Colony Forming Units (CFU)

Colony forming units assay was performed after different incubation time (0, 2, 4, 6, 8, and 24 h) with CBG (0, 2.5, 5, and 10 μg/ml). 10-fold serial dilutions of the untreated and treated samples were prepared by repeatedly transferring 100 μl from one sample to another tube containing 900 μl PBS. After vigorous vortex, 100 μl of the bacterial suspensions were spread on BHI agar plates and incubated overnight at 37°C in the presence of 5% CO₂. After incubation, the number of colonies was counted using the ImageJ software (Breed and Dotterrer, 1916). The following equation was used to calculate the CFU per ml in the original sample:

CFU per ml = Number of colonies × dilution factor × 1/volume seeded on the plates.