Rapid screening method of Saccharomyces cerevisiae mutants using calcofluor white and aniline blue

Fungal cell walls are composed of polysaccharide scaffold that changes in response to environment. The structure and biosynthesis of the wall are unique to fungi, with plant and mammalian immune systems evolved to recognize wall components. Additionally, the enzymes that assemble fungal cell wall components are excellent targets for antifungal chemotherapies and fungicides. Understanding changes in the cell wall are important for fundamental understanding of cell wall dynamics and for drug development. Here we describe a screening technique to monitor the gross morphological changes of two key cell wall polysaccharides of chitin and beta-1,3-glucan combined with polymerase chain reaction (PCR) genotyping. Changes in chitin and beta-1,3-glucan were detected microscopically by using the dyes calcofluor white and aniline blue. Combining PCR and fluorescence microscopy, as a quick and easy screening technique, confirmed both the phenotype and genotype of the wild-type, h chitin synthase mutants (chs1 Delta and chs3 Delta) and one beta-1,3-glucan synthase mutant fks2 Delta from Saccharomyces cerevisiae knockout library. This combined screening method highlighted that the fks1 Delta strain obtained commercially was in fact not FKS1 deletion strain, and instead had both wild-type genotype and phenotype. A new beta-1,3-glucan synthase knockout fks1::URA3 strain was created. Fluorescence microscopy confirmed its phenotype revealing that the chitin and the new beta-1,3-glucan profiles were elevated in the mother cells and in the emerging buds respectively in the fks1 Delta cell walls. This combination of PCR with fluorescence microscopy is a quick and easy screening method to determine and verify morphological changes in the S. cerevisiae cell wall.