Figure 3
Direct effect of actives on oral epithelial cells and multi-species biofilms. The oral epithelial cell line OKF6-TERT2 was seeded at 1×105 cells/ ml in 24 well plates for toxicity studies. Cells were treated with concentrations of CHX (0.01, 0.05, 0.2% v/v) (A) and RSV (0.01, 0.05, 0.5% w/v) (B) for 30 minutes before washing with PBS. Cell viability was assessed using the Alamarblue® assay with absorbance read at 570 nm and 600 nm. Following this concentrations chosen for the remainder of the study were CHX 0.2% (v/v) and RSV 0.01% (w/v). To investigate the role of actives in biofilm treatment biofilms were grown on coverslips in 24 well plates for 6 days as previously described. Mature biofilms were treated with 0.2% v/v CHX and 0.01% w/v RSV for 30 minutes before washing with PBS. Biofilm viability was measured using Alamarblue® read at 570 nm and 600 nm (C). Bacterial DNA was extracted and each species quantified using SYBR® GreenER™ based qPCR (D). Biofilms were also analyzed by SEM at both 2000x (E, G, I) and 5000× (F, H, J). Samples were processed and viewed on a JEOL JSM-6400 scanning electron microscope and images assembled using Photoshop software. Untreated biofilms (E, F) were compared with biofilms treated with 0.2% (v/v) CHX (G, H) and and 0.01% (w/v) RSV (I, J). Samples were assayed in triplicate on three separate occasions and data are mean ± SD (**p < 0.01, ***p < 0.001).