3.3. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MCB)

The minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial compound that is able to inhibit bacterial growth under determined conditions [62], which vary depending on the antimicrobial compound used.

To determine the MIC values of the antibiotics, we followed the broth microdilution method for antimicrobial susceptibility tests as established by the Clinical and Laboratory Standards Institute (CLSI) [63]. To achieve this, we added increasing concentrations of amoxicillin (0.5–32 µg/mL) and colistin (from 0.125 µg/mL to 8 µg/mL) to 96-well microtiter plates with 100 µL of MHB in each well.

To determine the MIC values of the commercial EOs and the ICs, we adopted the methodology widely agreed upon in previous studies [9,10,11]. We added increasing concentrations of AEN, COLIFIT, thymol, and cinnamaldehyde (50–500 µL/L with 50 µL/L intervals) to test tubes with 5 mL of MHB. Since EOs have low solubility in aqueous buffers, it was necessary to include a vigorous shaking step (Ika vortex 3, Genius, Königswinter, Germany) in order to obtain a homogeneous antimicrobial suspension [64].

In the two methods, each well or test tube was inoculated with E. coli MG1655 or S. Typhimurium LT2 stationary-phase culture, respectively, at an initial concentration of 5 × 10⁵ CFU/mL and incubated for 24 h at 37 °C under static conditions, in the case of microtiter plates, or at 130 rpm, in the case of test tubes. Positive controls (inoculated at 5 × 10⁵ CFU/mL without any antimicrobial compound) and negative controls (inoculated with the highest concentration of the antimicrobial compound in the absence of bacterial inoculum) were also included in each experiment. After the incubation time, we determined optical density at 595 nm (OD₅₉₅) (Genios, Tecan, Männedorf, Switzerland) in order to obtain an objective measurement of bacterial growth and thus be capable of determining which concentration was capable of inhibiting the respective bacterium’s growth. “Bacterial growth”, as such, was noted as the point in time when the OD₅₉₅ was ≥10% of the OD₅₉₅ of the positive control.

The minimum bactericidal concentration (MBC) is defined as the lowest concentration of an antimicrobial compound that is able to inactivate ≥99.9% of the initial bacterial concentration [62]. MBC determination was carried out in parallel to MIC determination. After the incubation of the test tubes, 100 µL of each one was spread out on MHA plates and incubated for 24 h at 37 °C. After the incubation time, colonies were counted, and CMB was determined. As in MIC determination, positive and negative controls were included.