TY - JOUR
T1 - High-resolution visualization of the microbial glycocalyx with low-voltage scanning electron microscopy
T2 - Dependence on cationic dyes
AU - Erlandsen, Stanley L.
AU - Kristich, Christopher J.
AU - Dunny, Gary M
AU - Wells, Carol L
PY - 2004/11
Y1 - 2004/11
N2 - 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.
AB - 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.
KW - Alcian blue
KW - Enterococcus faecalis
KW - Exopolysaccharides
KW - Glycocalyx
KW - Klebsiella pneumonias
KW - Low-voltage SEM
KW - Polycatioic dyes
KW - Safranin O
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UR - http://www.scopus.com/inward/citedby.url?scp=8744254706&partnerID=8YFLogxK
U2 - 10.1369/jhc.4A6428.2004
DO - 10.1369/jhc.4A6428.2004
M3 - Article
C2 - 15505337
AN - SCOPUS:8744254706
SN - 0022-1554
VL - 52
SP - 1427
EP - 1435
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
IS - 11
ER -