Different methods have been used to study biofilm formation including CLSM, AFM [19 (link)]. In our study, CLSM has been employed to reveal the biofilm structure as well as the cell viability within biofilms. Biofilms grown on the rough surface of the DBR discs were treated with Polident® experimental formula M138-12 (GlaxoSmithKline, UK) or Efferdent® antibacterial denture cleanser (Prestige brands, USA) for 5 min according to manufacturer’s recommendations, and untreated controls were stained with 10 μM of the cell-permeable fluorescent stain SYTO 59 (labeling of all cells) and 10 μM of the cell-impermeable fluorescent stain SYTOX Green (labeling of cells with compromised cell walls only) (Invitrogen, USA) in PBS buffer for 20 min at room temperature in the dark before visualization [17 (link)]. All biofilm images were collected with a Zeiss LSM 5 PASCAL CLSM using LSM 5 PASCAL software (Zeiss, Germany). Excitation at 633 nm with an argon laser in combination with a 650 nm band-pass emission filter was used for SYTO 59 fluorescence imaging. SYTOX Green signals were visualized using 488 nm excitation with a helium-neon laser and a 503-530 nm band-pass emission filter.