2.4.2. Seeding of the Dental Composites with Bacteria

Eighteen disc samples (three for each experiment) with a diameter of approximately 12 mm and a height of 1 mm were sterilized by immersing the specimens for 10 s in 70% ethanol and drying at room temperature. Dry samples were rinsed twice with 200 µL of phosphate buffered saline (PBS, Pan-Biotech, Aidenbach, Germany) and then placed in the wells of 12-well polystyrene plates. Next, 1000 µL of BHI broth were added to each well. Simultaneously, other plates with composite discs were filled up with 1000 µL of BHI broth with 0.25% sucrose, which is a main sugar responsible for biofilm formation on the dental materials. Sucrose was added to broth to determine the changes in bacterial biofilm formation on the surface of B, BM, F, FM samples dependent on the absence or presence of this sugar in the medium. Finally, an amount of 20 µL of bacterial inoculum (1.5 × 10⁸ CFU/mL) was transferred to each well. In the case of mono-species biofilm tests it was S. sanguinis or S. mutans. For mixed-species biofilm assays it was 10 µL of S. sanguinis (1.5 × 10⁸ CFU/mL) and 10 µL of S. mutans (1.5 × 10⁸ CFU/mL) added together. Sterility controls (only BHI or BHI+sucrose) and positive biofilm growth controls formed on the polystyrene surface (BHI or BHI + sucrose with bacterial inoculum) were included in all experiments. Plates were anaerobically incubated at 37 ℃ for 48 h.