2.5. Antibacterial Assay

For the antibacterial activity, E. coli NCTC 9001, S. Typhimurium NCTC 12023, and S. aureus NCTC 6571 (SIGMA-ALDRICH) were grown on Brain Heart Infusion Broth (BHI Broth), (OXOID), at 37 °C for 24 h. As for the antifungal activity, A. niger (SIGMA-ALDRICH) was grown on Potato Dextrose Agar (PDA), (BIOlab), at 22 °C for 5 days [35].

The assay was performed by the agar dilution technique against a group of bacterial strains, as recommended by the National Committee for Clinical Laboratory Standards [36].

After placing 5 μL of different dilutions of the EOs (1, ½, ¼) and the extracts (90%, 50%, 25%) on Brain Heart Infusion Agar (BHI) plates, plates were dried at room temperature. Then, 1 μL of the bacterial strain (10⁸ CFU/mL), in serial dilutions, was spread to the plates. Plates were left to dry at room temperature for 30 min, before the incubation. The incubation was implemented at 37 °C for 24 h. After incubation, there was a record of the presence or absence of microorganisms and a comparison with Control. All experiments were performed in triplicate at each concentration.