The direct contact test was performed as described by Serio et al. [32 (link)] and modified as follows. Stock emulsions containing 4% (v/v) EOs (CAR1 or THY5) were prepared in tryptone salt solution (8.5 g/L NaCl, 1.0 g/L tryptone; Oxoid) with 1% Tween 80 (Sigma-Aldrich) added as an emulsifying agent and vortexed for 10 min. The emulsions were filter-sterilized (0.22 μm filter pore size). To study the effect of different EO concentrations, appropriate aliquots of stock emulsions were diluted in 10 mL PBS, to obtain final concentrations of 0–0.12–0.25–0.50% EOs.
EO dilutions were inoculated with a standard bacterial suspension (1 × 108 CFU/mL) of L. monocytogenes OH or S. Typhimurium LT2 indicator strains and incubated at 37 °C for 30 min. Bacterial cells were also exposed to 0% EO (control, C) and to 0% EO plus 0.125% Tween 80 with or without 50 μg/mL ampicillin, to confirm test efficacy and to exclude any antimicrobial effect of Tween 80, respectively (CTw + Amp or CTw). Bacteria were harvested by centrifugation for 10 min at 1100× g at 4 °C and appropriate dilutions were plated on tryptone soy agar to evaluate bacterial cell viability by colony counting, after the incubation of the plates for 18 h.
EO dilutions were inoculated with a standard bacterial suspension (1 × 108 CFU/mL) of L. monocytogenes OH or S. Typhimurium LT2 indicator strains and incubated at 37 °C for 30 min. Bacterial cells were also exposed to 0% EO (control, C) and to 0% EO plus 0.125% Tween 80 with or without 50 μg/mL ampicillin, to confirm test efficacy and to exclude any antimicrobial effect of Tween 80, respectively (CTw + Amp or CTw). Bacteria were harvested by centrifugation for 10 min at 1100× g at 4 °C and appropriate dilutions were plated on tryptone soy agar to evaluate bacterial cell viability by colony counting, after the incubation of the plates for 18 h.