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

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. In this study, we systematically characterize a two-dye (SYTO 9 / Propidium Iodide) LIVE/DEAD assay coupled with flow cytometry to rapidly and scalably quantify post-stress survival in diverse yeast species. By optimizing staining buffer, dye concentrations and staining time, we minimized artifacts and improved reproducibility. Notably, we identify an “Intermediate” population containing damaged cells with enhanced SYTO 9 uptake but little propidium iodide (PI) accumulation under sublethal stress, providing finer gradations of cellular damage compared to CFU or PI staining alone. We demonstrate 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, the SYTO 9/PI staining effectively distinguishes sublethal from lethal doses, offering a valuable alternative for rapid, high-throughput survival quantification.