Figure 1.
Development of multi-species biofilm co-culture model. A. Multi-species biofilm culture protocol. Thermanox™ coverslips were placed in 24 well plates for biofilm culture. Bacteria were grown on appropriate agar plates and cultured in broth for 1–2 days. Cultures were then washed three times in PBS and standardized at 1×109 CFU/ ml, added to artificial saliva to make a 1×108 CFU/ml final volume and added to the biofilm on the appropriate days and cultured in aerobic and anaerobic conditions. After 7 days culture artificial saliva is removed from mature biofilms which are then washed with PBS and stored at -80°C. B. Hanging basket co-culture model. Multi-species biofilms were grown on Thermanox™ coverslips and previously described. Epithelial cells (OKF6-TERT2) were seeded in 24 well plates at 1×105 cells/ml in cell culture media. Biofilms were attached inversely to Millipore cell culture inserts using Vaseline® and placed over wells containing cells. Cells and biofilms were co-cultured for 4 and 24 h.