Groups of Extracellular Polymeric Secretions by Confocal Laser Scanning Microscopy

Extracellular polymeric secretions (EPSs) are a complex matrix of molecules secreted by bacteria and cyanobacteria in the construction of microbial biofilms. Biofilms are important in microbial disease processes, binding of antibiotics and metals, environmental contaminant cycling, and water purification processes (1). The EPS matrix of a biofilm consists mainly of polysaccharides, proteins, and lipids (2). Confocal scanning laser microscopy (CSLM) coupled with various probes has been used for localization of specific components of EPSs (3). Lectin-specific binding has made it possible to probe for the presence of specific sugar residues including mannose, glucose, galactose, glucosammine, and fucose. However, labeling of important functional groups such as carboxyl (OCOOH) and sulfhydryl (OSH) groups in EPSs has been difficult. Here we report a new method to visualize fluorescently labeled carboxyl and sulfhydryl groups through biotinylation of those functional groups followed by streptavidin-conjugated fluorescein and CSLM. We used marine stromatolites, Highborne Cay, Bahamas, containing filamentous cyanobacteria, Schizothrix sp., as a model. Schizothrix sp. is coated with sheath and EPSs containing uronic acids and acid polysaccharides. Carboxyl and sulfhydryl groups of acid polysaccharides in EPSs are believed to play important role in binding Ca in calcification of marine stromatolites (4). Freshly collected marine stromatolite samples, Highborne Cay, Bahamas, were sectioned using sterile razor blades. Immediately after collection, the sections