4.4. MIC and Minimal Fungicide Concentration (MFC) Determination

Minimum inhibitory concentrations (MICs) were determined by the broth microdilution technique, following the Clinical and Laboratory Standards Institute (CLSI) guidelines by diluting freshly prepared filipin derivatives in Roswell Park Memorial Institute (RPMI) 1640 medium with glutamine and 0.2% glucose, but without sodium bicarbonate (Sigma), buffered with 0.164 M MOPS pH 7.0 to concentrations of 60, 50, or 40 μg/mL, of which 100 μL was added to the first row of a round-bottomed, 96-well culture plate. This was followed by a 1:1 dilution series in medium. Yeast antifungal susceptibility testing was performed according to the M27-A3 protocol [34], whereas for filamentous fungi testing we used the M38-A2 method [35]. The MIC value was determined to be the lowest concentration of antibiotic that inhibited completely the growth of the fungi and could be determined by the eye on the 96-well plate after an incubation of 48–72 h at 35 °C. Commercial filipin III (Sigma) and amphotericin B (Sigma) were used as controls.

For minimal fungicidal concentration (MFC) determination, the total volume of each well used for MIC determination, starting from the last well in which growth was observed up to the highest drug concentration tested, was transferred onto agar plates. The MFC corresponded to the lowest drug concentration at which no colonies were observed after 48–72 h of incubation.

MIC and MFC values were determined as the average of three determinations from independent experiments. The MFC/MIC ratio was calculated to determine whether the substances exhibited fungistatic (MFC/MIC > 4) or fungicidal activity (MFC/MIC < 4) [36].