An Optimized FungaLight LIVE/DEAD Assay Quantifies Post-Stress and Antifungal-Treatment Survival in Diverse Yeasts Using Flow Cytometry

Quantifying post-stress survival in yeasts is crucial for biological, biomedical, and industrial research. Traditional methods like Colony Forming Unit (CFU) assays are labor-intensive and time-consuming, while dye-based survival assays lack standardized protocols and systematic characterization. This study optimizes a two-dye (SYTO9 / Propidium Iodide) FungaLight assay coupled with flow cytometry to rapidly and scalably quantify post-stress survival in diverse yeast species. By optimizing buffer conditions, dye concentrations, and staining time, we minimized artifacts and improved reproducibility. Notably, we identified an “Intermediate” cell population with enhanced SYTO9 uptake but little propidium iodide (PI) under sublethal stresses, providing finer gradations of cellular damage compared to CFU or PI staining alone. We demonstrated the assay’s applicability across Candida glabrata , Saccharomyces cerevisiae , and Candida albicans after hydrogen peroxide treatment and in C. glabrata after Amphotericin B exposure. While CFU is more sensitive at lower stress levels, FungaLight effectively distinguishes sublethal from lethal doses, offering a valuable alternative for rapid, high-throughput survival quantification.