Determination of the minimum bactericidal concentration (MBC)

The minimum bactericidal concentration (MBC) was also assessed according to the CLSI guidelines [27, 28]. After completion of the MIC assay, the 96-well microtiter-plates were further incubated for MBC testing. In brief, 10 μl from each well containing the tested plant extract concentration series were plated on CBA or HCB. In particular, E. coli, S. aureus and E. faecalis were plated on CBA at 37°C for 24 h, streptococci and C. albicans were incubated on CBA at 37°C and 5–10% CO₂ atmosphere for two days. The obligate anaerobes were cultivated on HCB at 37°C for five days. To create anaerobic conditions an anaerobic jar and the Anaerocult. A gas generator system (Merck, Darmstadt, Germany) were used. Finally, the CFU were determined visually. The MBC was defined as the concentration at which a three Log decrease in bacterial growth (= 99.9% inhibition) was detected compared to the growth control.