4.5. Minimum Inhibitory Concentrations (MIC)

Microbial inoculum was cultivated for 24 h in Mueller Hinton Broth (MHB, Oxoid, Basingstoke, UK) at 37 °C for bacteria and Sabouraud Dextrose Broth (SDB, Oxoid, Basingstoke, UK) at 25 °C for yeasts. An aliquot of 50 μL of inoculum with an optical density of 0.5 McFarland was added to a 96-well microtiter plate. Subsequently, the S. aromaticum EO was prepared by serial dilution to a concentration range from 400 μL/mL to 0.2 μL/mL in MHB/SDB, and 100 μL of suspension was thoroughly mixed with bacterial inoculum in wells. Bacterial samples were incubated for 24 h at 37 °C. Yeast samples were incubated for 24 h at 25 °C. MHB/SDB with EO was used as a negative control and MHB/SDB with inoculum was used as positive control, representing uninhibited growth.

For non-adherent microorganisms, the absorbance was measured after an incubation period at 570 nm by Glomax spectrophotometer (Promega Inc., Madison, WI, USA). The MIC of biofilm-forming bacteria was measured with the use of crystal violet. The suspension with non-attached cells was discarded, the wells were washed with distilled water three times, and left to dry at room temperature. Following the addition of 200 μL of 0.1% (w/v) crystal violet to the wells, the plates were incubated for 15 min. Subsequently, the wells were repeatedly washed and dried. Stained biofilms were solubilized with 200 μL of 33% acetic acid [48], and absorbance was measured at 570 nm. Minimum inhibitory concentration was determined as the concentration of CEO at which absorbance was lower than the absorbance of the maximal growth control. The test was performed in triplicate.