Biofilm formation assay

Biofilm formation assay was conducted using the crystal violet staining method (Holmberg et al., 2009). Bacterial strains were cultured overnight until the OD₆₀₀ reached 0.5, and then transferred to 96-well cell plate at 100 μl per well and cultured at 28°C for 72 h without shaking. Thereafter, the wells were washed with sterile water twice and stained with 125 μl 0.1% crystal violet (w/v) for 30 min to quantitatively measure the attached biofilms. After removing the dye, wells were thoroughly dried at 37°C, and 1% sodium dodecyl sulfate (SDS) was added to dissolve the biofilm at 200 μl per well. The quantity of biofilm formed was determined at an absorbance of 570 nm using a Multiskan™ FC microplate spectrophotometer (Thermo Fisher Scientific).