*2.7. Antimicrobial Activity Test*

The antimicrobial activity of the compounds was firstly tested by the agar diffusion method using 0.2% solutions of the investigated compounds in dimethyl sulfoxide (DMSO). Plates containing Mueller-Hinton agar (MHA) were inoculated with aliquots of suspensions of microbial cultures. An equal amount (30 μL, 0.06 μg) of each sample solution was introduced into wells (8 mm in diameter) punched in MHA following a sterile procedure. Standard commercial discs with gentamicin (G, antibacterial agent) and nystatin (Ns, antifungal agent) were used as references. A positive control using only inoculation and negative control using only DMSO in wells were also prepared. The plates were incubated at the appropriate temperature for 24−48 h, and the resulting inhibition zones (diameter, mm) were recorded.

Broth dilution method was performed for in vitro determination of the minimum inhibitory concentration (MIC) of the compounds [23]. Stock solutions of the investigated compounds (0.1% in DMSO) were serially diluted in MPB to final concentrations ranging from 2 to 200 μg/mL. After inoculation, the test tubes were incubated at the appropriate temperature for 24 h under shaking. Positive controls (compounds and MPB, without inoculum) and negative controls (MPB and inoculum, without compounds) were also prepared. Growth of the strains was assayed by monitoring the turbidity at 600 nm (OD600). Microbial growth (%) was determined on the basis of the positive control, which was considered as 100%. The MIC was considered to be the lowest concentration of the tested sample to inhibit the visible growth of microorganisms. All assays were performed in triplicate, and the average was taken; standard deviations were less than 5%.
