MBDS Solvent: An Improved Method for Assessment of Biofilms

Abstract

Biofilms are recognised as an important contributor to bacterial resistance towards traditional antimicrobial treatments. Assessment of biofilm formation currently relies on a 96 well microtitre plate assay, which usually involves the colourimetric detection of stain (typically crystal violet) removed from previously stained biofilm. The amount of crystal violet released is then used as a quantitative indicator of the amount of biofilm formed. Currently, this is achieved by solubilisation of the stain by ethanol which results in partial decolourisation of the crystal violet stained biofilm which impacts the accuracy and reproducibility of this method. Herein, we describe a modified biofilm dissolving solution (MBDS) which produces a more uniform and reproducible colour release from stained biofilm through solubilisation of the biofilm architecture itself. Here we use crystal violet stained biofilms of P . aeruginosa strain PA0-1, to demonstrate an approximate two fold increase in crystal violet release by MBDS, as compared to ethanol treatment. In addition, when ethanol decolourised biofilms were treated again with MBDS, an almost equal amount of remnant crystal violet was recovered by dissolving the biofilm and the stain trapped within it. These results were reflected in microscopic analysis of ethanol treated and MBDS treated biofilm. Similar results were obtained when MBDS was used to decolourise and dissolve the biofilms of a number of other bacterial species highlighting the advantages of MDBS as a universal solvent for the colour detection of biofilm.