3.5. Antimicrobial Assay Using Agar Diffusion–Dilution Test

Based on paper-disk diffusion and dilution assay [59,60], the antimicrobial activity of the isolated compounds was tested against two Gram-positive bacteria: S. aureus (ATCC 25923) and S. epidermidis (ATCC 12228); four Gram-negative bacteria: E. coli (ATCC 25922), E. cloacae (ATCC 13047), K. pneumoniae (ATCC 13883) and P. aeruginosa (ATCC 227853); and three pathogenic fungi: C. albicans (ATCC 10231), C. tropicalis (ATCC 13801) and C. glabrata (ATCC 28838).

The antimicrobial activity of the isolated compounds were further determined using the agar dilution technique [23]. For all the assays, stock solutions of the tested samples were prepared at 10 mg/mL concentration. Serial dilutions of the stock solutions in broth medium (100 μL of Muller-Hinton broth or on Sabouraud broth for the fungi) were prepared in a microtiter plate (96 wells). Then, 1 μL of the microbial suspension (the inoculum, in sterile distilled water) was added to each well. For each strain, the growth conditions and the sterility of the medium were checked, then the plates were incubated as referred above. MICs were determined as the lowest concentrations preventing visible growth. Standard antibiotic netilmicin and amoxicillin (at concentrations of 4 to 88 μg/mL) were used to control the sensitivity of the tested bacteria, with amphotericin B and 5-fluocytocine (at concentrations of 0.4 to 1 μg/mL) as a control against the tested fungi (Sanofi, Diagnostics Pasteur at concentrations of 30, 15 and 10 μg/mL). For each experiment, any pure solvent used was also applied as a blind control. The experiments were repeated three times and the results were expressed as average values.