4.2. Cariogenic Biofilm Challenge

Cariogenic bacteria used for biofilm challenge were Streptococcus mutans ATCC 35668 (American Type Culture Collection), Streptococcus sobrinus ATCC 33478, Lactobacillus rhamnosus ATCC 10863 and Actinomyces naselundii ATCC 12014 [26]. The microorganisms were cultured on blood agar plates for 2 days (37 °C, anaerobically). Then, a single colony was picked from each agar plate and transferred to tubes containing brain-heart infusion (BHI) broth with 5% sucrose to prepare 24 h broth cultures at 37 °C under anaerobic conditions. After that, the bacterial cell pellets were harvested by centrifugation (1500× g, 37 °C, 10 min). Bacterial suspensions were then prepared in BHI broth with 5% sucrose to a cell density of McFarland 2 (6 × 10⁸ cells/mL) [5]. Each restored tooth was soaked in a well of a 12-well plate containing 500 µL of each bacteria culture. The plate was maintained in an anaerobic chamber at 37 °C for 7 days. The medium was refreshed daily [7] and Gram stain test of the used medium was performed to check contaminants.