4.4. Determination of the Colony Forming Units (CFUs)

The CFUs were quantified as described in detail earlier [53]. In brief, after exposure in the oral cavity, the specimens were rinsed for 10 sec each with 1 mL sterile 0.9% NaCl to remove non-adherent microorganisms. Additionally, the reverse material surfaces and their upright side margins were cleansed using small, sterile foam pellets (Voco GmbH, Cuxhaven, Germany) after which each material was transferred into sterile Eppendorf tubes (Eppendorf GmbH, Wesseling-Berzdorf, Germany) and ultrasonicated for 2 min in 1 mL 0.9% NaCl on ice. After vortexing for 30–45 s, the suspensions of each material were then serially diluted up to 1:10³ in 0.9% NaCl. Aerobic and facultative anaerobic bacteria were cultivated on Columbia blood agar plates (CBA, Becton Dickinson, Heidelberg, Germany) at 37 °C and 5–10% CO₂ for 5 days. Yeast-cysteine blood agar plates (HCB, Becton Dickinson, Heidelberg, Germany) were used to cultivate anaerobic bacteria at 37 °C for 10 days (anaerobic chamber, Genbox BioMérieux SA, Marcy/Etoile, France). The number of CFUs per cm² was determined using a colony counter (WTW BZG 40, Xylem Analytics, Weilheim, Germany). All measurements were repeated twice.