Application of multiple parameter imaging for the quantification of algal, bacterial and exopolymer components of microbial biofilms

Techniques are required for the simultaneous or sequential determination of multiple parameters within microbial biofilms. Confocal scanning laser microscopy in combination with a range of fluorescent probes and markers offers an approach to quantitatively defining many aspects of biofilm communities. Ey applying multispectral imaging in conjunction with nucleic acid stains, fluor conjugated lectins, and autofluorescence we have developed a simple approach to evaluate biofilm community composition. Biofilms were treated with the fluorescent nucleic acid stain SYTO 9 to allow quantification of bacterial biomass and fluor conjugated lectins (i.e. Triticum vulgaris lectin)to identify and allow quantification of exopolymeric substances. Far red autofluorescence was imaged ts quantify algal biomass. Digital image analysis of the CSLM optical thin sections in each of the channels was used to determine such parameters as biofilm depth, bacterial cell area (biomass), exopolymer area and algal biomass at various depths and locations. In addition, three colour red-green-blue projections of the biofilms were computed. The method proved simple and effective for determining treatment effects such as grazing by invertebrates. (C) 1998 Elsevier Science BN.