575

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

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

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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 Andreasen, 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 importance 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 number of brewers' yeast cultures and have obtained evidence that these cultures 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 cultures 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 lyophilization. The destruction of such a high proportion of the population might be expected to show evidence of selection even though the degree of nonhomogeneity is not great. In order to determine whether any change in growth requirements 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

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L.

ATKIN,

W.

MOSE8,

[VOL. 57

AND P. P. GRAY

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-

7C219 (stock) ...............................

13.2

0.2

11.8

12.6

14.5

................... 7C219-Lla.1 (lyoph.) ..... 7C219-Lla.2 (lyoph.) ........................

12.8 12.6 12.8

1.1 0.6 0.1

5.1 2.2 9.2

10.8 7.4 12.6

8.4 6.8 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

7C219 (stock) ........

13.2

0.2

11.8

12.6

14.5

7C219.1 (S,)* ......................... 7C219.2 (S,)* ........

7C219.4 (So)* ........ 7C219.5 (S.)*..........

13.2 13.2 11.4 13:2 11.8

0.2 0.2 0.2 0.2 0.3

12.0 9.4 11.8 12.0 11.4

12.0 12.0 12.0 12.0 12.0

14.5 13.7 14.0 13.7 13.2

7C219-Lla.2 (lyoph.) ..............

12.6

0.6

2.2

7.4

6.8

................ 12.6 7C219-Lla.2a (L,)t .. .......... '12.8 7C219-Lla.2b (L.)t ................ 11.0 7C219-Lla.2c (L.)t ..... 7C219-Lla.2d (L.)t ................ 11.4 7C219-Lla.2e (L.)t ............. ...... 11.6

0.7 0.4 0.7 0.6 0.7

2.0 2.9 1.9 1.1 1.6

8.7 6.5 8.4 8.8 7.4

7.4 6.6 8.2 6.8 8.1

7C219.3 (S,)* ...............................

*

S.

Inositol B1 and Bs

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-

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7C219-Lla.3 (lyoph.) .

Inositol Bi and Bo

1949]

577

PRESERVATION OF YEAST CULTURES

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 culture 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)

None

Biotin

Pantot

Inositol

B1 and B6

7C219 (stock) .13.2 7C219.Lla.2 (lyoph.) .11.8

0.2 0.2

11.8 2.6

12.6 6.2

14.5

7C30 (stock) ................................ 17.2 7C30-L6a.5 (lyoph) .11.4

0.6 0.4

14.0 6.8

2.1 1.4

16.7 4.1

7C247 (stock) .13.2 10.8 7C247-LS5a.2 (lyoph.) .......

0.8 0.1

0.2 0.0

5.6 5.2

13.2 2.8

7C81 (stock) ................................ 11.4 7C81-L7a.1 (lyoph.) .11.0

0.7 0.9

0.7 5.7

12.8 5.7

12.6 7.8

12.6 8C166 (stock) .......................... 8C166-L9a.2 (yoph.) .9.0

0.7 0.5

0.5 0.7

0.6 9.8

12.8 9.0

...............

4.6

these examples are found cultures that have suffered a loss or marked curtailment 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 proportion 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, baking, 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

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BIOS FACTOR OMUTTED FROM MEDIUM CULTURE NUMBER

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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,

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100-123. WICKERHAM, L. J., AND ANDREASEN, A. A. 1942 The lyophil proces: its use in the preservation 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.