FIG. 3.

Quantitative assay of biofilm formation by wild-type (RB50) and Bvgⁱ-phased-locked (RB53i) B. bronchiseptica at different nicotinic acid concentrations. Bacteria were grown in 96-well polystyrene plates, and biofilm formation was quantified by absorbance of solubilized crystal violet stains, as described in Materials and Methods. Biofilm formation in the wild-type bacteria peaked in the Bvgⁱ phase (0.2 to 0.8 mM nicotinic acid). The Bvgⁱ-phase-locked mutant formed high levels of biofilm at all nicotinic acid concentrations. Bvg⁺-phase growth condition is 0 to 0.1 mM nicotinic acid, 0.2 to 1.6 mM is Bvgⁱ phase, and 4.0 mM (and above) is Bvg⁻ phase. OD₅₉₅, optical density at 595 nm.