Crystal-violet biofilm assay

A static biofilm formation assay was performed on six bacterial strains (MSSA 6538, MSSA 25923, MRSA MW2, S. epidermidis, Pseudomonas aeruginosa PAO1, and Escherichia coli O157:H7) in 96-well polystyrene plates (SPL Life Sciences, Korea), as previously reported⁴⁸. Briefly, cells were inoculated into LB medium (total volume 300 μl) at an initial turbidity of 0.05 at 600 nm. Antibiofilm agents were added at different concentrations at inoculation and cultured for 24 h without shaking at 37 °C. To quantify biofilm formation, biofilms were stained with crystal violet for 20 min, dissolved in 300 μl of 95% ethanol, and absorbances were measured at 570 nm (OD₅₇₀). Cell growths in 96-well plates were also measured at 620 nm (OD₆₂₀). Biofilm formation and static cell growth results are presented as the averages of two independent cultures of twelve replicate wells.