Hope Chris, University of Liverpool, United Kingdom

Cell viability within oral biofilms

Chris Hope
Unit of Plaque Related Diseases, School of Dental Sciences, University of Liverpool, Edwards Building, Daulby Street, L69 3GN Liverpool, United Kingdom, e-mail: chope@liv.ac.uk

Confocal laser scanning microscopy (CLSM), when used in conjunction with fluorescent indicators of membrane integrity, has revealed the spatial distribution of cell vitality within laboratory grown microcosm oral biofilms and demonstrated that dental plaque contains a large proportion of nonviable cells. Image analysis techniques were developed to elucidate depth related trends in cell vitality by comparing fluorescence profiles for viable and nonviable cells individually, an approach which minimises errors due to the variations in the confocal capture settings (i.e. offset, gain and laser power). Fluorescence intensity profiles through the depth of the plaques showed an offset between the position of the maximum viable intensity and the maximum nonviable intensity, suggesting that the outer layers of the biofilm contain proportionally more viable bacteria than the inner regions. It was proposed that these analyses could be used to evaluate the effectiveness of antimicrobial compounds against oral biofilm.
Conventional assays to establish the effectiveness of antimicrobial compounds upon oral biofilm typically involve exposing said biofilms to the agent and subsequently enumerating viable bacteria by traditional culture methods. This additional culturing step has an inherent weakness in that cells in a viable non-cultivable state will not be detected and closely associated cells, such as co-aggregating pairs or single-species microcolonies, may be counted as a single colony forming unit. These problems will be circumvented by CLSM employing vital staining. An antimicrobial compound can be added to the confocal viewing solution, which bathes the biofilms during scanning, to achieve the desired concentration of agent before a series of further CLSM images are captured. This technique was successfully used to demonstrate the ineffectiveness of 0.2% chlorhexidine against dental plaque and also showed that this commonly used oral disinfectant caused the biofilm to contract. CLSM has also been used to demonstrate the effectiveness of photodynamic therapy in real-time.