colony

Figure 6. R cells of Rugose colony

Figure 7. Rugose spreader colony

Figure 8. Rugose Spreader cells

Figure 9. A newly reverted S type that still retains

its oil drOps.

-5...
except for the presence of a large oil drop in many of the
cells. After four more transfers the oil drops disappeared.
It was found that unless the newly reverted 8 four was
rapidly transferred in malt extract broth, it tended to
revert to the spreader or R type (Figure 9).

In order to study the effect of an increased amount of
lithium chloride in the medium, the rough was inoculated
into a tube of 0.25 per cent lithimn chloride yeast extract
broth. The culture was set aside for 9 days and was then
plated. About 50 per cent of the resulting colonies were of
the type from which the culture was originally made; the
other 50 per cent did not ShOW’the characteristic radial
ridges, but had an even contour. Slender filaments extended
outward from the edge of the colonies into theznedium
(Figure 10). Both types of colonies were snow-white and
both types were of the same size, i.e., 4 to 10 millimeters
in diameter. The cells of this new R type were all
narrow rods having large vacuoles but only a few small
oil drops. Inany of the cells had no oil drOps whatever
(Figure 11).

When this R was grown twenty-four hours in malt extract
broth and then plated on the corresponding agar, a spread-
ing type of colony was produced. The spreader likewise was
not rugose. It had the same surface texture as the R from
which it was derived. The celonies grew to a size equal to
that of the rugose spreader referred to previously, and
similarly had an irregular periphery (Figure 12). The

cells were mainly rods, although some round cells were present.

Vacuoles were common, but oil drops were small and scarce

-6-
(Figure 12). This evenly cantoured spreader was just as
unstable as the rugose spreader. 0n continued transfer in
malt extract broth it reverted to the 8 form.

At the same time that the first inoculation of the S
form was made into the 0.10 per cent lithium chloride yeast
extract broth, another transfer from the amps culture was
inoculated into 0.25 per cent lithium chloride plain broth
and serially transferred. The growth.in this medium
was very slow. None of the serial transfers showed any
variation, and at the end of 50 days the colonies that were
produced still glistened. The cells were of the 8 type,

but were dwarfed in size.

 

Dissociation of Pichia alcoholophila Klbcker

The S form of Pichia alcoholophila Klbcxer grows at the
bottom in liquid media. The colonies are smooth and glisten—
ing. The cells are round to slightly elongated in sings and
frequently prodmze pseudo-mycelia. They average about six
microns in length. A stock culture showing a wrinkled growth
typical of R yeasts was examined and was found to contain
cells with small 011 drops and no vacuoles. An attempt was
made to dissociate this yeast by growing it in 0.10 per cent
lithium chloride yeast extract brotln but this yeast could
not tolerate it. Even 0.05 per cent lithium.chloride
inhibited its growth. It was found that yeast extract broth
is in itself a mild dissociating agent, and after a few
transfers in it colonies were produced which were extremely
rugose (Figure 14). Colonies two to six days old were

ccmposed of cells which were round to elongated in shape.

 

Figure 10 Figure 11

 

 

Figure 12 Figure 15
Figure 10. Evenly contoured R colony
Figure 11. Cells of the evenly contoured R colony
Figure 12. Evenly contoured spreader colony
Figure 13. Cells of the evenly contoured spreader colony

-7-
All cells had small vacuoles, and a few contained small
011 drops (Figure 15). They approximated the S cells in size.
When the colonies were six days old or older, the cells
became much larger, averaging 10 to 16 microns, with vacuoles
constituting most of the interior. Metachromatic granules
were easily visible in most of the cells, and oil drops
became very prominent. Pseudomycelial formation was also
common at this stage (Figure 16).

When giant colonies of this R form were grown on malt
extract agar and incubated for 10 to 15 days, secondary S
colonies appeared at several points on the periphery. When
these were subplated, they produced smooth colonies, the
cells of which had the appearance of the true S cells except
for the presence of small oil drops (Figures 17 and 18).

The oil drops disappeared only after many rapid transfers
in malt extract broth. As long as the S cells retained the
oil drops they could be brought back to the R form by

aging in malt extract broth for two weeks, at the end of
which time a thin pellicle fonned on the surface of the
medium. It was found that the R form.remains stable in

yeast extract broth.
Discussion

When the 8 form of S. aceris-sacchari was grown in

 

yeast extract broth containing 0.10 per cent 1ithium.chloride
the cells lost their homogeneous appearance. Oil drone were
numerous, nearly all of the interior of the cell was a vacuole,

and the cells grew mainly at the surface of liquid media

 

Figure 14 Figure 15

 

 

Figure 16 Figure 17

 

Figure 18 Figure 19

 

Figure 14. R colony of Pichia alcoholophilia
Figure 15. Cells of four day old R colony
Figure 16. Cells of seven day old R colony
Figure 17. S colony

Figure 18. 8 cells with oil drops

Figure 19. S cells without oil drops

-8-
rather than at the bottom as obtains with the S form. This
R form.produced a rugose, opaque colony. When grown in
yeast extract broth containing 0.25 per cent lithium.chloride,
this rugose R further changed into long slender rods having
large vacuoles and the oil drops had practically disappeared.
When plated, these cells produced opaque, evenly ccntoured
colonies in contrast to the rugose colonies previously produced.
Both tymes of the R form.when rapidly transferred in malt
extract broth soon produced unstable Spreading colonies and
later the S form of the yeast.

A stock culture of Fichia alcohOIOphila which was inter-

 

mediate between the S ain R form was made thoroughly R by
transferring it in yeast extract broth. The cells mmnposing
the R colony underwent a change in morphology, became larger
and developed more and larger oil drops as the colony became
older. When giant R colonies were grown and incubated for
10 to 15 days, secondary S colonies appeared around the
periphery. The cells of the secondary colonies cantained
oil drOps, but after several transfers in malt extract broth

the cells were typical of the S form.

{O

Summary

Rough yeasts have certain characteristics in common which
distinguish them from anooth yeasts;

1. Large oil drops are usually present in R yeasts.
Sometimes S cells show oil drops, but they are small in size
and few in number as canpared to the oil drOps present in
the R forms.

2. The R cells have very laroe vacuoles. When vacuoles
are present in S cells they are much smaller than those in
the R cells.

5. The R cell is larger than the correSponding S cell.

4. The R colony is larger than the corresponding S colony.

5. The R colony is opaque and rough, the S colony is
smooth and glistening.

6. The R cells do not tend to form "colonies" in liquid
media as do the S forms. The R daughter cells sqgarate
immediately from the mother cell.

7. The R cells come to the top of a hanging drOp and
adhere to the covergLass, making it possible to photograph
them readi y. Smooth cells go to the bottom of a hanging
drop and show'Brownian movement, which makes it difficult
to photograph them.

These characteristics were determined by observations
made on Fabian and.McCulloughs' R yeasts, on naturally
occurring R yeasts, and.on the R yeasts wkuch the writer has

produced.

-10-

Conclusion

The object of this study was to note in detail the
.changes that occur when a yeast dissociates. The photographs
as well as the descriptions already given fulfill this
objective. Two observations resulting from this work may
be mentioned here to advantage;

1. Dissociation in yeasts is a gradual process. That
is,none of the various stages arise spontaneously. There
is a gradual transition from one type of cell or colony to
another.

2. There is a marked tendency for the dissociants to

revert to the form from which they were derived.

l.

-11-

Literature Cited

Rabian, F. W. and thullough, N. B.
Dissociation in yeasts.

Jour. Bact. vol. 27, No.6, June, 1934, p. 583-623
Henrici, A. T. and Punicari, L.

A study of variation in a chromogenic
asporogenous yeast.

Jour. Bact., July, 935, p. 125

Studies on the Products of Fermentation of the S and
R Forms of Yeasts

Introduction

While working with dissociated forms of yeasts it was
noticed that during the process of fermentation certain
forms gave a pronounced odor of esters. The question naturally
arose as to whether the products of fermentation of the R
forms differed materially either qualitatively or quanti-
tatively from the products of fermentation of the correspond-
ing S forms. Therefore, an eXperiment was set up to determine
the amounts of certain products during the process of
fermentation. Yeasts which had been dissociated previously

were used in this experiment.

Description of Cultures Used

1

The cultures used were; Tue S form.of Saccharomyces

 

aceris-sacchari Fabian and Hall. This yeast produces a
glistening, moist growth on agar slants and convex colonies
on agar plates. The growth is mainly at the bottom in

liquid media although a thin film forms on the surface after
a few days growth. The cells average 3.5 to 7 microns in
diameter, are spherical, and are homogeneous in appearance.
The R form of S. aceris-sacchari. This yeast produces a
rugose, white, dry growth on agar slants and flat colonies

on agar plates. It produces abundant growth on the surface

of liquid media and a moderate growth at the bottom. The

cells average lo to 16 microns in length. 218 cells vary
in.morphology, some being round and others being elongated.
The individual cells do not p1esent a homogeneous appearance
as do the S cells, for the R cells contain one or more oil

drOps and a large central vacuole.

The S form of Pichia alcoholophila Klbcher. This yeast

 

produces a glistening, moist growth.on slanted agar and
convex colonies on agar plates. It is a bottom yeast. The
cells average 6 microns in diameter and are scherical or
slightly elongated in shape. The 3 cells occasionally show

small oil drops.

The R form of Pichia alcoholgphila. This yeast produces

 

a dull, wrinkled growth on agar slants and a raised, rugose
colony on agar plates. It produces an.abundant growth both
on the surface and at the bottom of liquid media. The cells
average 10 to 20 microns in length. They contain harge
vacuoles and oil drOps.

1‘!

The S form 01 Saccharomyces cerevisiae Hansen, Saaz

 

strainl. This is an industrial yeast that produces a
bottom alcoholic fermentation. The colonies on agar

plates are convex, and agar slant cultures have a moist,
glistening appearance. The cells are Spherical ”id average
6 microns in diameter.

The R form of S. cerevisiae Saaz produces a wrinkled,

 

1. For the saze of brevity this yeast will be hereafter

referred to as Saccharomyces cerevisiae Saaz.

 

white growth on agar slants and a raised, rugose colony.

The growth is mainly at the surface of liquidzmedia, although
there is moderate growth at the bottom. The cells vary in
length from 14 to 20 microns and vary considerably in shape.
As with the other R fonns, they contain oil drops and large
vacuoles.

The SR form of Willia anomala Saito. The S form of

 

this yeast was first obtained by Fabian and McCullough (1)
from the naturally oacurring R form.in 1953. It was put
away in a stock collection and when it was cultured again
for use in this eXperiment it showed p“‘ti&l reversion to
the R form. It produced a colony Miich was less wrinkled than
the true R, was gray instead of white, and had a slightly
moist appearance. It produced a thin scum on liquid media.
Because it had characteristics of both types it was
designated the SR form.

The R form of H. anomala. This yeast pooduces a white,
rugose growth on agar slants. The colonies are rugose and
raised. The cells range in length from 14 to 20 microns in
length. ‘The growth in liquid media is abundant at the sur-
face and moderate at the bottom. The cells show great
variation in morphology. Only a.small per cent of the

cells contain oil drops, but all contain large vacuoles.

-4-

Description of Media Used

The media used were; Cider. The cider had an
initial Balling reading of 12 degrees which was ingreased
to 22 degrees by the addition of sucrose in order to supply
sufficient sugar for a maximum.fermentation 3y all the
yeasts. Dextrose instead of sucrose was added to the cider

in which the S and R forms of 2. alcoholo iila were to be

FE

 

groum because this yeast fernents dextrose very slightly,
but does not ferment sucrose at all. After autoclaving
the cider for fermentation by the S and R forms of g.

alcoholophila showed a pH of 3.6, and 0.557 grams per 100

i.—

 

cc. of total acid calculated as malic acid. The cider for

fermentation by the two forms of S. cerevisiae Saaz had an

 

initial pii of 3.6 and contained 0.400 grams of acid per
100 cc. calculated as malic. The initial pH of the cider

for fermentation by the two fonts 0f.§° aceris-sacchari

 

and the two forms of E. anomala was pH 5.8 and contained
.340 grams of acid calculated as malic per 100 cc.

After the cider had been sterilized and before it
was inoculated with the yeasts, an analysis was made of it
to determine the presence of esters aid alcohol. The
results were negative. This procedure was followed with
all the other meoia used with negative results.

halt extract broth. This medium consisted of five
per cent sodium chloride, three per cent meat extract
(Liebig's), 10 per cent peptone (Bacto), 10 per cent

dextrose and two per cent mrommer's malt extract.

-L“"

Trommer's malt extract was used because it is high in total
solids and produces excellent growth. All of the malt

extract broth needed was prepared at one time. After auto-

J

per 100 cc.

U

ClSVlL: it ccufixxhied 0.159 Trams cu? total acid'

_.

\I

of medium calculated as lactic acid. The p1 was 5.2.
Synthetic medium. This medium contained 0.10 gram
monobasic potassium phCSphate, 0.10 gram monobasic ammonimn
phosphate, 0.10 gram calcium sulphate, 0.10 gram of magnesium
sulphate, 10.0 grams sucrose, and 90 cc. of distilled water.

The reaction of theznedimn was adgusted with concentrated

‘5

sulphuric acid. This medium was al

If)

0

'.

made up in one batch.
After autoclavin; the pi was 4.5 and it contained 0.105

grams per 100 cc. of medium of total acids when calculated

as sulphuric acid.

Inoculation of hedia

The media were .laeed in six liter Erlenmeyer flasks,

f

V

each flask receiving five liters of the medium. These flasns
were fitted with rubber stoppers through which a tube of glass
and a siphon assembly passed. The tube of gliss provided for
the interchange of gases and was plugged with cotton. The
siphon provided for the removal of samples of media. The
latter had two Hoffmann clamps separated by a glass tube thus
lessening the danger of contaninating the medium (See Eigure 1).
After autoclaving, the rubber stogpers were given a heavy
coating of paraffin.

The media in the flasxs were inoculated with tw) per cent

of 24 hour cultures of the various yeast f

0

rue, all due

precautions against contamination being observed. The

v

 

Figure 1. showing inoculated flasks of the different
media with the various yeasts

cut.)-

inoculation was mede by removing the plug from the glass tube

Opening into the flash and pourin_; the 111001111111; in. The
guard against the entrance of contamination, the flasks while
being inoculated were set inside a steamer wnich had been

running just previously for several minutes.

Chanical and Yeast Analysis

The methods of analysis used unless otherwise stated
were according to the Offical and Tentative Methods of Analysis
of the Association of Official Agricultural Chemists (1950)
(2). These analyses include pi, total acidity, volatile
acids, non-volatile acids, alcohol, and ester determinations.
At the same time that the chemical analysis was made, the
cultures were plated on malt extract agar in order to check

the cultures for contamination and to see whether any

dissociatior was taking place within the fermenting media,

and in case there was, to determine the extent of the
dissociation. The pH was determined by a quinhydrone electrcde.
Preparation of the sample was carried out according to

XVII, 25. Theruemiod used for determining the total acidity

is miat given under XVII, 44, except that ten cc. of the sample
was diluted with 90 cc. of distilled water before titrating

in order to give a clearer end point. Factors of 0.0067,
0.0049, 0.0090 were used in calculating the total acidity as
malic, sulphuric, and lactic, respectively. The volatile

acids were determined according to procedure given under

XVII, 47, except that they were eXpressed as malic,

sulphuric, and hectic acid and factors of 1.116, 0.817, and

-7-

1.500 were used respectively. Alcohol was determined by
neutralizing 100 cc. of the sample with l and l sedi"
hydroxide sslution, distilling, and then obtaining the per
cent by volume of alcohol by a Tag eubulliometer. The ester
determination is essentially the procedure given under

XVII, 65, however, it was necessary to use from 25 to 150

cubic cc. of excess alkali depending upon the amount of

esters in the sample. All analyses were run in duplicate.

Results.
The data given.in tables 5 and 5 shows that in the cider
medium and in the malt extract broth the S form of g. aceris-

sacchari reached a peak in volatile acid production by the

()1

4 th day. This peak was followed by a rapid decline in the
concentration of the volatile acids until approximately 50

per cent of the.maximum amount remained by the 75th day.

Table 1 shows that in the synthetic medium the volatile

acids increased steadily up to the 90th da,, however, the
concentration at that time equaled the concentration of the
volatile acids in the malt extract broth when at their peak.
It is interesting to note that although the maximum production
of volatile acids in the cider medium reached little more than
half the maximum production in either of the other two media,
a decline in ccncentration nevertheless took place. These
same tables sh‘w that by the 75th day the percentage of alcohol
was practically the same in the synthetic medium and in malt
extract broth, but that the cider medium contained 49 per cent

more. There was 24 per cent more esters in the cider medium

-8-

than in the synthetic medium, and 96 per cent more esters in
theInalt extract broth than in the synthetic mediume

During the 75 day fermentation period dissociation occured
in the yeasts. However, the increase in ester production is
directly attributable to the action of the S forms on this
medium rather than to the intermediate fonns since, as will
be shown later, the R form of this yeast produced less
esters than did the 3 form.

Figure 2 is a graph showing the relationship between
alcohol, volatile acids, and esters produced by the S form

of S. aceris-sacchari in cider medium. It will be noted that

 

the amount of volatile acids and alcohol increased rapidly
after the 5th day, yet the ester production was very scant
until approximately the 40th day after which tune they
increased rapidly.

Tables 2, 4, and 6 show that the volatile acids produced

by the R form.of g. aceris—sacchari, like the S form, howed

 

a continuous increase in the synthetic medium throughout the
entire experiment, but showed a decrease after approximately
50 days in cider medium.and malt extract broth. Unlike the
condition obtaining with the 8 form, the concentration of
volatile acids produced in the sgmthetic medium at the 75th

day was about six per cent

Jl

_reater than the greatest con-

f

centration of this product in either cider medium or malt
extract broth. At 60 days the alcohol production had reached
its peak in all three.media. The cider medium showed 31

alcohol concentration of 5.60 per cent, malt extract broth

4.80 per cent, and synthetic medium, 3.47 per cent by
volume. The ester production in all three media was the

same. Figure 5 shcws graphically the production of fermenta-

tion products by the R form.in cider. It will be noted that

l

t

\

er the ester started production that

L

er

within terldays a
1.1 . . , a. ' 'i - .' ‘ ,., “A. .' _,. ° ‘ ‘. . ' ”4.
there was rapid decrease in the volatile dCldb aLd later a
decrease in the adcohol.

The data prGSented in tables 7, 9, and 11 show that the
amount of volatile acids pro duced by the SR form of 3-1.

anomala was approximately equal in the cider medium and in

the malt extract broth at the 75th day, but there was 65 per

6

cent more in the synthetic.medium. Alcohol roduction

varied greatly in the different media. At the 75th day the
synthetic medium contained 1.40 per cent alcohol. the cider
medium contained 2.52 per cent alcohol, and the.malt extract
broth contained 5.45 per cent alcohol. The relatively
large amount of alcohol in the latter medium.may be due in
part to the presence of a large per cent of true S cells which
appeared in the medium during the latter part of the fermenta—
tion. The difference in ester production Was not large. The
cider medium showed the highest concentration of esters and
the synthetic medium showed the lowest.

Tables 8, lo, and 12 show that the R form 0f.1° anomala
reached a peak of approximately 0.180 grams volatile acids

ract broth and cider medium at the

ct

per 100 co. in malt ex
60th day. In synthetic medium a peak of 0.268 grams per

100 cc. was also reached at the same time. In the malt

-13-

extract broth 135 per cent more alcnhol was produced than in
either of the other two media. This large increase can only
be due to differences in carposition of the media, as
practically no dissociation took place in any of the three
media. The amount of ester production on the 75th day'was
Oéa6,oz,4 and 14,5 grams per 100 cc. in the synthetic medium,

cider medium, and malt extract broth, respectively.

Bone of the S and R forms of g. alcoholophili or 5.

 

cerevisiae Saaz grew in the synthetic medium. Therefore,

 

only the data concerning the fermentation of these yeasts
in cider radium and malt extract broth will be presented.
Tables 13 and 15 indicate the slight fermentation

produced by the 8 form of P. alcoholophila. In malt extract

 

broth the greatest concentration of volatile acids was 0.012
grams per 100 cc., while in cider it was 0.025 grams per 100
cc. The alcohol concentration in both media varied consider—
ably during the fermentation, but did not rise above 0.48
per cent. Only traces of esters were produced.

fables 14 and 16 show that the fermentation produced
by the R form of 2. alcoholOphila is nearly the same as the
fermentation produced by the S fo n, except that more volatile
acids are produced in the malt extract broth than in cider
medium. The slight fermentations produced by the S and R
forms were not due to poor growth of the yeast forms, since

both grew abundantly in the twoxnedia used.

-11-

Tables 17 and 19 show that the maximum vohttile acids

produced by the S fern of S. cerevisiae Saaz in the cider

 

medimn exceeded the amount produced in malt extract broth

by 57 per cent. The alcohol prodmition was much more rapid
in malt extract broth and reached a higher maxim'n alount
than in the cider medium. only a very small amount of esters
was procuced, the highest concentration being 0.02 5 ans per
100 cc. in malt extract broth. When one loopful of malt

extract broth was plated on the 45 day, no colonies resulted.

v a few colonies

0

At 60 days 5 cc. of this medium.produced onl
when plated. At the 75th day 5 cc. of the culture produced
about 400 colonies. The culture was dead in cider by the 55th
day. Plates inoculated with 5 cc. of the medium produced no
colonies. Figure 4 shove the course of fermentation by the

S fornlin cider.

Tables 18 and 20 show that the R form.of S. cerevisiae

 

Saaz produced 50 per cent more volatile acids in malt extract
broth than in cider medium. Lixewise 175 per cent more
alcohol was produced in the malt extract broth than.in the
cider. The ester production was nearly equal in both media,
1.07 grams per 100 cc. being produced in the malt extract
broth. Figure 5 shows a rapid decrease in alcohols and
volatile acids after the 57th day. The esters, however,
continued the rapid rise which Started about the 25th day.

Table 21 shows in which media dissociation tcok place
and the per cent of cells that had reverted at the end of

60 days. It may be noted that in no case was there any change

of form in the synthetic medium. Four of the fOfldS which
dissociated in malt extract broth did nOt dissociate in
cider. Furthermore, the.malt extract broth contained a
greater number of dissociated forms at 60 days than did
the cider in which the same species and form.of yeast was

growing.

Discussion

The camparison of the products of fermentation of the

 

S and R forms of g. aceris-sacchari as given.in tables 1 to
6 inclusive show that the medium is a more important factor
than the form of yeast in determining the amount of volatile
acids produced. Io illustrate, the R form.produced much
more volatile acids in the synthetic medium.and in cider
medium than the 3 form did, but the 3 form produced slightly
more in the malt extract broth ttmrxdid the R form, Without
exception the S form produced more alcohol than did the

R form in the same Kinds of media. In cider and malt extract
media the S form produced more esters than the R form; the
reverse was true in the synthetic medium.

Tables 7 to 12 show no significant different in the
production of volatile acids in the three media between the
SR and R forms of E. anomala. In all media the SR form produced
more alcohol than the R form. The difference was only slight
in the S3nthetic medium, but in malt extract broth the SR
fonn produced 80 per cent more than.the R form did. Ester

produ tion varied with the kind of?medium used. In malt

-13..

extract broth the R form produced 28 per cent more esters than
was produced by the SR form, but the SR form produced 60 per
cent more in the synthetic medium and 30 per cent in the cider
medium than the R form.

Tables 15-16 indicate that there was no significant differ~
ence between the products of fermentation of the S and R forms

of P. alcoholophila.

 

Tables 17 to 20 show that the R form.of S. cerevisiae

 

Saaz produced 22 per cent more volatile acids in malt extract
broth and 58 per centtmore volatile aci‘s in cider than did
the 3 form. The S form far exceeded the R form.in alcohol
production in both media, the ratio in cider medium was 5:1
and in.malt extract broth 6:1. The S form produced practically
no esters, but the R form produced approximately one gram per
100 cc. in both.media.

The S and SR forms of the four yeasts vary to atmarked
degree in their fermentations. Tables 5, ll, 19 and 15 show

that the ratio of the maximum production of volatile acids

in malt extract broth by the S or SR forms of_§. aceris-sacehari,

 

,3. anomala, S. cerevisiae Saaz, and P. alcoholophila is 24:17;

 

 

6:1, respectively.

The ratio for maximum alcohol production in the same
order is 28:52:34El, and the:maximum.ester ratio is 68:44;l;l.
Figure 2 shows graphically the fermentation of cider medium

produced by the 3 form of S. aceris-saechari which is a strong

 

ester Producer. It will be noted that about fifteen days
after the rapid formation of esters began, the Volatile acid

concentration dropped rapidly. E'gure 4 shows the fermentation

-14;-

Of the same medium.by The D form of S. cerevisiae Sssz whhzn

 

is a very weak ester producer. The volatile acids after 25

day of fermentation reached a rather constant concentration

U)

which we naintafined throughout the rest of the fermentation.

A

The dn>p in concentration an the volatile acids in the case

 

of the S form of g. aceris-sacch ri may have been due to the
combination of volatile acids and alcohol to form esters.
the alcohol COLCGLEPSBiOH would not be as notice-
able as the drop in volatile acids concertration due to the
comparatively large amount of the fonner in the media.
Eieures 5 and 5 which are graphs of fermentatiohs caused by

orms show the drop in volatile acid

Fh

ester-producing yea t

(I)

and alcohol which followed the rapid increase in ester
production. The cause of ester formation may be a: endo—
enzyme which acts upon the acids and alcohol after its
liberation by the decomposition of dead yeast cells.

Tables 6, 12, 18, and 16, show that the R forms differ
as much between each other as do the S forms. The ratio of
the maximum production of volatile acids by the R forms of

o 1 _ 9 " . -,,4 “ (W “ Y ' ’fi . {‘ - 5 P ’ '
S. aceris-saccuari, J. afogdfld, S. ceretisiae Saaz and P.

 

 

alcoholgphila is 21:19 lt-l, respectively; of alcohol,

 

24:15:26:l, rengctively; and of esters, 8:11:12:O,

respectively.

-15)—
sunmary and Conclusions

Detailed analysis of the ‘roducts of fe nentation of

 

the S and R ferns of the four yeasts studied, S. cerevisiae

Saaz, S. aceris-sacchari, R. alcoholophila and W. anomala

‘—
_

 

 

in three different media, cider, malt extract broth and a
synthetic medium in general show;

1. In some species of yeasts there is a marked differ-
ence in the fermentation properties of the S and R ferns While
in other species there is little or'no difference.

the S ferns produced more alcohol in a shorter

‘-

2. That
length of time than did the R forms.

3. The production of volatile acids was extremely
variable. The amount produced varied with the different
media, with the species of yeast and with the S and R forms
of the different species. For example in malt extract broth

the S form of S. cerevisiae Saaz produced considerably more

 

volatile acid than did the R form.while in cider the R form

of this suecies produced much more volatile acid than the

&

q

S form. In

cf-

he case of g. anomale there is but little differ-
ence between the S and R fonts in the amount of volatile acid
produced in any of the media. The S and R forms of the other
two species likewise exhibited differences in the amount of
volatile acid produced in the three media.

4. The production of esters by the S and R forms of the
four Species of yeasts studied does not show'as great a
variation as does the volatile acid content. There are some
exceptions to this. Theznost outstanding one is in the case

of the S form of S. cerevisiae Seaz in both malt extract broth

 

-16-

and cider where the amount of esters produced is practically
nil while the amount produced by the R form of the yeast
is one gram per 100 cc. Another exception is in malt

‘

-S."CC{l

.L

extract broth where the S form of 'ceri

(I)
s
w

ri.produzes

IU)
11"

 

28 per cent more esters than the 3 form. In the case of

51'.

W. alanola the R form produces e0 per cent more esters than

H

coholphila produced practically

 

the S form of this veast. P. a

no esters in any of the media.

5. In the yeasts which oroduced esters

‘ ’

t21e e

(D

ters did

Q .
I

not appear in aipreciaole quantities until between the
35 and 45 day after inoculation. Shortly after the esters

appeared there was a correspondin decrease in alcohol and

C;

‘—

volatile acids. In the case of the S form of S. cerevisiae

 

Saaz there was a moderate volatile acid and high alcoholic
content in malt extract broth, but practically no esters
were fonuai

6. The three media used affected the stability of the
yeasts differently. The S and R ferns of all four Species
were the most stable in the synthetic medium. In this medium
there was no tenden 3 for the S form to change to the R form
and vice versa. There was a greater tendency for than to
change in the cider, while in malt extract broth they were

the least stable.

 

 

 

 

 

 

 

 

 

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m OOH L mH .O L 001m L Hmm.o L LLOH .0. L Canoe L mom L mm
L L h? L L \P L
m 00H L mo.o L Hm.o L mon.o L mbo.o L mm¢.o L 5.9 L mm
L L iLI k L L h
m 00H L .«o.o L on.0 L Hmn.o L mno.o L nm¢.o L o.n L ma
L L L L L Phil L L
w 03 L 8.0 L 3.0 L mmné L 30.0 L mend L o4. L m
L L L L L L L
L 00.0 L 00.0 L Hnm.o L moo.o L o¢n.o L m.n L o
L L L P. L L L L
mempmom L QEBH0>L awed Lcfiow oapmowLuLom OLHQEL L
abflcoa ma. Hagme Ln LOLHQS mm oLomL mm mLomme hchLomL Lcounmanmm
maamo m onmme mnopmoL aonooawL mHLewHopnnonL maLpdao>LoHnmpwhpLuL mm L mhmc Ho
m mo ammo nmmL ammo nomLmnoo Lam. Limo nmmL ‘wqoo nmmL ammo nmmL L umpasz
dadfiond wLLHLa mo
gnom m 03p an :oprpqunmm mgansw omonodm ammo nmm 0H mnfiqflmpiuo nmcfio mo wLthmmm .OH manna

 

 

 

 

 

 

 

 

 

um o¢ Lm cm L mm.o L m¢.m L ¢¢¢.o L «La.o L mHL.o L o.n L LL
J F L P P L ,L L
Mm LN m mp. m¢.o L mw.o L ma¢.c L ¢>H.o L ¢LQ.O L L.& L 00
L L L ? L L L
mm LL 4m mm. Hm.o L mm.m L L L Hmo.o L m.n L m¢
L L L L L . L L
mm «mm modm m. oH.o L mm.& L mm¢.o L oLo.o L moo.o L m.m L cm
L L L L L P L
mm 00H L mo.o L Ho.H L mo¢.o L o¢o.o L mo¢.o L m.m L ma
L L L llr Ll L L
L 00.0 L 00.0 L omH.o L moo.o L mma.o L m.m L o
L iv L L I» L L L:
mewpmow L madao> L awed oaamaLcLom omeowLoHom OLLowHL L
.ESH@¢E Lamnpw mm. hp L mm cHomL mm cLowL mm mpfioflowL LcmpmmEnmm
ma mHHwo m cum. myopmm L HOSOOHwLmHprHo>onoqL mHmeHo>L mapmpmhprL mm L mhmo mo
mLmo puma AmmLPnoo LQMprnmo 9mm. Lama nme ammo MWWL ammo pmmL L Lonfisz
wmsaonm wHHHLB mo Show mm mnp Ln nofipmnnmahmw
msflnzo omonuxmc puma awn 0H muflnwmpqOO :ponp nownuxm Lama mo mfimhawnd .HH mapwe

 

 

 

 

 

 

 

 

 

 

 

 

. L
m 00 L0 0 L 00.0 L 00.0 L 00H.0 L 0H0.0 L 00L.0 L 0.0 L 0L
L IF L F L} F L
m 00 .m L L 00.0 . L NH.0 L LLN.0 L 000.0 L 000.0 L L.m L 00
L L L|I L L [L L
m Om 41m CH L OO .O L mH .O L mbmoo L LvHO .O L OmN.O L m.& L mm
L L L L P L L
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L L L L L? L P
0 0L .0 00 L 00.0 L 0H.0 L L00.0 L 0H0.0 L 000.0 L 0.0 L 00
L L L L L, F L
m 0m 40 0L L 00.0 L NH.0 L H00.0 L 0L0.0 L «00.0 L 0.0 L mm
L L L IF L L [r
m N Am 00 L 00.0 L 00.0 L 000.0 L 000.0 L Lon.0 “ L.¢ L 0L
L L L L Li L L
m 00H L 00.0 L :6 L 3nd L 0006 L «and L min L m
L L L P L L L
L 00.0 L 00.0 L Hmmu.0 L $00.0 L meo0 L m.& L O
L L L L L L L
L L 0530p. 300 0:08.300 03009300 0220. L
£3003 5 .3303 gnaw. Ln L 0.0 300. mm 30.0me Ling? Lompamanmm
0300 m 080 m. 00 0.3000. H3636. «HapflopnaonL 0393033909033. mm L 930 Mo
mo p900 nmmL L000 39580 nomr p000 3%. ammo ummr L000 3%? L .3352
waanmoaonooam 033m mo 050% m
03 L0. n3 panamanow mfipdc 0008230 300 90m 0H mfifiapgo L030 mo 03323 .3 03.09

 

 

 

 

 

 

 

 

 

 

\h L F‘
m 00H L 00.0 L 00.0 L 00H.0 L mH0.0 L bHN.0 L 0.0 L 0k
L L L L {L L L
m 00H L 00.0 L H&.0 L 0NN.0 L bH0.0 L mflN.0 L b.& L 00
L L IL F‘ L» F |L
m 00H L 00 .0 L 0H .0 L mumm .0 L 0H0 .0 L H0m.0 L 0.0 L mm
L L L L + L L
m OOH L 00.0 L 0H.O L mmm.0 L «H0.0 L bwm.0 L 0.0 L 0¢
L L; L L L L L
m 00H L 00.0 L NH.0 L N¢0.0 L NH0.0 L $00.0 L 0.0 L 00
L {P L L L L L
N 00H L 00.0 L 50.0 L mmm.0 L 0H0.0 L m¢&.0 L 0.0 L mm
L L L L L L L
m 00H L 00.0 L 00.0 L Nflm.0 L 000.0 L H0&.0 L 0.0 L 0H
.F rLt »; L L L L
m 00H L 00.0 L 0H.0 L $00.0 L 000.0 L «00.0 L 0.0 L 0
L L L L L L L
L 00.0 L 00.0 L H0&.0 L 000.0 L 000.0 L 0.0 L 0
L L L L L
L m deHo>u dwow OHHQEdeQw owpmomLcwow UHHQSL L
adfidma :LLmudumow HaguoL hp L mm cflowL mm cwowme hpacaod. Lcmanmenmw
maamo m 0:0 m. 00 mumpmmL HonnungoawumHo>unonL mafipwao>LmapwpmanpL mm L mmwc
00 Puma HWWL p200 umMLmqmo Hmmh. p200 AmmL ammo nmmL #:00 90m+[ Pmo 900852

 

 

 

adasmoaogooaw wfinofim mo Show m 039

hp nofipwpamanmm mnflndc amoupN00 anoo 00% OH mnfinwmpcoo nmoLo Lo wflmhfiwad mflpma

.wa

 

 

 

 

 

 

 

 

 

 

L L L
m 00H L 00.0 L 00.0 L 00H.0 L 0H0.0 L bHN.0 L 0.0 L 0b
L L L \L \L L L
m 00H L 00.0 L H0.0 L mNN.0 L bH0.0 L m¢m.0 L b.0 L 00
L L L T‘ Fl P‘ |L
m 00H L 00.0 L 0H.O L 000.0 L 0H0.0 L H00. L 0.0 L 00
L L L L L L L
m 00H L 00.0 L 0H.0 L mm0.0 L ¢H0.0 L b00.0 L v.0 L 00
L L L L L L L
m 00H L 00.0 L NH.0 L 000.0 L NH0.0 L $00.0 L 0.0 L 00
L xi? L L L L L
m 00H L 00.0 L 50.0 L 000.0 L 0H0.0 L mfi0.0 L 0.0 L mm
L L L L L L L
m 00H L 00.0 L 00.0 L N00.0 L 000.0 L H00.0 L 0.0 L 0H
L. Lt r» L L L L,
m 00H L 00.0 L 0H.0 L $00.0 L 000.0 L «00.0 L 0.0 L 0
L F Ly L L L L
L 00.0 L 00.0 L H00.0 L 000.0 L b00.0 L 0.0 L 0
L L L r> L L L
L L 0§HO>L dHOd OHHLNSLMLHOQ owumomewow OHHLQEL L
abwwma :LL0000000 azzp0L hp L 00 0L00L mm 0H00L00 hpaoaomL L00pm0en0m
mHHmo m cfiw 0. m0 mnmpmmL HOflnbAwLmHHuflfioblfiofiL mHprHOPLmHprmhpflpL mm L mhwc
Mo P000 meL 0:00 L0MLpn00 H0mLx 0:00 n0mL 0:00 n0mL LQ00 H0ML[ Lmo A0pfidz

 

 

0LL000L0000H0 0L00L0 00 0000 m 000

hp noH000u0an0m mnflndc 0monpx00 ps00 p09 OH mnfinampaoo n00L0 Lo mfimhamad magma

.fia

 

 

 

 

 

 

 

m ¢ .m L .mm mm. No.0 L om.o L mwm.o Loo.o L omm.o L m.« LL
b L r L [P
m OOH L H0 .O L NW .0 L 0HN.O 000 .0 L 000.0 L 0.¢ 00
L J L . Ll .L L r
mLLH am «H m NLLL HO .O L mmd L Hmmd L mH0.0 L mwmd L mew L ma.
L L b L L L
m m «Mm mm. OOLO L wH.O L OLH.O L mO0.0 L HmH.O L m.¢ L on
LI L L L L L L
m OOH L 00.0 L Om.O L OMHO L mO0.0 L H¢H.O L H.m L mH
L p L L! L L L
L 00 .O L 00 .O L mmH .O L mOO .O L mnH .O L Nd L O
L b L L L L L
L mumpmow L oasHoLLL gum 33.3.33 033933 oHpowHL L
€53an LS. HLEpoL Lap L mm god. ma uHowL ma L333? Lcmpdmahmm

oHLLmLOLL .LpapanLp. ma . wage Lo
psoo nmmr LL80 nmmr L .3252

mHHmo m cam <Lmd mumpmmL H0£00HwLmHHpmH0>InonL
mo puma nmmL Lame nanHuoo 9mm. puma nme

 

 

mLanoaonooaa aLnOLm Lo anon m osL_Lp
nogmpgmmfiwm mansw mmonpxmc ammo neg OH mnanamfibo L393 pownpwo pHmE Ho £anng .mH mewa

 

 

 

 

 

 

 

 

m 00H L 00.0 L 00.0 L mom.0 L 0H0.0 L mmN.0 L 0.¢ L 0b
L - L L L. L L L
m 00H L H0.0 L 00.0 L $00.0 L 6H0.0 L 000.0 L m.¢ L 00
L L IF L LLF L L
m OOH L H0.0 L Hm.O L LON.O L ON0.0 L me.o L m.¢ L mw
L L L P I! LI L L .
m OOH L 00.0 L mm.O L L L LOm.O L O.¢ L om
L r L LL L F L
m OOH L 00.0 L NH.O L mHH.O L NO0.0 L me.O L H.m L mH
p? L L L L L L
m OOH L O0.0 L 00.0 L OmH.O L mO0.0 L OMH.O L m.m L O
Llrf L? L L P p L
L mpwpmom L mafiHOLL cfiow oLpowHLvLom owpmowLoaow owpowHL L
agacma Lthpm ma L hp L mm OHom L ma nHomL mm LpLOLomL acmLQmanmm
2H mHHmo m and m. mumpmmL HOQOOHmLmHmeHopnnonL oHmeHo>L anwpwanpL mm L mhmo Ho _
mo Lama 9mm. Lame AmmLpgmo nmmL Lama nmmL Mums nmmL Pfimo nmmr. L nmafism

 

 

LLLELLOLESE .9303. mo ago“ m 2:. LE
qoflpwpqmanmw mqfinso mmonpmmo ammo nmm OH mnanfiwpnoo apomp pomnpxm.pHma Mo mammHmnd .OH oHnwa

 

 

 

 

 

 

 

 

 

 

 

 

.00 m L 00.0 L H¢.m L No¢.0 L $00.0 L 0m¢.0 L Mom L mb
5 LLLLLLonm ELL L L L L L L L
.00 m L O0.0 L Lm.m L mmm.0 L HO0.0 L ¢O¢.O L m.» L mm
LLH spacnm oz. k L L .L L .L
HSHQUOH OQOL H0.0 L moon L b0¢.0 L bbo.o L mmfloo L m.m L am
3,. fisonm oz. L , k L L r L
m OOH L Hooo L acom L H©¢.O L Omo.o L Ommoo L m.n L md
L L \b b} L L P
m OOH L H0.0 ,L N¢.¢ L >&¢.O L Obo.o L mam.o L m.n L on
L L L L L r L
m OOH L H0.0 L mm.¢ L mH¢.O L mL0.0 L OO¢.O L O.n L mm
L L L L L L V
m OOH L L fim .H L 00m .0 L L mdo .0 L bfim .O L mom" L mH
L; \r I» L L L L
m OOH L 00.0 L mm.o L OL¢.O L OO0.0 L OO¢.O L O.& L m
L L L [L Ll L L
L 00.0 L 00.0 L mmm.o L OO0.0 L oo¢.o L 0.9 L O
L LIII P L F L r
L 3325. masHobL god OHHwaLcHow 0.33.». How oHHdaL L
85258 LHhspo mm. ho. L 3 god. mm 33me L333». 63:3an
qH mHHmo m and. magma L HonoLLHmLoHL pwHoLTnoLLL oHHpmHomeHnwpmanpL mm L 93c Ho
‘mlho ammo nEnmo 90%“ng nom% ammo non. ammo mom. ammo mm? L Meagan
«3m omHmHLLonmo mogogoodm no Show m
23 L3 no“ pgnmanmm mfifidc amongm ammo 9mm OH manHmunoo .393 no 393mg .LH 39me

 

 

 

 

 

 

 

 

 

 

m OOH L OOoH L mb.O L w0¢.O L $00.0 L mb¢oo L Hon L mb
L L L! . L L P L
m OOH L mmoo L HN.H L ¢O¢.O L mOH.O L Hmm.O L m.m L no
L L L {L L L \P
m OOH L on.O L Om.H L m$oo L ONH .O L Hmm.O L mim L mm
L L L tL L‘ . L L\
m OOH L mm .0 L ®¢.H L ¢m¢.O L NHH.O L mbm LLO L mom L mHV
L L L tr L L \F
m OOH L wH .O L QNH L m$ .O L OLLO.O L «mm.o L O.mLL L m&
L L L L P .r L
m OOH L LO .0 L mm .H L O$.o L LLHVO .O L mmuv .O L 03b; L mm
L L r L L L L
m OOH L no.0 L L>.O L Om¢.o L ON0.0 L mm¢.O L m.m L mH
F L L L L L P
m OOH L OO .0 L ASN.0 L 00$ .0 L NH0.0 L Mbfi .O L m.& L m
Ir L L L F L L
L 00 .O L OO .O L mm &.O L 000 .O L cow .0 L O .n L 0
TI\ L LI r L L L
L mpwpmow L ®§HO>L CHOG OHHQHHHLOHOQ OHPOOGLUHOQ OHHGHLLL L
533 L HELL? E L 8 3%. mm 33me L330? Lumpumanmm
HHH mHHOO m EQLmd mHOPmOL HOQOOHGLOHHPQHOLVIQOHLL GHHPmHOPLQHQQpQHPHPL mg L thc MO
m MO EOO HOPL PQOO HOPLvQOO H0? #900 HONL WHHQO HOWL ”CHOU .HQW— L H0952

 

 

uwmm memH>mnmo mwohaonwnoowm mo Show w
map hp noflpwpsoanmm mafinsc omOLOdm ammo mom OH mnHmepaoo anHo Ho mHmmHmnd .OH mHnwe

 

 

 

 

 

 

 

 

 

.00 m :H neaonm. No.0 L m>.o L Lom.o L mmo.o L omm.0 L H.¢ L mp
ouwhwcofi mxmm LMm W L L L L L L L
.00 m SH nuBOM_L H0.0 L ON.® L mON.O L 000.0 L mmN.O L n.¢ L 00
anom mlm .m mm» L L L . L. L}
LLLLwOL mac. Lo.o L «L.o L mum.o . «no.0 L mon.o L n.¢ L ma
an...” EPBOLHM 02» L L L{ L L L
m 00H L 00.0 L mm.m L Omm.o L «no.0 L H¢n.o L m.¢ L on
L L P L I.[ L L
m 00H L 00.0 L oo.¢ L mmm.o L L¢o.o L mam.o L n.¢ L ma
P L L L L L L
L OO .0 L 8 .O L ONH .O L N00 .0 L 09H .0 L N. m L O
F L L L L L L
L L meLSHOPL dHod OHpOwHLGHod OHPQOQLGHOG OHpOmHL L
.55Luma Lopmpmom Hanna. ha L mm dflomL ma cLowL ma hchLowL Lompnmanmm
ma maamo m and. mm mgopmm. HonoonLmHLLmHo>un0nL mawpwao>L manwpwnaapL mm L mama mo
m molwnoo nmmL ammo ummLpzmo ummL Puma HmWL ammo new». name ammm L nmnfisz
udwm mwflmwbmnmo mmohfionwgoowm mo Show m map an
HOLpapnmanmm mawnsc omonpxmd ammo nag 0H mqanflwunoo nuoun pomnpxm pads mo mflwhamq¢ .mH magma

 

 

 

 

 

 

 

 

 

_
m OOH . bO.H . mHom . HON.O . MOHoo . fiomoO . mom . mb
Ir — b Ll p u u
m 00H . m>.o . am.¢ . mmm.0 . mmH.o . mmm.o . m.m _ om
» L F b r Ir -
m 00H . um.o _ mm.¢ . bam.o . boo.o . o>¢.o _ o.¢ . m¢
- L » pl b L h
m 00H _ mo.o . oo.m ; wom.o . mbo.o . wbm.o . H.¢ . om
P b . p b r .
m 00H . No.0 . m¢.H . fiom.o . Ono.o . m¢m.o . ¢.¢ . ma
» h P I»! L {P L
m 00H . 00.0 _ 00.0 . mmH.o . moo.o . mnH.o . m.m . o
b p L n L p —
asflcmfi . . wadao>. cfiow capowa.caow oaumom.cfioa oagowa. .
:H mHHmo_mpapmom flagpo. an _ mm ”How. mm cfioa. mm haacaod. .cmpnmaumm
m and m mo. ma mumpmn. Honooam.maaumao>::on. oaapmao>. mflnmpmnpwp. um . mama mo
ammo ppm r, pawn nmm.pnmo yam; Fame 9mm» ammo mam. mama umm. [3 909852
uwmm owfiwfi>mumo mmohfionwzoowm Ho Show m map an
godpmemaumH mnflmsc mmonpwmc ammo Hog 0H mnHuHMpnoo sponp pomnpxo pama mo mammamnd .om manna

Table 21. Showing

stability of the yeast forms

influence of various media upon the

 

lgedium.

r
I
I
I
S; ithe ti o j'S
I

Yeast forge 'Ko.of”deys
'bcfcre rever-‘cells reverted

'SiQL began
I

form of S.aceris-sacchari' Kc chagge

I

'Per cefitiof

' at 60 daysfig

 

I
I
I
I
I
I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cider " " " " " " ' R0 cherne
_ T I V
Malt extract'" " " " " " ' 0 80 3
gynthetic 'R fonn of S.aceris-saccheri' No change '
I r I
Cider III II II II II II I NO C‘nar,ye I
I r ' fl
Malt extract'" 1 " " " " ' 15 ' 97 a
Sznthetic 'SR form of W. ancmala ' No change '
I , I fl
Cid er I II II II II I 35 I 50
F T —T
Malt extract' " " " " ' 15 ' 75
T —L I :
Slnthetic 'R fern of W. ancmala ‘ No change '
fi r— r 3
Cider '" " " " " ' No change ‘ No change
I T‘ T
Malt extract'" " " " " t 45 t 4
Cider '8 form of P. alcoholcphila ' 5 ' 91
I T r
Malt extract'" " " " " ' 15 I 100
Cider 'R form of P. alcoholophila ' No change '
7' T I
Malt extractl" " " " " ' Kc change '
Cider 'S fern of S.cerevisiae Saaz' No change
I 'T
Malt extract”' " " " " " ' 45 ? 5
Cider 'R form

 

I
Halt extract'"

II

of S.cerevisiae 8832' No change
fir

II

II

" " ' No change

Ifl-I-JI

 

1.3. E 35... 16% 5 39E.

 

 

  

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‘Acflwnnllll tin: \mufluntlwl.‘
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-17-

Literature Cited

Fabian, F. W., and thullough, N. B.
Dissociation in yeasts.
Jour. Bact., vol. 27, No.6, June, 1934, p. 583-625

Official and Tentative Rethods of Aralysis of the
“ssociation of Official Agricultural Chemists,
Third Edition, 1930, Washington, D. C.

A013: 01'! LED Gil-LEN T
The writer wishes to eXpress his appreciation
to Dr. F. W. Fabian‘wio first interested him
in the dissociation studies of yeasts, to
Dr. W. L. Kallmann who taught him the technique
of taking photomicrographs, and to the men
of the chemistry deparmment who advised him

concerning the analytical work.