Broth Microdilution Assay of the Two Essential Oils

The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined by a microdilution method in 96-well plates according to Clinical and Laboratory Standards Institute (CLSI), with a slight modification (Sader et al., 2006; Meng et al., 2016; Wu et al., 2018; Wu et al., 2021). A series of diluted essential oils were prepared with DMSO as the solvent by two-fold serial dilution starting from a stock solution of 512 mg/mL. The final concentrations of the test essential oils were obtained in a range between 1 mg/mL and 512 mg/mL. Each well received 5 μL of a specific concentration of the essential oils and 195 μL of MHB inoculated with the test microorganism (1.5 × 10⁵ CFU/mL); the final concentrations of the examined essential oils were reached. Gatifloxacin was used as positive control and DMSO was used as negative control (Wu et al., 2021). The microplates were incubated in a bacteriological oven for 24 h at 37°C, and the susceptibility results of tested samples were monitored by measuring the absorbance at 600 nm using a Multimodel Plate Reader (Infinite 200). The lowest concentration without visible growth was defined as the MIC (Wu et al., 2021).

The minimum bactericidal concentrations (MBCs) were determined based on the MIC results (Jabrane et al., 2010; Chouaib et al., 2015; Wu et al., 2018; Wu et al., 2021): serial sub-cultivation of a 5 μL aliquots near the MIC with 195 μL fresh MHB were uniformly inoculated onto MHA solidified in 90 mm Petri dishes; incubation for 24 h at 37°C. The lowest concentration of antimicrobial agent that killed at least 99.9% of the starting inoculum was defined as the MBC endpoint, which was determined as the lowest concentration with no visible growth of bacterial colony on the Petri dishes (Wu et al., 2021). All experiments were conducted in triplicate. The final concentration of DMSO in the 96-well plate had no effect on bacterial growth.