Optimization of resazurin-based viability staining for quantification of microbial biofilms

The resazurin-based viability staining is often used to quantify viable biofilm cells grown in microtiter plates (MTP). The non-fluorescent resazurin is reduced by metabolically active cells to resorufin which is fluorescent. The amount of fluorescence generated is related to the number of viable cells present. Unfortunately, the linear range of the method is restricted and the lower limit of quantification is approximately 106 colony forming units (CFU) per biofilm. The goal of the present study was to optimize this method to broaden its applicability. We added fresh growth medium and resazurin to mature Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cenocepacia and Candida albicans biofilms formed in MTP. Subsequently, the increase in resazurin-based fluorescence was followed over time and we determined the time needed to reach a specific value of fluorescence as well as the time to reach the maximum fluorescence. These time points correlate with the number of viable cells that were initially present and results were compared to plate counts. Using these alternative read-outs, we were able to extend the linear range from 106-108 to 103-108 CFU per biofilm, meaning that lower numbers of viable cells can be measured and the effect of anti-biofilm treatments can be quantified more accurately. Moreover, this approach is less expensive and less laborious than conventional plating techniques.