FIGURE 6. 8 hr pH of biofilm spent media is altered by HOSCN treatment.

Biofilms of <i>S. sanguinis</i> or <i>S. mutans</i> were grown in 24-well plates (Corning Incorporated Costar cat. # 3524) using DROOL containing 1% sucrose with no amylase added. Overnight cultures were first rinsed and resuspended with 10 mM Tris-HCl (pH 7). 2X biofilm DROOL and 10 mM Tris-HCl (pH 7) (with bacteria and either with or without HOSCN), were added in equal amounts for a total volume of 1 ml, an initial A₆₀₀ = 0.04, and 0-500 µM HOSCN at t = 0 hr. Biofilms were grown at 37ºC and 5% CO₂ for either 8 or 24 hr then gently rinsed with phosphate buffered saline (PBS) before being subjected to crystal violet (CV) staining or CFU counting and determining pH of spent biofilm media. Biofilms were CV stained as described earlier. Biofilms subjected to CFU counting were vigorously resuspended in 1 ml PBS by pipetting and by scraping the well bottom with a 200 µl pipette tip. Serial dilutions of resuspended biofilms were plated on BHI and incubated overnight for subsequent CFU counts. Spent media pH was determined by interpretation of at least three separate spots on Hydrion pH paper.