Characterization of Bacterial Biofilm Composition

Bacterial strains were grown in TSBYE in a sterile 8-well treated μ-Slide (Ibidi) under 5% CO₂ at 37°C for 16 h. The biofilm samples were gently washed with PBS three times to remove unattached cells followed by incubation in 5% bovine serum albumin (BSA) in PBS for 1h. S. parasanguinis was incubated with a polyclonal antibody that recognizes the surface amylase binding protein (AbpA) [17 (link)] or a monoclonal antibody that recognizes the surface fimbriae protein Fap1 [20 (link)]. P. aeruginosa was incubated with a monoclonal antibody that recognizes an outer membrane protein (Omp) (Abcam). Alginate was probed using a polyclonal antibody that was provided as a gift from Dr. Gerald B. Pier at Harvard University [55 (link)]. The biofilms were washed 3 times with PBS to remove the unattached primary antibodies and then incubated with fluorescent-conjugated secondary antibodies (Molecular Probes) for 30 min. Alexa Fluor 594 (red)-conjugated goat anti-mouse IgG and Alexa Fluor 488 (green)-conjugated goat anti-rabbit IgG were used to stain bacterial cells and alginate. The stained samples were then washed 3 times prior to detection using a fluorescence (Nikon X-Cite series 120 PC) or a Nikon A1+ confocal laser scanning microscope (CLSM) (Nikon Instruments Inc.). NIS Elements microscopy imaging software was used to calculate biofilm biomass and co-localization.