*2.4. Quantitative Determination of Biofilm*

Bacteria used in this study were biofilm-proficient *S. aureus* ATCC 6538 and *E. coli* K-12 strains. Biofilm formation was measured under static condition by adopting quantitative crystal violet (CV) binding assay of Kwasny and Opperman with modifications [22]. In the current study, the flat inner surface of glass petri dish coated with prepared composites and resin was overlaid with 4 mL of sterile nutrient broth (composition is tabled in Supplementary Materials), so that the total area of the coating was covered. Then, 0.1 mL of logarithmic phase cultures of either *E. coli* or *S. aureus* grown over night to an optical density of *ca.* 0.1, at 595 nm in the appropriate growth media, were inoculated into sterile media in coated bottom plates prepared as above. Inoculated bottom plates were incubated in a bacteriological incubator at 37 °C under UV-A irradiation with intensity of 0.2 mW/cm2 with *Ȝ*max around 365 nm (which is harmless to cause bacterial reduction), for different exposure durations. Later, the broth with planktonic cells was discarded by decantation. The plates were washed twice by gentle swirling with 2 mL of sterile phosphate-buffered saline to remove any non-adherent cells. Cells which remained adherent (biofilm mass) to the surface of polymer coated bottom plate were fixed by heating in a hot air oven at 60 °C for 60 min. Later plates were cooled to room temperature and stained with 1 mL of 0.06% (w/v) solution of crystal violet which was allowed to stand at room temperature for 5 min. Then plates were washed several times with phosphate-buffered saline to remove excess CV staining. Biofilm bound CV was eluted by vortexing with 1 mL of 30% acetic acid (destaining solution) for 10 min. The 0.2 mL aliquots of the wash solution with eluted crystal violet were transferred to 4 different wells of 96-well microtiter plates for the purpose of measuring the absorbance at 600 nm. Results were expressed as inhibition percentages of biofilm development. The percent inhibition of biofilm growth produced by each nanocomposite surface was calculated with the formula,

$$\left\{ 1 \cdot \left[ \text{CV OD}\_{600} \text{(composite)} \right] \Big\arrow\_{\text{average CV OD}\_{600} \text{(negative control)}} \right\} \times 100 \tag{l}$$

where CV OD600 is OD of crystal violet destaining solution obtained at Ȝmax 600 nm. The results are presented as the average of four individual replicates. To check the binding affinity of CV to the prepared composites and neat epoxy, a similar assay with 48 h of UV exposure was conducted as above with the plain broth which was not inoculated with bacteria. The OD of destaining solution when measured was found to be insignificant to interfere with the experimental results. Then, the resulting silver concentrations in the same plain broth were also quantified by atomic absorption spectroscopy (AAS) analysis, released into the exposed media by the composites of different Ag-TiO2 loadings. AAS analysis of released silver concentration was carried out with a 7700X instrumentation (Agilent, Santa Clara, CA, USA), using different standard concentrations.

The reduction in biofilm colonization on composite was also determined in terms of CFU (colony forming unit), by sonicating assayed composite plate with 5 mL PBS for 5 min to remove adherent bacteria. The PBS suspension of released cells was then diluted appropriately, and spread on nutrient agar plate. The bacterial CFUs per milliliter of PBS that formed upon the medium was determined after incubation for 48 h at 37 °C. The experiment was repeated two times under identical conditions along with negative control (neat epoxy). The biofilm log reduction values were determined as difference between Log10 CFU/plate recovered from the treated plates and Log10 CFU/plate recovered from control plate (neat epoxy). Each experiment was conducted with three replications for each composite plates and colonies were enumerated to obtain the log reduction.
