A multi-species cariogenic biofilm was developed using a method modified from our previous study . Briefly, Streptococcus mutans from the American Type Culture Collection (ATCC) 35668, Lactobacillus acidophilus ATCC 9224, and Actinomyces israelii were anaerobically cultured on blood agar plates at 37 °C for 48 h. A single colony was picked from each plate to prepare 24-h broth cultures in brain heart infusion (BHI) supplemented with 5% sucrose at 37 °C under anaerobic conditions (95% nitrogen and 5% carbon dioxide). Subsequently, bacterial cell pellets were harvested and resuspended in the BHI to a cell density of McFarland 2 (106 cells/mL). A 500-µL aliquot of each bacteria culture was mixed and inoculated on each dentine block sitting in a well of a 24-well plate with BHI. The dentine blocks were aerobically incubated in 1 mL of mixed BHI suspension (adhesion phase) at 37 °C for 90 min. After rinsing with PBS to remove non-adherent cells, the dentine blocks were aerobically incubated in the BHI at 37 °C (biofilm phase). The dentine blocks were taken out after 48 h for analysis; the BHI was refreshed after 24 h .