3.7. Minimal Inhibitory Concentration (MIC) Determination

MICs were determined by broth micro-dilution method according to the guidelines of the Clinical and Laboratory Standards Institute in cation-adjusted Mueller-Hinton broth (caMHB) [51].

Briefly, bacteria were cultured on tryptic soy agar (TSA) (Difco, Richmond, CA, USA), incubated overnight at 37 °C and adjusted to a bacterial density of 10⁶ CFU/mL as starting inoculum. Triterpenic acids were solubilized in dimethylsulfoxide (DMSO) (Sigma, St. Louis, MO, USA) and then diluted in the nutrient broth to obtain a final concentration of 256 mg/L. Serial two-fold dilutions were made to obtain a concentration range from 0.25 to 128 mg/L and 7.81 to 500 mg/L respectively. After overnight incubation, 30 µL of a 0.02% resazurin (Sigma, St. Louis, MO, USA) solution was added to the wells (in order to facilitate the detection of bacterial growth) [10] and the plates were incubated during 1 h at 37 °C in the dark. The MIC corresponds to the lowest concentration of compound for which no metabolization of blue resazurin in pink resorufin was observed by sight. All tests were made in triplicate.