Biofilm assays.

The effect of phenazines on biofilm formation was examined in B. lata ATCC 17760 and its phenazine-deficient isogenic mutant B. lata phzA and in an overproducing derivative carrying the pBBR1MCS-all plasmid. The ability to form static biofilms was determined using the crystal violet biofilm assay developed by O’Toole (88). Briefly, cultures were grown on LB plates supplemented with appropriate antibiotics for 24 h, bacteria were scraped off the agar, and the OD₆₀₀ was adjusted to 0.1. The normalized suspensions were diluted 1:100 in KMB broth and 100 μl aliquots were dispensed into 96-well U-bottom Costar PVC microplates (Corning, Corning, NY). The inoculated microplates were incubated for 48 h at 27°C, after which all wells were gently rinsed with water to remove the unattached cells and media components. Crystal violet (0.1%) was added to each well, and after 15 min of staining the microplates were rinsed and dried and the retained dye was solubilized with 30% acetic acid and quantified by measuring absorbance at 550 nm. The experiments were repeated 4 times with 60 replicates per strain, and differences between treatments were assessed by Kruskal-Wallis rank test followed by Dunn’s multiple comparison test (P < 0.05).

For flow cell biofilms, the bacteria were cultured overnight at 27°C on one-third-strength King’s medium B (1/3 KMB) plates and then scraped off the surface of the agar and suspended in 1/3 KMB broth at a density of approximately 10⁸ CFU ml⁻¹. The standardized bacterial suspensions were inoculated into a 24-well two inlets one outlet flow cell plate that was connected to a BioFlux 200 microfluidic system (Fluxion Biosciences, Alameda, CA). The inoculum was first pumped into the flow cell at 1.0 dyn/cm² for 6 sec, and bacteria were then allowed to attach to the channel surface for 1 h at 27°C. Initially, the flow rate of 0.16 dyn/cm² was maintained for the first 12 to 14 h, and after that, a flow rate of 0.44 dyn/cm² was set for 36 h to continuously pump fresh 1/3 KMB through channels containing bacteria. The development of biofilms was monitored by collecting bright-field images every 20 min with an LS620 digital microscope (Etaluma, Carlsbad, CA). The acquired images were merged into a time-lapse movie using BioFlux software (Fluxion). The images were also normalized using the threshold and slider tools of the BioFlux Montage software (Fluxion) and used to quantify the biofilm growth by calculating total percentage of area covered by the biofilm and plotting it against the time frames.