Agar Disk Diffusion Assay of the Two Essential Oils

The antimicrobial of essential oils were determined according to the standard agar disk diffusion method with a slight modification (Luangtongkum et al., 2007; Gaudreau et al., 2008; Benamrouche et al., 2014; Wu et al., 2018; Wu et al., 2021). A 0.5 McFarland (1 × 10⁷ to 1 × 10⁸ CFU/mL) concentration of the bacterial suspension was uniformly inoculated onto MHA solidified in 120 mm Petri dishes. Once the dishes were prepared, 6 mm-diameter discs of filter paper containing 5 μL of the examined essential oil solutions, which had been diluted with dimethyl sulfoxide (essential oil: DMSO 1:9 v/v), were pressed gently against the surface of the agar. Discs containing gatifloxacin (1 nmol) was used as positive control, while DMSO was used as the negative control. The dishes were incubated in a constant temperature incubator at 37°C for 24 h. The inhibition zone diameter was measured by a vernier caliper. All the experiments were performed in triplicate (Wu et al., 2021).