The effect of stresses on the growth of yeast cells was measured by spotting assays. Each of the tested yeast strains (BY4741, MTCC4780, MTCC4781 and MTCC4796) was grown in YEPD medium to achieve a cell concentration of 2 × 108/ml. Each culture was then diluted to an OD600nm of 1, and serial 10-fold dilutions were prepared. Thereafter, 5 μl of each dilution was spotted on a YEPD plate (as a control), as well as YEPD supplemented with ethanol (8%), furfural (0.5 g/l) and 5-HMF (0.5 g/l). The thermotolerant phenotype was tested by incubating the spotted YEPD plate at 40°C.