Biofilm composition by CLSM

The composition of 48 h biofilms was observed by CLSM, exposed to three types of dyes: (i) SYTO dye that stains nucleic acids; (ii) FilmTracer SYPRO Ruby Biofilm Matrix stain (Invitrogen, Paisley, UK), which labels most classes of proteins (Berggren et al., 2000); (iii) wheat germ agglutinin (WGA) conjugated with Oregon Green (Invitrogen), which stains N-acetyl-D-glucosamine residues (Wright, 1984). The fluorescence of dyes was detected using the following combination of laser excitation and emission band-pass wavelengths: 476 nm/500–520 nm for SYTO, 405 nm/655–755 nm for SYPRO and 459 nm/505–540 for WGA. After each staining step, the biofilms were gently rinsed with sterile water. The biofilm images were acquired in an Olympus^TM FluoView FV1000 confocal laser microscope and biofilms were observed using 40x water-immersion objective. The images were analyzed sequentially using two virtual channels. Three stacks of horizontal images (640 × 640 pixels) were acquired for each biofilm at different areas in the well. Two surfaces of two independent replicates were observed in each CLSM experiment.