Antifungal assay: Minimal inhibitory concentration (MIC).

Two pathogenic fungi namely, C. albicans (ATCC 10231) and M. furfur (ATCC 14521) were used in this study. They were maintained and cultured according to the CLSI recommended methods. The micro dilution method was used to determine the MIC of tested samples (24). A stock solution of 1.0 g/ml for each tested sample was prepared in dimethyl sulfoxide. These stock solutions were two fold serially diluted with the RPMI 1640 medium (Life Technologies, Gibco®) into five different concentrations (50, 25, 12.5, 6.45, 3.125, 1.562 mg/ml). Subsequently, these tested samples in different concentrations were evaluated in 96-well plate for antifungal activity. Briefly, 40 μl of the tested samples were placed in each well and 10 μl of fungal suspension was added to each well. The positive control comprised of 40 μL of RPMI medium and 10 μL of fungal suspension, whereas the negative control contained 50 μL of RPMI medium. The micro plates were incubated at 37°C for 24 h (24). After that, 10 μL p-iodonitrotetrazolium violet (Sigma Aldrich, USA) (2 mg/mL, in water) was added to each well and the micro plates were further incubated at 37°C for 48 h. MIC was defined as the lowest concentration that inhibited the colour change of p-iodonitrotetrazolium violet (25).