Determination of the minimum bactericidal concentration (MBC)
The assessment of the minimum bactericidal concentration (MBC) was also
conducted following the protocols outlined by the CLSI. Following the
completion of the MIC assay, the 96-well microtiter plates were
subjected to further incubation for MBC testing. In a concise manner, 10
µl from each well, containing the respective concentration series of the
tested plant extracts, were plated onto CBA or HCB plates. Specifically,E. coli , S. aureus , and E. faecalis were plated on
CBA agar plates and then incubated at 37°C for 24 hours. Streptococci
and C. albicans were placed on CBA agar plates and incubated at
37°C in a 5%-10% CO2 atmosphere for 2 days. On the
other hand, the obligate anaerobes were cultivated on HCB agar plates at
37°C for a duration of 5 days within an anaerobic chamber (Anaerocult,
Merck Chemicals GmbH, Darmstadt, Germany). Ultimately, a visual
determination of colony-forming units (CFU) was performed. The MBC was
defined as the concentration at which a three-log decrease in bacterial
growth (equivalent to 99.9% inhibition) was observed in comparison to
the growth control.