Died or Not Dyed: Assessment of Viability and Vitality Dyes on Planktonic Cells and Biofilms from .

Viability and vitality assays play a crucial role in assessing the effectiveness of novel therapeutic approaches, with stain-based methods providing speed and objectivity. However, their application in yeast research lacks consensus. This study aimed to assess the performance of four common dyes on planktonic cells as well as sessile cells that form well-structured biofilms (treated and not treated with amphotericin B). Viability assessment employed Syto-9 (S9), thiazole orange (TO), and propidium iodide (PI). Metabolic activity was determined using fluorescein diacetate (FDA) and FUN-1. Calcofluor white (CW) served as the cell visualization control. Viability/vitality percentage of treated samples were calculated for each dye from confocal images and compared to crystal violet and PrestoBlue results. Heterogeneity in fluorescence intensity and permeability issues were observed with S9, TO, and FDA in planktonic cells and biofilms. This variability, influenced by cell morphology, resulted in dye-dependent viability/vitality percentages. Notably, PI and FUN-1 exhibited robust staining, with FUN-1 vitality results comparable to PrestoBlue. Our finding emphasizes the importance of evaluating dye permeability in yeast species beforehand, incorporating cell visualization controls. An improper dye selection may lead to misinterpreting treatment efficacy.