Cell density and cell count measurements
We grew yeast strains from frozen glycerol stocks overnight in 5 mL liquid YNB complete medium (0.67% Yeast Nitrogen Base; 2% glucose). OD600 was measured with a spectrophotometer (BioTek Epoch 2) in 96-well plates with 200 µL yeast culture per well, in four dilutions with miliQ water (2, 2.5, 3.33, 5, 10 and 20-fold dilution) to provide a range of OD readings. Raw OD measurements were blank-corrected. The average OD of each strain was calculated from six technical replicates per dilution, with three independent read-outs obtained from the plate reader. To maintain linearity between cell number and OD600 values, OD must be within the dynamic range of equipment. However, OD frequently reaches values near 10 after 24 hours of cultivation at 25°C. In the current study, we therefore used an OD600 of 0.4 as the upper limit.
For flow cytometry, we used the same cultures that we had prepared for the OD600 measurements, and diluted them further in phosphate-buffered saline (PBS) with a 50X dilution factor in 96 well plates. The plate layout, i.e. the position of strains and replicates on the 96-well plates, were identical in both the spectrophotometer and flow cytometry runs.
Linear regressions between cell density (OD from spectrophotometry) and cell count (from flow cytometry) were performed. Goodness of fit (R2) and 95% confidence intervals were calculated and plotted along the slope in GraphPad Prism 10. Species and strain-specific slopes were extracted as the change of Y per one unit increase in X (i.e. the cell count increasing per OD600unit). We tested for differences between species in slopes, using ANOVA with Tukey HSD tests for pairwise comparisons in JMP (v17.2.0). To assess how much of the variance in slope is explained by differences between strains, we applied a mixed model using ‘species’ as fixed and ‘strain’ as random effect.