Minimum Inhibitory Concentration and Minimum Bactericidal Concentration

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of thyme oil for S. enterica were evaluated using a broth dilution assay (32). One milliter of filter-sterilized thyme oil was added to 99 mL of sterile BHI broth (pH 7.4) containing 0.5% (v/v) yucca extract to give an initial concentration of 1.0% (v/v). Two-fold dilutions of thyme oil (1.0%) were prepared in BHI broth with added yucca extract to obtain the following thyme oil concentrations: 0.5, 0.25, 0.125, 0.062, 0.031, and 0.015% (v/v). The S. enterica cell suspension (9.0 Log₁₀ CFU/mL) in saline was diluted in fresh saline to obtain 7.0 Log CFU/mL. Aliquots (0.1-mL) of the diluted cell suspension were used to inoculate tubes of sterile BHI broth (10 mL/tube) containing various concentrations of thyme oil to obtain an initial S. enterica concentration of ~5.0 Log₁₀ CFU/mL. Inoculated and non-inoculated BHI broth with yucca extract served as positive and negative control, respectively. All tubes of BHI broth were incubated at 35°C and checked for turbidity after 24 h. The MIC of thyme oil was determined as the lowest concentration at which no turbidity was observed after 24 h. To determine the MBC, 0.1 mL aliquots from the tubes showing no turbidity were surface plated on TSAYE. The TSAYE plates were then incubated (35°C) and bacterial colonies were counted after 48 h. The lowest concentration of thyme oil that demonstrated ≥99.9% (3-log) kill of the pathogen was deemed as the MBC (33).