MIC Determination of Antibacterial Oils

The minimum inhibitory concentration (MIC) of the oils and those of the reference standards (azithromycin and cinnamaldehyde) were determined using the broth dilution method [17 (link),25 (link)]. Briefly, using 96-well microtiter plates (Merck Sigma-Aldrich, Johannesburg, South Africa), 100 µL of Mueller–Hinton broth (MHB) was transferred into each well, after which 200 µL two-fold serial dilutions of the antibacterial agents (oils (concentration ranging from 0.72 to 367.2 mg/mL) and positive controls (azithromycin and cinnamaldehyde (0.031–4 mg/mL)) and 2% DMSO (negative controls) was transferred into the wells. This was followed by the addition of 100 µL of standardized bacterial inoculum (1.33 × 10⁸ CFU/well), and each treatment was performed in triplicates. After 24 h of incubation at 37 °C, 40 µL of P–iodonitrotetrazolium (INT, 0.2 mg/mL) was added and incubated for another 45 min. Bacterial growth inhibition (clear wells with no color change) was observed and recorded visually. The MIC of the oils was recorded as the lowest concentration that inhibited bacterial growth with no visible growth [26 (link)].