Imaging Demonstrates Biofilm Volume Reductions After Treatment with Oral Rinses

Objectives: Compare effect of oral rinses on biofilm removal in a flow cell via analysis of confocal laser scanning microscopy (CLSM) images.
Methods: Hydroxyapatite coupons in CDC biofilm reactors were inoculated with pooled human saliva (IRB-approved) and biofilms grown 72hr. Biofilms in microscopy flow cells were stained with SYBR® Green (SG; stains nucleic acids) and Concanavalin-A conjugated to rhodamine-B (ConA; stains polysaccharides). 0.12% chlorhexidine gluconate (CHG), 0.07% cetylpyridinium chloride (CPC), 0.2% delmopinol (DEL), essential oil (EO), 3M^TM Experimental Rinse (EXP), or phosphate-buffered saline (PBS) flowed through the cell at 1 ml/min (n=5). CLSM was conducted before treatment and at about 3min and 6min of treatment in separate channels for each stain. Biofilm volumes were calculated by analysis of image stacks for each stain. Biofilm percent volume reduction (PVR) was calculated: PVR=(pretreatment volume -treated volume)/(pretreatment volume). Univariate ANOVA models applied to log-transformed PVR values adjusted for differences in treatment and imaging times; two-sided Tukey tests (P<0.05) were used to compare PVR.
Results: PVR results are shown in Table 1; superscript letters in each column denote groups that are not statistically significantly different. At 3min EO removed significantly more biofilm than other rinses for SG and ConA stains; EXP removed significantly more biofilm than all rinses except EO with SG, and more than DEL or PBS with ConA. At 6min CHG, CPC, EO, and EXP removed significantly more biofilm than DEL or PBS with ConA. At all timepoints, PVR for DEL and PBS were not significantly different.
Conclusions: These results demonstrate that CLSM image analysis is a viable method to show differences in biofilm removal among oral rinses. There are differences in PVR calculated with nucleic acid or polysaccharide staining, suggesting differences in removal of bacterial cells versus the biofilm matrix during treatment with rinses.