3.6.2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of essential oils and hydroethanolic dry extracts were determined using microdilution assay [88]. At first, preparation of stock solution of essential oils and extracts were prepared. Essential oils were dissolve in sterile Müller-Hinton Broth (MHB) containing DMSO (40 µL) with 2% of Tween 80, and the final stock solutions were 64 mg × mL⁻¹ Extracts were dissolved directly in sterile MHB and the final stock solutions were 64 mg × mL⁻¹.

Next, a series of twofold dilutions of tested essential oils and extracts in the concentration range from 64 to 0.125 mg × mL⁻¹ were prepared in MHB, using sterile 96-well microtiter plates (Brand, Wertheim, Germany). To each well of plates 10 µL of bacterial inoculum was added and the final concentration of bacteria suspension was about 5 × 10⁵ CFU × mL⁻¹. The well with medium and inoculum without essential oil or extract was also prepared and served as a negative control. In turn, wells with medium and essential oils or extracts without inoculum served as positive controls. The plates with bacteria were incubated at 37 °C for 24 h. The MIC value was defined as the lowest essential oil/extract concentration, in which no visual growth of bacteria was noted. To determine MBC value, 10 µL of broth was taken from the well showing no bacterial growth and spot-inoculated onto plates with Müller-Hinton Agar medium. The plates were incubated at 37 °C for 24h. The MBC was defined as the lowest concentration of essential oil or extract, which resulted in complete reduction of bacteria. Each experiment was performed in triplicate.