Confocal imaging of biofilm formation process using fluoroprobed Escherichia coli and fluoro‐stained exopolysaccharide

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Abstract
We developed a novel method of evaluating biofilm architecture on a synthetic material using green fluorescent protein‐expressing Escherichia coli and red fluorescence staining of exopolysaccharides. Confocal laser scanning microscopy observation revealed the time course of the change in the in situ three‐dimensional structural features of biofilm on a polyurethane film without structural destruction: initially adhered cells are grown to form cellular aggregates and secrete exopolysaccharides. These cells were spottily distributed on the surface at an early incubation time but fused to form a vertically grown biofilm with incubation time. Fluorescence intensity, which is a measure of the number of cells, determined using a fluorometer and biofilm thickness determined from confocal laser scanning microscopy vertical images were found to be effective for quantification of time‐dependent growth of biofilms. The curli (surface‐located fibers specifically binding to fibronectin and laminin)–producing Escherichia coli strain, YMel, significantly proliferated on fibronectin‐coated polyurethane, whereas the curli‐deficient isogenic mutant, YMel‐1, did not. The understanding of biofilm architecture in molecular and morphological events and new fluorescence microscopic techniques may help in the logical surface design of biomaterials with a high antibacterial potential. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 70A: 274–282, 2004

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