The Preservation of Yeast Cultures by Lyophilization.

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