4.7. Determinations of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The determination of the MIC was carried out according to the method described by Tan et al [3]. Stock solutions of CA were kept in 25% (v/v) ethanol to enhance their solubility in suspension. Antimicrobial effects of CA were evaluated using a serial twofold dilution method. CA was initially diluted in 25% (v/v) ethanol (1:100 v/v) and then in TSB medium. Serial twofold dilutions for CA were prepared to obtain concentrations ranging from 10 to 0.15 mg/mL. A quantity of 100 μL of bacteria at a final concentration of 1 × 10⁵ CFU/mL in TSB was added into the wells of 96-well microplates. Free CA (10, 5, 2.5, 1.25, 0.62, 0.31, 0.15 or 0 mg/mL) was added to each well. The microplate was incubated at 37 °C for 24 h at 150 rpm. MIC refers to the lowest drug concentration that inhibits the growth of bacteria in culture medium after 18 to 24 hours of culture in vitro. MBC refers to the minimum drug concentration capable of killing cultured bacteria after 18 to 24 hours in vitro.