Time-Kill Assay

After overnight culture, C. albicans was diluted with YPD medium and the culture was incubated at 37°C until the log-phase of growth was achieved. C. albicans suspension (5 × 10⁵ CFUs) was transferred to the flasks containing 20 mL of YPD and FLZ (8 μg/mL) or Meth-Gin (2 mg/mL) or a combination of FLZ (8 μg/mL) and Meth-Gin (0.25, 0.5, 1 and 2 mg/mL). The flasks were kept in a shaker bath and samples (0.5 mL) were taken in duplicates at baseline and 1, 3, 6, 12, 24 hours. Samples were centrifuged at 5000 rpm for 15 minutes and reconstituted with sterile PBS to the original volumes to minimize any drug carryover effect. The numbers of CFUs were quantified by plating the serial dilutions onto SDA plates and incubated for 48 hours. The fungal density of each sample was determined by counting the CFUs.