4.5. Minimal Inhibitory and Fungicidal Concentrations

The values of the minimal inhibitory concentration (MIC) were obtained using the micro-dilution broth method, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (www.eucast.org; accessed on 1 November 2023), using the RPMI medium with MOPS (RPMI 1640 broth with 2% dextrose) and a spectrophotometric prominent inhibition of growth using Asys Hitachi 340, Driver version 4.02 (Biogenet, Poland) (optical density (OD) λ = 590 nm reduction of ≥90%) (MIC₉₀), for the growth of fungi [57]. The sterile 96-well polystyrene microtitrate plates (Nunc, Denmark) were prepared by dispensing 100 µL of the appropriate dilution of the tested compounds in a broth medium per well by serial two-fold dilutions to obtain the final concentrations of the tested compounds that ranged from 4096 to 0.06 µg/mL. The inoculums that were prepared with fresh microbial cultures in sterile 0.85% NaCl to match the turbidity of the 0.5 McFarland standard were added to the wells to obtain a final density of 5 × 10⁴ CFU/mL for yeasts (CFUs: colony-forming units). An appropriate DMSO control (at a final concentration of 5%), a positive control (containing the inoculum without the tested compounds), and a negative control (containing the tested compounds without the inoculum) were included on each microplate.

The minimal fungicidal concentration (MFC) values were determined by inoculating 10 µL of the inoculum onto the YPG medium plate from the wells (MIC; 2 × MIC; and 4 × MIC) from a 96-well titration plate. The MFC was expressed as the concentration of the compounds that reduced the number of colony-forming units on the YPG medium (CFUs) by 99.9% after 24 h of incubation at 37 °C. Additionally, to confirm the MFC, the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT reagent was used, adding 10 µL to the wells at a final concentration of 5%. The lack of a color reaction (formazan formation) confirmed the result.