Tang Hanxi, Liang Jinye, He Bin Z
Department of Biology, the University of Iowa, Iowa City, IA, 52241, USA.
bioRxiv. 2025 Jun 2:2025.04.14.648826. doi: 10.1101/2025.04.14.648826.
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 , , and after hydrogen peroxide treatment and in 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.
量化酵母应激后的存活率对于生物学、生物医学和工业研究至关重要。传统方法如菌落形成单位（CFU）测定法既耗费人力又耗时。在本研究中，我们系统地表征了一种双染料（SYTO 9 / 碘化丙啶）活/死测定法，并结合流式细胞术，以快速且可扩展地量化不同酵母物种应激后的存活率。通过优化染色缓冲液、染料浓度和染色时间，我们将假象降至最低并提高了重现性。值得注意的是，我们识别出一个“中间”群体，该群体包含在亚致死应激下SYTO 9摄取增强但碘化丙啶（PI）积累很少的受损细胞，与单独的CFU或PI染色相比，能提供更精细的细胞损伤分级。我们证明了该测定法在过氧化氢处理后的酿酒酵母、粟酒裂殖酵母和白色念珠菌中以及两性霉素B暴露后的白色念珠菌中的适用性。虽然CFU在较低应激水平下更敏感，但SYTO 9/PI染色能有效区分亚致死剂量和致死剂量，为快速、高通量的存活率量化提供了一种有价值的替代方法。
