Quantifying Bacterial Adhesion to Abiotic Surfaces

Adhesion to abiotic surface (polystyrene) was analyzed using 96-well plates as described previously [26 (link)]. Overnight cultures of bacteria grown in LB broth (10 μl) were added to 1 ml of LB. This volume was distributed in quintuples (200 μl per well) into a 96-well plate and incubated at room temperature for 24 h. Unbound bacteria were removed by washing the wells three times with PBS, and bound bacteria were stained with 1% violet crystal (CV) for 20 min. Wells were thoroughly rinsed thrice with PBS, and the dye in the adhered bacteria was solubilized with 100 μl of ethanol 70%. Finally, the amount of extracted violet crystal was determined using an enzyme-linked immunosorbent assay (ELISA) and measuring the OD₆₀₀ in a multiskan plate reader (Thermo Scientific).