A SPHEROPLAST RATE ASSAY FOR DETERMINATION OF CELL-WALL INTEGRITY IN YEAST

The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measure d spectrophotometrically in hypotonic solution in the presence of Zymo lyase, a mixture of cell wall-digesting enzymes. The optical density o f the cell suspension decreases as the cells lyse. We optimized this a ssay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1-5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5-15% for different cultures of the same strai n. This assay can quantitate differences in cell wall structure (1) be tween exponentially growing and stationary phase cells, (2) among diff erent S. cerevisiae strains, (3) between S. cerevisiae and Candida alb icans, (4) between parental and mutated lines, and (5) between drug- o r chemically-treated cells and controls. (C) 1998 John Wiley & Sons, L td.