3.9. Antifungal Assay

The fungus Candida albicans ATCC 10231 was streaked onto a Sabouraud agar plate and was incubated at 37 °C for 48 h. One colony was then transferred to fresh Sabouraud broth (15 mL) and the cell density adjusted to 10⁴–10⁵ CFU/mL. Test compounds were dissolved in DMSO and diluted with H₂O to give a 600 µM stock solution (20% DMSO), which was serially diluted with 20% DMSO to give concentrations from 600 µM to 0.2 µM in 20% DMSO. An aliquot (10 µL) of each dilution was transferred to a 96-well microtiter plate, and freshly prepared fungal broth (190 µL) was added to each well to give final concentrations of 30–0.01 µM in 1% DMSO. The plates were incubated at 37 °C for 24 h and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate (BMG LABTECH, Offenburg, Germany). Amphotericin B was used as a positive control (30 µg/mL in 10% DMSO). Where relevant, IC₅₀ values were calculated as the concentration of the compound or antifungal drug required for 50% inhibition of the fungal cells using Prism 7.0 (GraphPad Software Inc., La Jolla, CA). The antifungal results are presented in Figure S32 (Supplementary Materials).