2.3. Minimum Inhibitory Concentration

The broth microdilution assay was used to determine the minimum inhibitory concentration (MIC) of antifungals and polyphenols against the Candida strains according to the Clinical & Laboratory Standards Institute (CLSI) document M27-A2 [45] in 96-well flat-bottom microtiter plates (Corning no. 353072). Briefly, colonies of each strain were inoculated in 5 mL of YPD overnight at 37 °C in an orbital shaker at 225 rpm. The cells were harvested by centrifugation at 2700× g for 5 min, washed twice with sterile phosphate-buffered saline (PBS), and the final inoculum size was adjusted by hemocytometer to 1.0 × 10³ cells/mL in RPMI 1640. Antifungal agents and compounds were serially diluted in 50 μL, and 50 μL of cells were added. The concentration range tested was from 128 to 0.06 μg/mL for phenolic compounds. The plates were incubated at 37 °C for 24 h. Amphotericin B, caspofungin and fluconazole belong to the three major classes of antifungal agents (polyenes, echinocandins and azoles, respectively), and were used as positive controls. The concentration range tested was from 128 to 0.06 μg/mL for AMB and CPF, and 256 to 0.125 μg/mL for FLC. The least concentration of the compound that inhibited the visible growth was considered the MIC, and the most active compounds were selected and used for further studies.