4.6. Fluconazole Synergy Test—Checkerboard Method

The synergy tests were performed according to the checkerboard method [61,62], for each tested solution and each Candida strain. The checkerboard microplates were designed to include, on the same microtiter plate, all the EUCAST E.DEF 7.3.2 [63] clinical breakpoints for the tested strains (where available). One hundred microliters of two folded dilutions from the tested solutions were performed in each of the 8 rows of a 96-microtiter plate, in distilled water (on the first wells of each row, the final concentration of the solution was 50%; the last wells contained the lowest concentrations, 0.03%). Two folded dilutions of the fluconazole (Sigma-Aldrich) stock solution were prepared in RPMI 2X and 50 µL from the diluted solution were distributed in all the wells of each row of the microtiter plate (in the first row, the fluconazole final concentration was 16 µg/L in all twelve wells, in the second row the finale concentration was 8 µg/L, and in the 8th row, 0.125 µg/L). The fungal inoculum (0.5 McFarland) was diluted 1:10 in RPMI 2X and 50 µL from this suspension was distributed in each well of the microtiter plate. The plates were incubated for 36 h at 37 °C. Positive control well was considered the last well of the last row, the well where the fluconazole and the tested solution concentration were the lowest, while the negative control was considered the first well of the first row, where the fluconazole and the tested solution concentration were the highest. The fractional inhibitory concentration (FIC) was calculated with the following formula: FIC = FIC 1 + FIC 2, where FIC 1 was fluconazole MIC from the studied well/fluconazole MIC, and FIC 2 was the tested solution MIC from the studied well/tested solution MIC. The MIC for the fluconazole was considered the first well where fungal growth was 50% inhibited, at the lowest tested solution concentration, while the tested solution MIC was considered the first well where fungal growth was inhibited, at the lowest fluconazole concentration. If the FIC value was ≤0.5, the studied substances exerted a synergic effect with fluconazole. If the FIC value was between 0.5 and 2, the effect was considered indifferent, and if the FIC value was >2, the effect was considered antagonistic [63].