4.4. Screening of Antibacterial Activities of EOs by Disc Diffusion Method

As a preliminary screening step, the antibacterial activities of all seven EOs was determined using agar disc diffusion, according to the Kirby-Bauer method [37] with some modification. EOs were diluted in analytical grade sterilized dimethylsulfoxide (DMSO) (Sigma Aldrich, St. Louis, MO, USA) and stock solutions of each of the oils at a concentration of 0.7% and 1.4% were prepared. We filter sterilized the prepared stock solutions by sterile syringe filter 0.2 µm (Corning, New York, NY, USA). Using the culturing method (detailed in Section 4.2), we prepared cell suspensions of S. epidermidis and P. acnes cultures at bacterial density adjusted to 10⁸ CFU/mL. We spread bacterial suspensions on BHI or RCM agar plates for testing EOs against S. epidermidis or P. acnes, respectively. We immersed sterile filter-paper discs in 20 µL (EOs and DMSO) at either 0.7% or 1.4% and placed discs on the surface of the agar until dry; they were then incubated under appropriate conditions detailed for each bacterium above. We used two standard reference antibiotics clindamycin (2 µg/disc) and erythromycin (15 µg/disc) as reference controls, and we used DMSO as a negative control. We evaluated the antibacterial activity of each EO at each concentration by measuring the zone of inhibition diameter by Vernier’s caliper expressed in millimeters (mm). The assays were performed in triplicate and repeated as three independent experiments.