2.9. Crystal Violet Biofilm Assay

Biofilm formation in microtiter plates was quantified by crystal violet (CV) staining, essentially as described by O’Toole and Kolter [26]. An S. mutans overnight culture grown in THB was diluted to an OD₆₀₀ of 0.02 in THBS, and 100 μL of the diluted culture was added to the wells of a 96-well microtiter plate, which was subsequently incubated at 37 °C for 24 h. The wells were aspirated and remaining planktonic bacteria were removed by addition and removal of 120 μL Milli-Q water (MQH₂O). The biofilms were stained for 15 min with 120 μL 0.4% CV. CV quantification was done by washing the wells twice with 150 μL MQH₂O, solubilizing the CV with 30% acetic acid for 30 min, and measuring the absorbance at 590 nm.