2.9.2. Microdilution Method

The modified microdilution technique was used to evaluate the antimicrobial activity. Cell suspensions were adjusted to a concentration of approximately 2 <sup>×</sup> 105 CFU/mL in a final volume of 100 μL per well. The inoculum was stored at 4 ◦C for further use. Minimum inhibitory concentrations (MICs) were performed using 96-well plates. Dilutions of the extracts were carried out in wells containing 100 μL of Muller-Hinton broth and 10 μL of inoculum was added to all the wells. The microplates were incubated for 24 h at 37 ◦C. The MIC was detected following the addition of 20 μL (0.2 mg/mL) of resazurin solution to each well, and the plates were incubated 2 h at 37 ◦C. The MIC was identified as the lowest concentration that prevented the color change from blue to pink. The minimum bactericidal concentrations (MBCs) were determined by serial subcultivation of 2 μL into 96-well plates containing 100 μL of broth per well and further incubation for 48 h at 37 ◦C. The lowest concentration with no visible growth was defined as MBC, indicating 99.5% killing of the original inoculum.
