Microdilution Assay for MIC Determination

Determination of minimum inhibitory concentration (MIC) was performed by an adapted binary serial microdilution standard method [46 (link),47 (link)] in liquid medium (NB for bacteria and YPG for yeasts), using 96-well microtiter plates. From each BPE sample, serial two-fold microdilutions were achieved in 150 μL of corresponding broth medium seeded with the standard inoculum of 1.5 × 10⁸ CFU/mL (for bacterial strains) and 3 × 10⁸ CFU/mL (for yeast strains). Due to the BPE samples containing ethanol, a control (C_Et) was performed. The negative control was considered the sterile liquid medium, and the positive control (C+) was the broth medium inoculated with microbial suspensions, following the same conditions described before. The dishes were incubated at 37 °C for 24 h for bacterial strains and 48 h for yeast strains. The MIC values were established by visual analysis and spectrophotometric measuring of absorbance at 620 nm using a BIOTEK SYNERGY-HTX ELISA multi-mode reader (Winooski, VT, USA).