4.6. Minimum Biofilm Inhibitory Concentration (MBIC)

We determined MBIC for EOs against S. epidermidis biofilm using the microtiter plate method, as described previously [39], with some modifications. Briefly, we incubated 100 µL of S. epidermidis at 10⁸ CFU/mL in BHI–1% glucose (w/v) with 100 µL of the EO concentrations 0.00875–0.56% (v/v) at 25 °C for 24 h. Following incubation, we removed the contents of each well and gently rinsed the wells twice with 300 µL of PBS. We air dried the plate for 30 min and stained the formed biofilm with 0.1% (w/v) crystal violet (CV) for 30 min at room temperature. We then washed the excess CV from the plate with 200 mL of PBS per well, repeated washing three times and air-dried the plate. To measure the biofilm stained with CV, we solubilized CV using 95% (v/v) ethanol and measured the CV color intensity at OD_595nm using a Microplate reader (Infinite F50 Tecan). We determined MBIC for oregano, thyme, lemongrass, tea tree EOs, and thymol. We used BHI broth as a negative control and S. epidermidis cell culture without EOs as a positive control. We determined MBIC as the EO concentration at which the OD_595nm is equal to that of the negative control. Experiments for each EO concentration were performed in triplicate and the assay was repeated three independent times.