Gaspak jar system

Brain heart infusion (BHI) broth was used to grow S. sanguinis and T. denticola ATCC 35405 and the bacteria were incubated at 37°C in an anaerobic atmosphere for 24h using GasPak jar system (Becton Dickinson, Franklin Lakes, NJ, USA). The bacterium culture in broth medium was measured at 600nm and diluted to obtain a suspension of McFarland standard 0.5 (equivalent to 1,5x10 8 CFU/mL). After homogenizing, the bacterium suspension was distributed into a 96-well plate, and incubated at 37°C for 24h in an anaerobic atmosphere to formed biofilm. The supernatant was removed and the biofilm attached on the bottom of well plate was rinsed with phosphate buffer saline (PBS). 200 uL M. pendans extracts with different concentrations (100%, 50%, 25%, 12.5% and 6.25%) were distributed into the well plate. Chlorhexidine (1.2%) was used for the positive control and biofilm wells without herbal tested was used for the negative control. Inhibitory effects were observed after incubation at 37°C for 1h, 3h 6h, and 24h in an anaerobic atmosphere. Crystal violet dye (0.5% w/v) was distributed into the well plate and incubated for 15 min. The extraction of the remaining crystal violet dye in the well plate was measured after the addition of 200 µL 90% ethanol and the absorbance was measured using a microplate reader at 595 nm.