A fluorescence assay to determine the viable biomass of microcosm dental plaque biofilms

Dental plaque bacteria form complex and robust cell aggregates which cannot be counted accurately using epifluorescence microscopy. This causes a significant problem for quantifying their viability. The aim of the investigation was to develop a fluorescence assay to quantify the viable biomass of dental plaque biofilms. Using an artificial mouth system, microcosm plaques were grown under a range of fluoride and mineralizing conditions, and were treated with the oral antiseptics chlorhexidine (CHX) and Listerine. Plaques were harvested, made into suspension and stained in microtitre plates with a di-chromatic fluorescent stain (Live/Dead BacLight). The percentage of viable biomass was calculated from the regression data generated from a viability standard. The standard was constructed using different proportions of viable (green fluorescence) and non-viable (red fluorescence) plaque bacteria, and growth conditions for optimizing green fluorescence were investigated. The results from the assay showed that fluoride at 1000 and 3000 ppm promoted plaque viability by at least 15%, from approximately 45 to 60%, and at 5000 ppm to approximately 87% (P<0.05). Plaques treated with Listerine and CHX from d 0 yielded insufficient biomass to be tested for viability, however 14 d post-treatment, viability was comparable to untreated plaques (approximately 55%, P>0.05). Treatment with Listerine and CHX from d 3 reduced biomass but not viability. Development of this assay enabled viability of plaque bacteria which cannot be resolved with epifluorescence microscopy to be evaluated. It offers a rapid alternative to epifluorescence microscopy and could be applied to nonoral bacteria.