A method for quantitative determination of biofilm viability

In this study we present a scheme for quantitative determination of biofilm viability offering significant improvement over existing methods with metabolic assays. Existing metabolic assays for quantifying viable bacteria in biofilms usually utilize calibration curves derived from planktonic bacteria, which can introduce large errors due to significant differences in the metabolic and/or growth rates of biofilm bacteria in the assay media compared to their planktonic counterparts. In the presented method we derive the specific growth rate of Streptococcus mutans bacteria biofilm from a series of metabolic assays using the pH indicator phenol red, and show that this information could be used to more accurately quantify the relative number of viable bacteria in a biofilm. We found that the specific growth rate of S. mutans in biofilm mode of growth was 0.70 h-1, compared to 1.09 h-1 in planktonic growth. This method should be applicable to other bacteria types, as well as other metabolic assays, and, for example, to quantify the effect of antibacterial treatments or the performance of bactericidal implant surfaces.