4.4. Crystal Violet Biofilm Assay

Three hundred microliters of 100-fold diluted overnight culture was added per well in 24-well assay plates and incubated for 24 h at 25 °C. After removing planktonic bacteria from the plate, biofilms were evaluated by the crystal violet assay. An amount of 0.3 mL of 0.5% crystal violet was added to each well of the plate. The plate was incubated for 30 min at 25 °C before removing the staining solution, and then it was washed three times with 350 μL phosphate-buffered saline (PBS, pH 7.5). After removing the washing solution, 300 μL 96% EtOH was added per well to dissolve the biofilm-bound crystal violet by gently knocking the plate. Absorbance was measured at 595 nm.