4.3.2. Antibacterial Activity

Disc diffusion assay. The antibacterial test was carried out following the disc diffusion method [40,41] using the King B nutrient media (KB) [42]. A bacterial suspension of each tested bacteria was prepared in sterile distilled water adjusted at 106 CFU/mL (OD ≈ 0.2 nm) using a turbidimetry instrument (Biolog, Hayward, CA, USA). Four millilitres of bacterial suspension mixed with soft agar (0.7%) at ratio 9:1 (*v/v*) were poured over each plate (90 mm diameter). Blank discs of 6 mm (OXOID, Milan, Italy) were then placed over the KB-plate surfaces and about 20 μL from each tested EO concentration at 0.1, 1 and 10 mg/mL was carefully applied over discs. Tween 20 was added to each tested EO concentration at 0.2% for accelerating the oil solubility. Tetracycline (1.6 mg/mL) was used as a positive control. The antibacterial activity was estimated by measuring the diameter of inhibition zone in mm ± SDs around each treated disc compared to the positive control ones.
