Visualizing Pseudomonas Biofilm Formation

Inoculation of flow cells was done by normalizing overnight cultures to an optical density at 600 nm (OD₆₀₀) of 0.05 and injecting into an Ibidi μ-Slide VI^0.4 (catalog no. 80601; Ibidi). To seed the flow cell surface, the medium flow was suspended, and the bacteria were allowed to adhere at room temperature for 1 h. Flow of 5% (vol/vol) LBNS with 0.1% arabinose was initiated at a rate of 0.15 ml/min and continued for 48 h. Following the biofilm growth period, the flow was terminated, and the biofilms were fixed with 4% paraformaldehyde. Psl was stained with a cocktail of three monoclonal antibodies provided by MedImmune (33 (link)). The antibodies were directly labeled with Alexa Fluor 647 using the Alexa labeling kit (Life Technologies). Confocal images were obtained on a Nikon A1R live-cell imaging confocal microscope. Images were obtained with a 20× oil immersion objective. Images were processed using the FIJI software (45 (link)). Quantitative analyses were performed using the COMSTAT2 software package (46 (link)). Total biomass was determined from Z-stack images using the BIOMASS command with the threshold set at 25. Three independent biofilms were imaged and analyzed. Statistical significance was determined by a one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test (**, P ≤ 0.01; ***, P ≤ 0.001).