Combinatorial Identification and Differentiation of Brewing and Wild Yeast Strains Using Phenotypic and Molecular Techniques

Accurate identification of yeast strains is essential for quality assurance in brewing and fermentation industries. In this study, we employed a multi-technique strategy to identify four unknown yeast strains initially submitted as two brewing strains ( S. cerevisiae and S. pastorianus ), a petite mutant, and a non-Saccharomyces wild yeast. Labeling errors during transit obscured the identity of the strains, prompting a rigorous evaluation using complementary phenotypic and genotypic techniques. The identification pipeline included growth analysis on WLN medium, cell morphology assessment via microscopy, detection of petite mutants using the TTC overlay method, differentiation between S. cerevisiae and S. pastorianus through melibiose metabolism in an X-α-gal assay, and PCR-RFLP analysis of the internal transcribed spacer (ITS) DNA region. Growth patterns and colony color on WLN medium offered preliminary phenotypic clues, while TTC overlay and YPG/YPD growth assays enabled detection of respiratory-deficient (petite) mutants. X-α-gal results aided differentiation based on melibiose metabolism. Final strain confirmation was achieved through PCR-RFLP of ITS DNA, which provided species-level resolution. Strain identities were determined as S. cerevisiae (A and D), S. pastorianus (C), and Brettanomyces bruxellensis (B). Strain D was further identified as a petite mutant. While most techniques yielded consistent results, minor discrepancies were addressed through the molecular data. Our findings emphasize the necessity of a multi-prong approach for strain validation in industrial applications, especially when labeling or storage errors arise. We also propose practical solutions to prevent sample misidentification during transport and handling.