THE PRESERVATION OF YEAST CULTURES BY LYOPHILIZATION

LAWRENCE ATKIN, WILLIAM MOSES, AND PHILIP P. GRAY

Wallerstein Laboratories, 180 Madison Avenue, New York 16, N. Y.
Received for publication February 25, 1949
Since the pioneer work of Rogers (1914) on drying cultures from the frozen
state and the wide application of this procedure to bacteria, the lyophil process
has been shown to be a successful method for the preservation of yeast cultures
in a viable state over extended periods (Elser et al., 1935; Wickerham and Andrea-
sen, 1942; Wickerham and Flickinger, 1946). The fundamental value of this
method of yeast culture maintenance depends upon the retention by the yeast
of all of its properties unchanged. Such a procedure would be of paramount im-
portance in the fermentation industries, and of no less importance for scientific
collections.
We have recently had occasion to study the influence of lyophilization on a

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number of brewers' yeast cultures and have obtained evidence that these cul-
tures may undergo certain marked changes as a result of lyophilization. In
several instances, the bios or growth factor requirements of cultures isolated
after lyophilization showed changes suggestive of a mutation or segregation of
characteristics.
Incidentally, Dopter (1948) has recently reported changes in morphology and
sporulation tendency in cultures of Saccharomyce8 ellip8oideus that had been
preserved by desiccation in a vacuum. His method of preservation, however,
is not lyophilization as ordinarily defined, since the cultures were not evacuated
and dried in the frozen state. He notes that no signs of sporulation could be
detected in his desiccated cultures and thus the changes could not be ascribed
to segregation.
Our own work followed the standard lyophilization technique as described by
Wickerham and Andreasen (1942). Yeast was suspended in normal horse serum,
frozen quickly, dried under a high vacuum, and finally sealed off. All the cul-
tures thus prepared have been found to be viable up to periods of three months,
at the present writing.
It was observed, however, that a very low survival rate was the general rule.
Upon investigation by the plate count method, it was found that as many as
99.98 per cent of the cells of the original yeast suspension fail to survive lyophili-
zation. The destruction of such a high proportion of the population might be
expected to show evidence of selection even though the degree of nonhomo-
geneity is not great. In order to determine whether any change in growth re-
quirements had occurred as a result of the lyophilization, the revitalized cultures,
which had been allowed to develop in test tubes of liquid malt wort, were plated
out on agar, and random isolated colonies were selected for testing according to
the bios technique described by Atkin, Gray, Moses, and Feinstein.' The parent
1 Paper to be presented at the Second International Congress of the European Brewery
Convention in Lucerne, Switzerland, from May 29 to June 4, 1949.
575
576 L. ATKIN, W. MOSE8, AND P. P. GRAY [VOL. 57
stock cultures were also tested in the same way, both after initial isolation from
brewery yeast and later after subcultures had been lyophilized.
A surprising number of variants were observed in the revitalized cultures.
In table 1 are shown the growth patterns of representative subcultures that were
TABLE 1
Distribution of bios patterns after lyophilization of a yeast culture
(Growth at 40 hours, 30 -mg moist yeast per ml)
BIOS FACTOR OMITTED FROM MEDIUM
CULTURE NUMBER
None Biotin Panto- Inositol Bi and Bo

7C219 (stock) ............................... 13.2 0.2 11.8 12.6 14.5

7C219-Lla.1 (lyoph.) ..... ................... 12.8 1.1 5.1 10.8 8.4
7C219-Lla.2 (lyoph.) ........................ 12.6 0.6 2.2 7.4 6.8

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7C219-Lla.3 (lyoph.) . 12.8 0.1 9.2 12.6 14.0

TABLE 2
Constancy of bios patterns of yeast cultures upon reisolation.
(Growth at 40 hours, 30 C-mg moist yeast per ml)
BIOS FACTOR OMITTED ROM MEDIUM
CULTUE NUMBERl
None Biotin Pantc Inositol B1 and Bs

7C219 (stock) ........ 13.2 0.2 11.8 12.6 14.5

7C219.1 (S,)* ......................... 13.2 0.2 12.0 12.0 14.5
7C219.2 (S,)* ........ 13.2 0.2 9.4 12.0 13.7
7C219.3 (S,)* ............................... 11.4 0.2 11.8 12.0 14.0
7C219.4 (So)* ........ 13:2 0.2 12.0 12.0 13.7
7C219.5 (S.)*.......... 11.8 0.3 11.4 12.0 13.2
7C219-Lla.2 (lyoph.) .............. 12.6 0.6 2.2 7.4 6.8
7C219-Lla.2a (L,)t ................ 12.6 0.7 2.0 8.7 7.4
7C219-Lla.2b (L.)t .. .......... '12.8 0.4 2.9 6.5 6.6
7C219-Lla.2c (L.)t ..... ................ 11.0 0.7 1.9 8.4 8.2
7C219-Lla.2d (L.)t ................ 11.4 0.6 1.1 8.8 6.8
7C219-Lla.2e (L.)t ............. ...... 11.6 0.7 1.6 7.4 8.1
* S. Subisolate from stock culture.
t L. = Subisolate from lyophilized stock culture.
isolated from one parent after lyophilization. It will be seen that one isolate
(Lla. 1) appears to have lost the ability to synthesize pantothenate at the same
rate as its parent, and it also grows more poorly in the absence of B1 and BR.
Another isolate (Lla. 2) has lost the ability to grow in the absence of panto-
1949] PRESERVATION OF YEAST CULTURES 577

thenate, inositol, and B1 or Bo as well as did the parent strain. On the other
hand, the third isolate shown (Lla. 3) resembles the parent culture quite closely.
The bios pattern that is used as a cultural characteristic is a relatively stable
and reproducible property of the cultures that have been used in this work.
As a measure of this reproducibility, five isolates were made of the parent cul-
ture described above and five isolates were made of one of the altered progeny
(Lla. 2). The bios patterns of the 10 subcultures are shown in table 2, from
which it can be seen that the pattern is quite reproducible. Judged by this
technique, the two cultures are distinctly different strains of yeast.
Variants have been found in more than 50 per cent of the revitalized cultures
examined by this technique. In table 3 the growth patterns of five different
cultures are shown with the pattern of a typical variant in each case. Among
TABLE 3
Effect of lyophilization on bios patterns of brewers' lager yeast cultures
(Growth at 40 hours, 30 0-mg moist yeast per ml)

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BIOS FACTOR OMUTTED FROM MEDIUM
CULTURE NUMBER
None Biotin Pantot Inositol B1 and B6

7C219 (stock) .13.2 0.2 11.8 12.6 14.5

7C219.Lla.2 (lyoph.) .11.8 0.2 2.6 6.2 4.6
7C30 (stock) ................................ 17.2 0.6 14.0 2.1 16.7
7C30-L6a.5 (lyoph) .11.4 0.4 6.8 1.4 4.1
7C247 (stock) .13.2 0.8 0.2 5.6 13.2
7C247-LS5a.2 (lyoph.) ....... 10.8
............... 0.1 0.0 5.2 2.8
7C81 (stock) ................................ 11.4 0.7 0.7 12.8 12.6
7C81-L7a.1 (lyoph.) .11.0 0.9 5.7 5.7 7.8
8C166 (stock) .......................... 12.6 0.7 0.5 0.6 12.8
8C166-L9a.2 (yoph.) .9.0 0.5 0.7 9.8 9.0

these examples are found cultures that have suffered a loss or marked curtail-
ment of the synthetic ability for each of the bios factors and also two apparent
reverse mutations or changes, i.e., the gain of the ability to grow in the absence
of a particular growth factor.
It is recognized that the bios typing technique measures only a minute propor-
tion of the total nujmber of synthetic functions that characterize a yeast cell, and
furthermore a difference in bios requirement is not usually regarded as sufficient
to characterize different yeast species according to the classical procedures. It
should be emphasized, however, that the industrial use of yeast in brewing, bak-
ing, distilling, and wine making depends upon a host of characteristics, many so
subtle as to have eluded the taxonomist entirely. As well as can be judged from
the literature, no one has made a critical study of the influence of lyophilization
578 L. ATKIN, W. MOSES, AND P. P. GRAY [VOL. 57

upon the character of the surviving yeast cells. In view of the results described
in the present communication, it is suggested that lyophilization as a method of
yeast culture preservation should be viewed more critically and tested carefully
before being adopted to the exclusion of other methods.
REFERENCES
DOPTER, P. 1948 Observations sur la resistance de la levure a la dessiccation dans le
vide (Observations on the resistance of yeast to desiccation in vacuo). Ann. inst.
Pasteur (Lille), 1, 161-174.
ELSER, W. J., THOMAS, R. A., AND STEFFEN, G. I. 1935 The desiccation of sera and other
biological products (including microorganisms) in the frozen state with the preservation
of the original qualities of products so treated. J. Imrmunol., 28, 433-473.
ROGERS, L. A. 1914 The preparation of dried cultures. J. Infectious Diseases, 14,
100-123.
WICKERHAM, L. J., AND ANDREASEN, A. A. 1942 The lyophil proces: its use in the preser-
vation of yeasts. Wallerstein Labs. Commun., 5, 165-169.
WICKERHAM, L. J., AND FLICKINGER, M. H. 1946 Viability of yeasts preserved two
years by the lyophil process. Brewers Digest, 21, 48T-52T, 58T.

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