Dual-Species Biofilm Formation Assay

P. gingivalis-S. gordonii dual-species communities were produced as previously described (Jung et al., 2019 (link)). Briefly, hexidium iodide (15 μg/ml, Molecular Probes)-labeled S. gordonii (2 × 10⁸ cells) was deposited on a glass coverslip anaerobically at 37°C for 16 hr in PBS. Unattached S. gordonii cells were removed by washing, and 5-(and-6)-carboxyfluorescein, succinimidyl ester (CFSE, 4 μg/ml, Molecular Probes)-labeled P. gingivalis parental or mutant strains (5 × 10⁷ cells) were added in pre-reduced PBS. After anaerobic incubation at 37°C for 24 hr, communities were washed with PBS, fixed with 4% paraformaldehyde and examined on a confocal microscope (Leica SP8) using 488 and 552 nm lasers for CFSE and hexidium iodide respectively. Volocity software (Perkin Elmer) was used for three-dimensional reconstruction and quantification of the volume of P. gingivalis and S. gordonii fluorescence.