To test for biofilm formation in the absence of saliva as reported previously (16, 50), bacteria in BHI broth were grown at 37°C for 16 to 18 hours in 5% CO2 in 96-well plastic plates (Corning). The plates were inverted and shaken to remove excess liquid growth medium, stain, and buffer. The retained biomass was stained for 15 min with 50 μl of 0.1% (w/v) crystal violet. The wells were then washed four times with sterile PBS. Excess PBS was removed by rapping the plate onto paper towels and air-drying for 2 min. To quantify sessile biomass, 200 μl of acidified ethanol (4% 1 N HCl/EtOH) was added to each well. In each well, the ethanol and crystal violet were gently mixed, 125 μl of the extracted crystal violet was transferred to a new flat-bottom well in a polystyrene microtiter plate, and the absorbance at 570 nm was determined with a BioTek Synergy 2 reader.

To test for biofilm formation in the presence of saliva, pooled human saliva was centrifuged at 4300 rpm (Beckman SX4750 rotor) for 5 min at 4°C, and supernatants were aliquoted into 1.7-ml microfuge tubes and centrifuged at 15,000 rpm (Eppendorf 5415D) for 20 min at 4°C. Saliva aliquots were repooled, and 200 μl of clarified saliva was added to each microtiter plate well. Plates were incubated for 1 hour at room temperature on an orbital shaker, and then the saliva was aspirated from each well and replaced with growth medium and bacteria. Negative control saliva-coated wells produced biofilms ≤5% of wild type in assays.

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