Quantifying Biofilm Biomass via Crystal Violet

To quantify the biofilm biomass, the washed biofilms were stained with 200 μl of a 0.1% crystal violet solution (1:4 dilution in DDW of the Gram's crystal violet solution, Merck) for 20 min at room temperature^{57 (link)}. The stained biofilms were washed twice with DDW, and after drying the wells, the stain was dissolved in 200 μl of a 33% acetic acid solution, and quantified by reading the OD at 595 nm in the M200 Tecan microplate reader. Different dilutions of the test compounds in the absence of bacteria were used to measure background signals. The percentage biofilm formation was calculated by dividing the OD of treated samples on OD of control samples, multiplied with 100%.