2.7. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)

Microdilution broth method according to the Clinical and Laboratory Standards Institute (CLSI) reference protocol M38-A2 for filamentous fungi with modifications [37] was used to evaluate the Minimal Inhibitory Concentrations (MICs) of the extracts that showed antifungal activity. Briefly, dilutions of test essential oils at various concentrations ranging from 0.0098 to 5 mg/mL were prepared with RPMI-1640 medium (with L-glutamine, without bicarbonate) and buffered to pH 7 with 0.164 M MOPS buffer (Sigma-Aldrich, St. Louis, MO, USA) and DMSO (Panreac, Barcelona, Spain) which final concentrations never exceeding 5% v/v. A 100 microliters solution of each essential oil concentration and 100 µL of tested molds suspension (1–2 × 10⁶ CFU/mL) were added to each well of the 96-well microtiter plates and then incubated at 25 °C for 72 h. Wells containing mold inoculum and RPMI 1640 instead of essential oils served as growth control. MIC was defined as the lowest concentration of the essential oils at which no visible growth of the fungal strain could be detected compared to their growth in the negative control well. Tests were performed in duplicate and visual interpretation was carried out by two qualified individuals. The Minimum Fungicidal Concentrations (MFCs) were determined according to Espinel-Ingroff et al. [38] by subculturing 10 µl of the wells with no mold growth onto plates containing PDA agar and incubating at 25 °C for 72 h. The lowest concentration of EOs with negative growth was considered to be the MFC. The isolates were tested in duplicate.