*2.5. Biofilm Formation*

The effect of culture medium on biofilm formation was determined by the widely used polystyrene crystal violet assay [22,23], with some modifications. Exponential phase cultures were prepared as described above, harvested by centrifugation, suspended in sterile water to remove residual medium, harvested, and resuspended in fresh PSY or SESOM to absorbance of 0.100 at 600 nm. Nunclon Delta Surface Polystyrene 96-well plates (Thermo Scientific, Waltham, MA, USA; catalog 167008) were loaded with 150 μL of cells in 7 replicates. The sterile culture media were loaded into additional wells to serve as blank. After 24 h incubation at 30 ◦C, the planktonic cells were removed by pipetting. Each inoculated well of the plate was washed twice by pipetting using sterile water to facilitate removal of all planktonic cells. Biofilms were stained by adding 200 μL of 0.1% aqueous crystal violet solution, shaking at room temperature for 20 min. The crystal violet solution was removed from the wells by pipette, followed by two cycles of careful washing with water by pipetting. The plate was left open for 30 min to dry and 200 μL of 95% ethanol was added to dissolve the crystal violet. The plates were incubated for 10 min at room temperature with shaking and absorbance determined at 570 nm.
