Towards standardised yeast viability measurement: A critical evaluation and systematic comparison of staining parameters

Determining yeast cell viability is critical, as yeast cells serve as both model and production organisms. Viability, defined as the proportion of living cells to the total cell count, is essential for process monitoring as well as optimising processes and product quality. Despite their widespread use, dye-based viability assessments lack standardised protocols, and literature reports inconsistencies and debated mechanistic foundations. To address this gap, we systematically evaluated the effects of staining solution pH (3.0, 7.8 and 10.6), dye concentration, and yeast strain (baker’s yeast Saccharomyces cerevisiae, and brewer’s yeasts W-34/70, and WB-06) on the reliability of image-based methylene blue (MB) and trypan blue (TB) viability assays. Highly alkaline conditions (pH 10.6) produced unreliable results, while higher dye concentrations improved discrimination between viable and non-viable cells without shifting mean viability. MB and TB exhibited broadly similar behaviour, indicating that reported MB inaccuracies are likely method-related rather than dye-specific. Across multiple strains, no single parameter set was universally optimal, but pooled analysis identifi ed MB at pH 3.0 as the most robust and reproducible condition. These findings support acidic MB staining at higher concentrations for automated, cross-strain viability assessments and reinforce models in which dye exclusion, rather than intracellular decolourisation, determines viability-dependent contrast.