Biofilm assay.

Overnight cultures (1.5 μl) were inoculated in U-bottom 96-well plates (Costar) containing 100 μl M8 salts minimal medium (Na₂HPO₄, KH₂PO₄, NaCl) supplemented with glucose (0.2%, vol/vol), Casamino Acids (0.5%, vol/vol), and MgSO₄ (1 mM), subsequently referred to as M8 medium. Biofilm assay plates were then stained with 100 μl of 0.1% crystal violet in water for 20 min at room temperature and destained for 20 min with 125 μl destaining solution (40% glacial acetic, 40% methanol, and 20% [vol/vol] H₂O). Absorbance was read at an optical density at 550 nm (OD₅₅₀), and destaining solution was included as the blank. Biofilm assays were done similarly to those in published work by the O’Toole group (52, 53).