FIG. 8.

Bacterial aggregation (A) or adhesion (B) assay to analyze the interaction of S. mutans with salivary preparations. In bacterial aggregation assays, cell suspensions were mixed with SAG, UWS, or PBS and transferred to cuvettes. The OD₆₀₀ of the samples was recorded at 10-min intervals at 37°C for 120 min in a spectrophotometer equipped with a temperature-controlled multicuvette positioner. Percentage aggregation (percent decrease in OD₆₀₀) was calculated as follows: [(OD₆₀₀ at 0 min − OD₆₀₀ at 120 min)/(OD₆₀₀ at 0 min)] × 100. For bacterial adhesion, aerobically or anaerobically grown cells were harvested at an OD₆₀₀ of 0.5 and stained with SYTO 13. Adhesion assays were performed in two different ways: salivary preparations were added to each well with the cell suspensions (fluid-phase salivary preparations), or wells were first coated with salivary preparations before inoculation with cell suspensions (surface-adsorbed salivary preparations). For the experiments with surface-adsorbed salivary preparations, each well was conditioned with 100 μl of SAG, UWS, or PBS for 2 h, washed, and air dried. For the experiments with fluid-phase salivary preparations, 150 μl of the cell suspension was inoculated into the wells concurrently with 15 μl of SAG, UWS, or PBS. Plates were incubated for 3 h in an aerobic or anaerobic environment and washed, and adherent bacteria were measured using a BioTek microplate scanning spectrophotometer. The data represented herein were from incubation under aerobic conditions, because there was no significant difference in bacterial adhesion to polystyrene wells between aerobic and anaerobic incubation. The error bars represent standard deviations. Data presented here are representative of at least three independent experiments.