4.5. Determination of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

Evaluation of MIC and MBC of the pathogenic bacteria E. coli O157:H7 and MRSA, was carried out following the broth microdilution protocol standardized in the GIBIM [34], which is based on the protocol of the Clinical and Laboratory Standards Institute (CLSI) document M100 S20 [72].

Values of MIC₅₀ and MBC were defined as the minimum concentration of EO being able to inhibit 50% and totally inhibits bacterial growth, respectively [73]. Additionally, it was determined if the activity of EOs was bacteriostatic; it was considered bacteriostatic if the value of the MBC was four times higher than the value of the MIC (MBC/MIC > 4) [74].

For MIC determinations, a pre-inoculum was prepared from fresh culture of tested strains using TSB and TSB supplemented with 0.25% (w/v) glucose for E. coli O157:H7 and MRSA, respectively. The pre-inoculums were incubated during 12 h at 37 °C and 200 rpm, until reaching a bacterial concentration equivalent to 4.4–5 × 10⁹ CFU/mL, using the Mcfarland scale as a reference [75]. Inoculum was prepared from the pre-inoculum, taking it to a final volume of 10 mL with sterile medium, until obtaining an absorbance between 0.07 and 0.1 (equivalent to ~5 × 10⁵ CFU) [35].

Subsequently, bacterial growth kinetics was monitored in 96-well ELISA microplates (Bio-Rad, Imarck), where 100 μL of the bacterial inoculum was plated along with 100 μL of each EO in serial concentrations from 0.18 to 3 mg/mL for both strains. These microplates were incubated at 37 °C with constant stirring (200 rpm), and microbial growth was evaluated measuring the absorbance every hour up to complete 24 h using an ELISA microplate reader at 595 nm. Wells containing bacterial cultures without EO were used as negative controls.

MBC was determined once cell growth kinetics of the pathogenic strains were evaluated. Aliquots of 100 μL from each well containing different EO concentrations were taken and plated in 2 mL Eppendorf tubes containing 900 μL of BHI; subsequently, these tubes were incubated at 37 °C for 24 h. To corroborate the bactericidal effect, an aliquot of 10 μL was taken from each tube and was transferred to BHI agar plates at 37 °C for 24 h.