2.9.3. Antifungal Activity

Antifungal activities were investigated by using the following fungi: *Candida albicans*(ATCC 10231), *Candida parapsilosis* (ATCC 22019), and *Candida zeylanoides* (ATCC 20356). Spore suspension (1.5 <sup>×</sup> 105) was obtained by washing agar plates with sterile solution (0.85% saline, 0.1% Tween 80 (v/v)), then added to each well for a final volume of 100 μL. The minimum inhibitory (MIC) and minimum fungicidal (MFC) concentrations assays were performed using the microdilution method by preparing a serial of dilutions in 96-well plates. The extracts were diluted in 0.85% saline (10 mg/mL), then added to microplates containing Broth Malt medium with inoculum and incubated for 72 h at 28 ◦C on a

rotary shaker. The lowest concentrations without visible growth (at the binocular microscope) were defined as minimal inhibitory concentrations (MICs). The fungicidal concentrations (MFCs) were determined by serial sub-cultivation of 2 μL of tested extracts dissolved in medium and inoculated for 72 h, into microtiter plates containing 100 μL of broth per well and further incubation for 72 h at 28 ◦C. The lowest concentration with no visible growth was defined as MFC indicating 99.5% killing of the original inoculum. The fungicide fluconazole (Sigma F 8929, Santa Clara, CA, USA) was used as positive control (1–3500 μg/mL). All the experiments were performed in duplicate and repeated thrice. Water was used as a negative control.
