Removal of Tongue Biofilms With a Tounge Cleaner: Microscopic Evaluations

Objectives: Bacterial biofilms that grow on the dorsum of the tongue are a recognized source of oral malodour and may serve as microbial reservoirs for the recolonisation of recently cleaned teeth.  Removal of biofilms from the tongue therefore enhances dental hygiene by reducing the oral bioburden.  The cheek and tongue cleaner on the Colgate 360 toothbrush is designed to remove these biofilms.  In this investigation, methods were developed to visualize this process.  Methods: Ex situ dental plaques and Klebsiella aerogenes expressing fluorescent proteins were used to develop biofilms on bovine tongue tissue or on membranes resting on agar.  The tongue cleaner was applied to the biofilm in a single line and the resulting furrows were visualised by; i) scanning electron microscopy, ii) epifluorescence microscopy by means of expressed fluorescent proteins or fluorescent dyes or iii) confocal laser scanning microscopy (CLSM) coupled with specialist image processing.  Results: The tongue cleaner efficiently removed biofilms from either substratum.  Epifluorescence microscopy produced clear images of biofilms, which were combined to produce composite aerial images of biofilm clearance. Biofilms expressing fluorescent proteins produced sharper and brighter images than those stained with exogenous dyes. CLSM visualised furrows within the biofilm in three dimensions to demonstrate biofilm removal. Biofilm thickness was reduced from 200-210 μm to 0-35 μm and biomass density was reduced from116 μm³/μm² to 0.07 μm³/μm². Scanning electron microscopy produced high-resolution images confirming these observations but required additional sample preparation steps. Conclusions: Results confirm that the cheek and tongue cleaner on the Colgate 360 toothbrush effectively removes ex situ dental plaque biofilms.  By employing a combination of methods, the removal of biofilms by the tongue brush was visualised.  Confocal laser scanning microscopy, combined with image processing allowed flexible and three-dimensional visualisation and quantification of the effect of tongue brush material on intact and hydrated biofilms.