Time-Kill Kinetic Analysis

Exponential-phase Candida cells (1 × 10⁶ cells/ml) were incubated with different concentration of protolichesterinic acid (0.5× MIC, MIC, and 2× MIC) and amphotericin B (MIC). Control cells were incubated with DMSO and sterile deionized water. About 10 μl samples from each treatment were collected at 0, 1, 2, 4, 6, 12, 24, and 48 h and serially diluted in PBS, and 50 μl aliquots were gently plated on the surface of PDA plates. The viable Candida colonies were counted after incubation at 37°C for 48 h. All of the experiments were performed in triplicate.

After the incubation period, the number of viable Candida cells was counted and expressed in log CFU/ml. The results were scrutinized and represented graphically, that is a fungal death curve vs. function of time. Data analysis for the protolichesterinic acid was measured as showing fungicidal activity when there was a decrease greater than or equal to 3 log₁₀ CFU/ml of the initial inoculum, resulting in the reduction of 99.99% or more CFU/ml in 48 h compared with the initial inoculum. Fungistatic activity was considered as the decrease in growth lower than 99.99% or <3 log₁₀ in CFU/ml from the original inoculum (Leite et al., 2015).