Polypyrrole

Biofilms formation was developed in 96-well polystyrene microtiter plates (Sumitomo Bakelite, Tokyo, Japan), which were previously coated with human saliva, BSA, anti-PAc serum [38 (link)], anti-PAc monoclonal antibody [39 (link)] and anti-GbpC serum [40 (link)] for 1h at 4°C. After pre-coating, wells were washed with phosphate buffered saline (PBS) 2 times. Biofilm formation assays were performed using a previously described procedure [36 (link)]. Wells containing 180 μl of TSB with 0.25% sucrose were inoculated with 20 μl of S. mutans UA159 and gtfBC mutant, S. sanguinis, S. mitis and S. gordonii from a cell culture with an optical density (OD₆₀₀) of 0.4. The plates were then incubated with various concentrations of polypyrrole (Sigma-Aldrich, Co., St. Louis, MO) or hyaluronic acid (MW 5,000–150,000, Tokyo Chemical Industry, Co. LTD, Tokyo, Japan) at 37°C with 5% CO₂ for 16 h. To observe the effects of hyaluronic acid on polypyrrole-dependent biofilm formation, polypyrrole was pretreated with 7 mg/ml hyaluronic acid for 1 h at 37°C and applied to the biofilm formation assay. After incubation, the planktonic cells were removed by washing with distilled water (DW), and the adherent cells were stained with 0.25% safranin for 15 min. After washing with DW 2 times, safranin was extracted from biofilm with 70% (v/v) ethanol, and quantitatively measured by the absorbance at 492 nm.