Fig. 4

Quantification of sustained oral biofilm after cleansing. Oral biofilms were formed on plastic discs by anaerobic cultivation in BHI broth containing sucrose (1 %) and human saliva (10 %). After a 72 h cultivation at 37 °C, the discs were washed with 1 ml PBS (pH7.4). The discs were further washed with 1 ml saline, 10 mM citrate (pH3.5) or 0.5 M L-arginine (pH3.5) for 30 min before fixing with 2 % glutaraldehyde in 0.2 M cacodylate buffer for scanning electron microscopy. Biofilm areas were measured using a color-based auto-selection tool in Photoshop CS6, and the ratios relative to the total observation field were calculated. At least five randomly selected fields were examined. Data are expressed as mean ± standard deviation, and the statistical analysis was performed by ANOVA followed by Tukey’s test. The p-values less than 0.05 were considered to be significant