Hexidium iodide

Communities of P. gingivalis and S. gordonii were generated as described previously (Kuboniwa et al., 2006 (link)). S. gordonii cells (2 × 10⁸) were stained with hexidium iodide (15 μg/mL, Invitrogen) and deposited on glass coverslips for 16 h anaerobically at 37°C. P. gingivalis cells (2 × 10⁷) were labeled with 5-(and-6)-carboxyfluorescein, succinimidyl ester (FITC, 4 μg/mL, Invitrogen) and reacted with S. gordonii for 18 h in prereduced PBS anaerobically at 37°C with rocking. After washing with PBS, coverslips were examined on a Leica SP8 confocal microscope using 488 nm (FITC) and 558 nm (hexidium iodide) lasers. XYZ stacks were digitally reconstructed using Volocity software (Perkin Elmer). Quantitation of the volume of P. gingivalis fluorescence was obtained using the Find Objects algorithm in the Volocity program. This process analyzed all P. gingivalis fluorescence in the 3D digitally recreated confocal images. To estimate microcolony formation, the Find Objects process was used with a threshold for 3D objects greater than 30 μm³.

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.