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DOI: 10.1369/jhc.4A6428.2004
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Journal of Histochemistry and Cytochemistry
Volume 52 (11): 1427-1435, 2004
Copyright ©The Histochemical Society, Inc.

High-resolution Visualization of the Microbial Glycocalyx with Low-voltage Scanning Electron Microscopy : Dependence on Cationic Dyes

Stanley L. Erlandsen, Christopher J. Kristich, Gary M. Dunny and Carol L. Wells

Departments of Genetics, Cell Biology, and Development (SLE), Microbiology (CJK,GMD), and Laboratory Medicine and Pathology (CLW), University of Minnesota Medical School, Minneapolis, Minnesota

Correspondence to: Dr. Stanley L. Erlandsen, Dept. of Genetics, Cell Biology, and Development, 6-160 Jackson Hall, University of Minnesota Medical School, Minneapolis, MN 55455. E-mail: erlan001{at}umn.edu

The microbial glycocalyx is composed of a variety of polyanionic exopolysaccharides and plays important roles in microbial attachment to different substrata and to other cells. Here we report the successful use of low-voltage scanning electron microscopy (LVSEM) to visualize the glycocalyx in two microbial models (Klebsiella pneumoniae and Enterococcus faecalis biofilms) at high resolution, and also the dependence on fixation containing polycationic dyes for its visualization. Fixation in a paraformaldehyde-glutaraldehyde cocktail without cationic dyes was inadequate for visualizing the glycocalyx, whereas addition of various dyes (alcian blue, safranin, and ruthenium red) to the aldehyde cocktail appeared necessary for stabilization. The cationic dyes varied in size, shape, and charge density, and these factors appeared responsible for different phenotypic appearances of the glycocalyx with each dye. These results suggest that aldehyde fixation with cationic dyes for high-resolution LVSEM will be a useful tool for investigation of microbial biofilms as well as investigation of the extent and role of the glycocalyx in microbial attachment to surfaces. (J Histochem Cytochem 52:1427–1435, 2004)

Key Words: glycocalyx • polycatioic dyes • alcian blue • safranin O • low-voltage SEM • Enterococcus faecalis • Klebsiella pneumoniae • exopolysaccharides


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