*2.4. Quantification of Biofilm*

The PMMA slides were collected after 24 h to 96 h and adhesion was assessed using crystal violet binding assay. Bacterial growth was fixed with acetic acid, followed by staining with 3% crystal violet (Ezzy Stain) and washing with PBS at 7.0 pH. Heat fixation was performed for 30 s followed by acetone washing. For accuracy of assessment and measurements, the procedure was repeated twice (per sample).

#### *2.5. Scanning Electron Microscopy*

Scanning electron microscopy was carried out for the analysis of the production of extracellular matrix material and to observe the bacterial presence and disinfection on acrylic slides. Biofilm slides were divided into 4 mm sections and washed with distilled water to remove the debris and were then negatively stained with 0.02% Uranyl acetate for 30 s. These 4 mm slides were platinum coated in a coating machine (JEOL 3000 FC, Tokyo, Japan), and the sections showed the presence of biofilm material when examined directly in SEM (JOEL-JSM IT-100, Tokyo, Japan).
