Crystal Violet Staining

S. aureus (5 × 10⁷ CFU/ml) was grown in Luria-Bertani (LB) broth on 96-well plates at 37°C for 24 h in the presence or absence of LTA. Planktonic bacteria were removed by gentle washing twice with phosphate-buffered saline (PBS). Biofilms were stained with 0.1% crystal violet solution for 30 min at room temperature, followed by washes with PBS to remove non-specific stain. The adhering dye was dissolved in solution (95% ethanol and 0.1% acetic acid), and absorbance was measured at 600 nm in a microplate reader (Molecular Devices, Sunnyvale, CA, United States).