3.10. Antifungal Assay

Similar to antibacterial activity test method. The MIC was performed by broth microdilution methods asper the guidelines of Clinical and Laboratory Standard Institute [72,73] with RPMI 1640 medium containing L-glutamine, with-out sodium bicarbonate and buffered to pH 7.0. Two-fold serial dilutions of the peptide compounds were prepared in media in amounts of 100 μL per well in 96-well microtiter plates. The test fungal suspensions were further diluted in media, and a 100 μL volume of this diluted inocula was added to each well of the plate, resulting in a final inoculum of 0.5 × 10⁴ to 2.5 × 10⁴ CFU·mL⁻¹ for test fungi. The final concentration of the peptide compounds ranged from 0.5 to 1024 μg·mL⁻¹. The medium without the agents was used as a growth control and the blank control used contained only the medium. Amphotericin B and Bavistin served as the standard drug controls for against medically important fungi and against agriculturally important fungi, respectively. The microtiter plates were incubated at 35 °C for 48 h for Candida species and 30 °C for 72 h for other fungi. The plates were read using ELISA, and the MIC was defined as the lowest concentration of the antifungal agents that prevented visible growth with respect to the growth control. The experiments were repeated at least thrice.