4.6.2. Determination of the Minimum Inhibitory Concentrations (MICs)

The MICs of C. cernuum essential oils against the selected Gram-positive and Gram-negative bacteria were determined using a broth microdilution method, according to the recommendations of the Clinical and Laboratory Standards Institute (protocol M07-A9) [45]. Bacterial suspensions in Mueller-Hinton broth (MHB; Merck, Darmstadt, Germany) at the final concentrations of 10⁶ CFU/mL were used in all experiments. Stock solutions of the essential oils (250.0 μL/mL) were prepared with 1% Tween 80 (v/v; Merck, Darmstadt, Germany) and a reference compound—thymol (Ernesto Ventos S.A., Barcelona, Spain)—was solubilized using 2% dimethyl sulfoxide (DMSO; v/v; Loba Chemie, Mumbai, India). Series of dilutions (from 1 to 250 μL/mL) were prepared by diluting the stock solutions with MHB. To each well in a 96-well microplate, containing 50 μL of the essential oil or thymol solution, 50 μL of the bacterial suspension was added. After 18 h of incubation at 37 °C, each well was spiked with 20 µL of 0.02% resazurin (Merck, Darmstadt, Germany) solution. The color change from blue to pink, after 3 h of incubation with resazurin, indicated the presence of viable bacteria. To exclude an inhibitory effect of Tween 80 and DMSO on the bacterial strain’s growth, control assays with MHB and MHB containing 1% (v/v) Tween 80 or 2% (v/v) DMSO were performed, as well as the MHB sterility control. All tests were run in duplicate.