Demonstration of yeast bud scars with the electron microscope

Barton (1950) clearly demonstrated yeast bud scars with the optical microscope. These scars are important since they enable one to determine the number of buds formed from a single cell and hence its comparative age. It is obvious also that if a bud is never formed at the site of an old bud (Barton, 1950) then the reproductive age of a cell is definitely limited. Methods designed to demonstrate yeast bud scars therefore should be of interest to cytologists. The present authors (Bartholomew and Mitt-wer, 1952) have used ultraviolet photolytic methods to obtain good electron microscope pictures of internal yeast structure. These same procedures slightly modified also clearly demon-strate yeast bud scars. This paper presents some of the pictures obtained. METHODS Saccharomyces cerevisiae (American type cul-ture no. 4110) was grown on wort agar, and cells of various ages were used for this study. Cells were fixed by suspension in Bouin's solution or by osmic acid vapor when the cells were in a wet film. Irradiation was accomplished at a dis-tance of 12 cm from a Westinghouse WL-15 sterilamp which is a low pressure quartz mercury vapor lamp emitting the bulk of its energy at 2,537 A. The irradiation treatment consisted of drying a thin film of yeast onto the surface of a watch glass, and exposure to the ultraviolet lamp for 48 hours. A small amount of distilled water was added then to the watch glass and the cells resuspended. Irradiation of the cells in suspension was continued then for an additional 48 hours. Following irradiation the cells were placed on the usual collodion membrane and observed in an RCA model EMU 50 kv electron microscope. Observations were made with and without chro-mium shadowing; however, shadowing gave the best results. Ascospores were induced by the use of a modi-fied Henrici vegetable juice medium (Wicker-ham, Flickinger, and Burton, 1946). Ascospores appeared in from five to seven days. The pres-ence of ascospores was determined first by stain-ing methods before electron microscope observa-tions were made. The bacterial spore stain of Bartholomew and Mittwer (1950) was observed to produce beautiful ascospore stains