Determination of carbon assimilation patterns of yeasts by replica plating.

For the differentiation of species of yeasts, considerable value is attached to the ability of aimilating various carbon sources for growth. Beijerinck (1889) introduced for this general purpose the auxanographic plate technique which was adapted by Lodder (1934) for the study of sugar assimilation in the nonfermenting strains of the asporogenous yeasts. Diddens and Lodder (1942) expanded its use to all species (fermentative as well as nonfermentative) of the genera Candida, Brettanomycem, and Trichosporon. Lodder and Kreger-Van Rij (1952) finally applied the method to all species of the perfect and imperfect yeasts. These authors modified the synthetic mineral medium previously used by adding a trace of yeast extract or a mixture of the B vitamin to insure better growth of those yeasts possessing one or more growth factor requirements. Only glucose, galactose, sucrose, maltose, and lactose were tested by the auxanographic procedure. Okunuki (1931), who used a synthetic agar in test tubes, was the first to recommend the use of a much larger assortment of carbon sources. The amount of growth after 5 days was taken as the criterion for utilization of the carbon source. Wickerham and Burton (1948) reviewed the work of a number of other investigators and pointed out the lack of a well defined, universal and complete basal medium. Such a medium was developed by these authors and was termed yeast nitrogen base. It is used in liquid form and is supplied with 0.5 per cent of the carbon source to be tested. Turbidity readings are made after 7 and 24 days to determine the amount of growth. The number of carbon compounds used was greatly expanded, and the procedure was applied successfully in a study of yeasts belonging to the genus Hansenula (Wickerham, 1951). Lederberg and Lederberg (1952) developed a method of replica plating to permit the transfer of a pattern of microbial growth from one initial agar plate to a series of others. In the procedure, sterilized velveteen is used which is stretched over the top of a cylindrical wooden block. The agar plate carrying the initial colonies is inverted onto the fabric and pressed down gently to transfer the growth to the cloth. The fabric then provides the inoculum for transfer to subsequent plates impressed in the same way. The Lederbergs recommended their method in general for all routine tests involving repetitive inoculation of many isolates on different media. Because of the extreme rapidity and simplicity of the procedure, we felt that it might be useful in the study of carbon assimilation in yeasts. When surveys of yeast floras are made, involving the handling and identification of large numbers of strains, the method should allow a great saving in time, especially when using a great number of carbon compounds. The present paper gives the results of our application of the replica plating method to the study of carbon assimilation by yeasts.