Pure ascospore suspensions of three strains of Saccharomyces cerevisiae have been prepared by sequential treatment with 2-mercaptoethanol, Glusulase, and sonication. Light microscopy and acid-fast staining revealed the presence of only ascospores. Various germination media tested using the ascospore preparation revealed different nutritional requirements for the germination of auxotrophic and prototrophic yeast strains. Acid-fast staining of the germinating ascospores was shown to be an effective method for studying the kinetics of ascospore germination. The anaerobic nature of ascospore germination was demonstrated using this staining technique.