Minimum Bactericidal Concentration (MBC) Assay

The minimum bactericidal concentration (MBC) determination was conducted using the broth microdilution method,¹³ where 100 μL of double strength Mueller Hinton broth was placed in each well of a sterile 96-well plate and 100 μL of the tested extract (100mg/mL) or DMSO (negative control) was then added to the first well of each row. Two-fold serial dilutions were done from one row to the next till the eighth row (50–0.391 mg/mL). The wells were then inoculated with 10 μL of the tested bacterial suspension (10⁸ CFU/mL). One row was used as a sterility control (neither bacterial suspension nor tested extract was added) and another row was used as a growth control (inoculated with bacterial suspension without adding the tested extract). Plates were incubated at 37°C for 24 h after which 5 μL of each well was spotted on Mueller Hinton agar plate then incubated at 37°C for 24 h. MBC was determined as the lowest concentration with no detectable colonies. The experiment was repeated three independent times.