Growth Curve Measurements by Microcultivation

Growth curve measurements by microcultivation were performed as previously described (44 (link)). Briefly, the log-phase cells were centrifuged and resuspended with fresh medium with or without stresses to get a starting OD₆₀₀ of 0.05. One hundred microliters of cell culture was added to each well of Costar clear polystyrene 96-well plates. Three or four biological replicates were analyzed for each strain. The 96-well plate was sealed with Breathe-Easy membrane (Sigma, lot number MKBZ0331) and then cultivated in an Epoch2 microplate photometer (BioTek) at 30 °C for 36 h for YPD and for 72 h for stress conditions. The OD₆₀₀ values of cultures were recorded every 10 min after 30 s of shaking at 282 rpm. The data were analyzed using the GraphPad Prism 8 software to generate growth curves and doubling time. Concentrations for liquid microcultivation were as follows: 2% galactose, 3% glycerol, 2 µg/mL Nocodazole, 8 mM DTT, 1 M NaCl, 6% ethanol, and 0.3% acetic acid.