Biofilm Formation

A microtiter dish assay with crystal violet (CV) was used to evaluate biofilm formation, as described by O’Toole (2011). Briefly, 200 μL of LB with or without the tested compound were inoculated with bacterial suspension containing 1 × 106 CFU in 96-well microtiter plate and incubated at 28°C for 72 h without shaking. The suspensions were discarded carefully and gently washed twice with double-distilled H₂O (DDW) to remove any unattached cells. Then, 200 μL of 0.1% of CV was added to each well. The plate was incubated for 20 min at room temperature and CV from each well was discarded carefully. Each well was washed twice with DDW and the plate was dried before adding 200 μL of 30% acetic acid to solubilize the CV and incubated for 15 min at room temperature. The biofilm was quantified by measuring absorbance at 550 nm in a microplate reader (Spectra MR, Dynext Technologies, VA, United States).