Microtiter plate assays to assess antibiofilm activity against bacteria

Bacterial biofilms demonstrate high broad-spectrum adaptive antibiotic resistance and cause two thirds of all infections, but there is a lack of approved antibiofilm agents. Unlike the standard minimal inhibitory concentration assay to assess antibacterial activity against planktonic cells, there is no standardized method to evaluate biofilm inhibition and/or eradication capacity of novel antibiofilm compounds. The protocol described here outlines simple and reproducible methods for assessing the biofilm inhibition and eradication capacities of novel antibiofilm agents against adherent bacterial biofilms grown in 96-well microtiter plates. It employs two inexpensive dyes: crystal violet to stain adhered biofilm biomass and 2,3,5-triphenyl tetrazolium chloride to quantify metabolism of the biofilm cells. The procedure is accessible to any laboratory with a plate reader, requires minimal technical expertise or training and takes 4 or 5 d to complete. Recommendations for how biofilm inhibition and eradication results should be interpreted and presented are also described. This protocol outlines simple and reproducible methods for assessing the biofilm inhibition and eradication capacities of novel antibiofilm agents against adherent bacterial biofilms grown in 96-well microtiter plates.