Live and dead bacterial cell staining

The 46-h-old biofilms were stained at room temperature in the dark for 30 min using a FilmTracer LIVE/DEAD Biofilm viability kit {"type":"entrez-nucleotide","attrs":{"text":"L10316","term_id":"309487","term_text":"L10316"}}L10316 (Invitrogen, Molecular Probes Inc., Eugene, OR, USA). The final concentrations of SYTO9 and propidium iodide (PI) were 6.0 μM and 30 μM, respectively. The excitation/emission wavelengths were 480/500 nm for SYTO 9 and 490/635 nm for PI for collecting the fluorescence. The stained live and dead bacterial cells were observed with an LSM 510 META microscope (Carl Zeiss, Jena, Germany) equipped with argon-ion and helium–neon lasers. All confocal fluorescence images were taken with an EC Plan-Neofluar 10x/0.30 M27 objective lens. Three independent experiments were performed, and five image stacks per experiment were collected (n = 15). A stack of slices in 6.4 μm step sizes was captured from the top to the bottom of the biofilm. The biovolume and thickness of live and dead cells were quantified from the entire stack using COMSTAT image-processing software²¹. The biovolume is defined as the volume of the biomass (μm³) divided by the substratum (hydroxyapatite surface) area (μm²). The three-dimensional architecture of the biofilms was visualized using Imaris 8.0.2 (Bitplane, Zurich, Switzerland). The original confocal data was uploaded to Imaris 8.0.2 software and the intensity of green and red fluorescence in full thickness of biofilm layers were captured automatically. The software reconstructed the 2-dimentional intensity of fluorescence in all the layers to a 3-dimentional volume stack²².