Biofilm Formation Assay for Vibrio anguillarum

The biofilm formation assay was performed in 96-well polystyrene microtiter-plates, as previously described [30] (link) with some modifications. Overnight cultures in LB₂₀ were diluted with fresh LB₂₀ medium to OD₅₅₀ = 0.1 and inoculated into a 96-well plate (200 µl per well). The plate was incubated at 28°C for 48 hours, after which wells were washed three times with 300 µl sterile physiological saline to remove all non-adherent bacteria. The remaining attached bacteria were fixed with 200 µl of 99% methanol per well for 2 hours, and the plate was emptied and left to air dry overnight. Then, the plate was stained for 20 min with 200 µl of 1% crystal violet per well. Excess stain was rinsed off by placing the plate under running tap water. After the plate was air dried, the dye bound to the adherent cells was resolubilised with 200 µl of 95% ethanol per well. The absorbance of each well was measured at 570 nm. For the quantification of exopolysaccharides, a Calcofluor white staining (Sigma-Aldrich) was used as previously described [30] (link). For each assay, a minimum of three different V. anguillarum cultures were used for each treatment. The reported data are representative of three independent experiments.