4.5. Checkerboard Method—Fluconazole Synergy Test

For each Candida spp. and tested AgNP SBEs, the checkerboard method [31] was performed, to evaluate if the AgNP SBEs might exert synergistic activity with different concentrations of fluconazole. The plates were designed to include (on the same plate) the clinical breakpoints of the tested Candida spp. (were available, according to EUCAST recommendations) [49]. From the first wells of the horizontal axis of the microtiter plates, 200 µL of AgNP SBEs were serial binary diluted in 100 µL distilled water. From a fluconazole (Sigma Aldrich, St. Louis, MO, USA) stock solution of 132 mg/L, two folded dilutions were prepared in RPMI 2X, and 50 µL from each of these dilutions were added on all the wells from each row of the vertical axis of the microtiter plates (the final concentration of the fluconazole in the first row was 16 mg/L, while in the last row was 0.125 mg/L). Fifty microliters of 0.5 McFarland fungal inoculum (1:10 diluted in RPMI 2X), were added in all the wells of the microtiter plates. The positive and negative controls were considered the wells from opposite corners of the microtiter plate, where the concentration of both fluconazole and AgNP SBEs were highest, respectively, lowest. After the plates were incubated at 37 °C, for 48 h, the MICs were assessed at over 50% growth inhibition (by visual examination), and the fractional inhibitory concentration was calculated for all the wells where the fungal growth was inhibited over 50%, using the following formula:

FIC values ≤ 0.5 were interpreted as synergic effect of the tested solutions with fluconazole; FIC values between 0.5–2, were considered as an indifferent effect; if FIC values were > 2, the effect of the tested solutions was considered antagonistic [50].