3.4.2. Preparation of the Media

The bacterial species were resuscitated by inoculation into Brain heart infusion (BHI) broth (Oxoid UK, Cat. no. CM1135) and incubated at 37 ◦C for 24 h after which, each strain was streaked aseptically onto Tryptone soya agar for single colony formation and incubated at 37 ◦C for 24 h. The cell suspensions were performed in sterile saline, standardized at 0.5 McFarland standard (Remel™, Kansas, Cat. no. R20410) at 1.5 × 108 colony forming units (CFU)/mL. Then, the working suspensions were obtained by a second 1:100 dilution onto BHI to approximately 106 CFU/mL.

## 3.4.3. Broth Microdilution Susceptibility Assay

The broth microdilution test was performed as previously described by Lourens et al. [63] and Sartoratto et al. [64] with slight adjustments. An EO stock solution of 51.2 mg/mL was prepared with a BHI:dimethyl sulphoxide (DMSO) (1:1) solution. In a 96-well plate, 100 μL of BHI was added to the experimental wells in triplicate except in well 1. Then, 200 μL of EO stock solution was added to well 1, from which a serial dilution was performed to the last experimental well. Subsequently, 100 μL of cell suspension was added to establish the two-fold 25.6–0.2 mg/mL sample concentration range and a bacterial cell suspension of approximately 5 × 105 CFU/mL. The plate was incubated at 37 ◦C for 20 h. After incubation, the antimicrobial activity was detected by adding 40 μL of 0.2 mg/mL INT (Sigma-Aldrich®, Cat no. I10406) aqueous solution. The plates were incubated at 37 ◦C for 1 h. The MICs were defined as the lowest concentration of essential oil that inhibited visible growth, as indicated by the color change of INT. Ampicillin (Sigma-Aldrich®, Cat no. A9393) was used as a positive control.
