Crystal violet staining.

We used the crystal violet method to stain the streptococcal biofilms on the plastic surface (69). Each well containing S. mutans biofilms was stained with 125 μl of a 0.1% solution of crystal violet (Sigma) for 15 min. Microtiter wells were rinsed three times with distilled water (dH₂O), flipped over, tapped vigorously on a stack of paper towels to remove all excess dye, and air dried overnight. Next, a 125-μl suspension of 30% acetic acid (Thermo Fisher) in dH₂O was added to each well to solubilize the crystal violet, followed by a 15-min incubation at room temperature. Finally, a 100-μl suspension of solubilized crystal violet was transferred to a clean microtiter plate and measured in a microtiter reader at 550 nm using 30 % acetic acid in dH₂O as a negative control.