Mixed Biofilm Formation of Oral Bacteria

S. gordonii cells (10⁸) were stained with hexidium iodide and aerobically cultured in CDM containing 0.025% glucose for 16 h using an 8-well saliva-coated LAB-TEK Chamber Slide System (Nalge Nunc International, Naperville, IL). For analysis of mixed biofilm formation, S. gordonii, an early colonizer of the tooth surface, was first cultured the same way as described above, washed gently twice by PBS, and then co-cultured with 1.5 × 10⁷F. nucleatum cells labeled with 5-(and-6)-carboxyfluorescine succinimidyl ester, as described previously (5 (link)). The bacterial cells (1.5 × 10⁸) were then used in an arginine-supplemented mixed biofilm experiment. S. gordonii-P. gingivalis-mixed biofilms were also generated in the same manner. As for the F. nucleatum mono-species biofilm formation, FITC-labeled organisms (2.5 × 10⁷ cells) were cultured in PBS with specific metabolites for 24 h. Biofilms were observed with a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss MicroImaging GmbH, Germany). Obtained images were analyzed using the Imaris 7.0.1 software package (BitplaneAG, Zurich, Switzerland), as described previously (38 (link)).