High-resolution Visualization of the Microbial Glycocalyx with Low-voltage Scanning Electron Microscopy : Dependence on Cationic Dyes
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
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Summary |
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Key Words: glycocalyx polycatioic dyes alcian blue safranin O low-voltage SEM Enterococcus faecalis Klebsiella pneumoniae exopolysaccharides
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Introduction |
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Ultrastructural detection of the glycocalyx has been difficult because of its high polysaccharide content, which does not interact with the common postfixation stain osmium. Therefore, the glycocalyx scatters few electrons and is relatively indistinguishable in conventionally processed samples for transmission electron microscopy (TEM). Detection of the glycocalyx has been facilitated by development of stabilization methods using cationic probes such as ruthenium red (Luft 1971) and alcian blue (Behnke and Zelandeer 1970
). Both probes facilitate deposition of osmium, which increases the electron density of the anionic polysaccharides. Detection of microbial glycocalyx with these methods at the TEM level has been reviewed (Erdos 1986
; Fassel and Edmiston 1999
). Several ultrastructural studies have used conventional scanning electron microscopy (SEM) to investigate the glycocalyx, but these studies (Marshall et al. 1971
; Costerton et al. 1981
; Fassel et al. 1991
) were hampered by low resolution and also by the inability to use low voltages (<5 keV), which yield increased information from small topographical features (Pawley and Erlandsen 1989
). The development of field emission SEM, together with the improvements in producing thin metal coatings of
1 nm, has greatly improved SEM so that resolution in the range of 34 nm can be obtained on conventional specimens.
The aim of this study was to investigate the presence of the glycocalyx using cationic probes and high-resolution field emission SEM at low voltages. Two model microorganisms producing a prominent glycocalyx were selected for investigation: a gram-negative bacterium, Klebsiella pneumoniae, which produces a mucoid capsule, and a gram-positive bacterium, Enterococcus faecalis, which produces an extensive glycocalyx when forming biofilms on cellulose catheters (Erlandsen et al. 2004). Our results demonstrate that field emission SEM permits evaluation, previously unparalleled by TEM, of the extent and nature of the bacterial glycocalyx. In addition, the phenotypic visualization of the EPS was shown to vary with the type of cationic probe and the length of fixation.
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Materials and Methods |
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Bacterial Strains
The OG1RF strain of E. faecalis was cultivated in tryptic soy broth in 24-well microtiter plates as described (Kristich et al. 2004). Hollow cellulose tubing (Spectra/Por; Spectrum, Houston, TX), 200 µm in diameter and 35 mm in length, was immersed in
0.6 ml tryptic soy broth, and the medium was inoculated with E. faecalis. Medium was changed daily and fixation of biofilms on cellulose tubing was performed after 4 days of culture at 37C (Erlandsen et al. 2004
). K. pneumoniae (ATCC 13,883) was cultured overnight at 37C in tryptic soy broth, and stationary phase cells were concentrated and washed with sterile PBS. Cells were attached for
2030 min to glass chips pretreated with a thin film of 0.1% poly-L-lysine to increase adhesion, and then were immersed in the desired fixative.
Fixation Protocol
Multiple experiments (four to six) were performed to investigate the role of cationic probes in visualizing the bacterial glycocalyx. Either cellulose tubing with biofilms of E. faecalis or glass chips with adherent K. pneumoniae were immersed for 422 hr in a mixture of 2% paraformaldehyde and 2% glutaraldehyde in 0.15 M sodium cacodylate buffer, pH 7.4, containing no cationic additives. Probes used to stabilize the anionic glycocalyx included the cationic dyes alcian blue, safranin O, and ruthenium red, and also the diamine L-lysine hydrochloride. The properties of these probes are listed in Table 1. To test the effect of different cationic probes, the same aldehyde fixative was modified by adding one of the following probes: 0.15% alcian blue 8GX, 0.15% safranin O, 0.15% ruthenium red, 0.15% lysine monohydrochloride, a mixture of 0.15% alcian blue and 0.15% lysine, or a mixture of 0.15% alcian blue and 0.15% ruthenium red. Lysine added to the aldehyde cocktail at a concentration of 0.15% tended to polymerize, producing loss of sample, so the concentration was reduced to 0.0075%.
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Results |
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Immersion fixation of K. pneumoniae in the paraformaldehyde-glutaraldehyde cocktail without added cationic probes revealed a complete lack of detectable glycocalyx on the cell surface (Figures 1A and 1B) . Addition of alcian blue to the aldehyde fixation and 4 hr of fixation produced an entirely different appearance. Thin spike-like processes extended from the cell circumference to the substratum (Figures 1C and 1D). The surface of the cells exhibited a rugose-like appearance of anastamosing folds. Extending the fixation time in alcian blue from 4 to 22 hr also produced a different appearance (Figures 1E and 1F). The attached cells still possessed spike-like processes, but the surface of the substratum was covered with a web of thin filaments originating from the surface of the K. pneumoniae. Close examination of the rugae on the cell surface (Figure 1F) also revealed a fine meshwork of filaments extending between the rugae. Aldehyde fixation in the presence of 0.0075% lysine resulted in a surface topography that was distinct from that shown using alcian blue. Fixation for 4 hr in the presence of lysine resulted in retention of the spike-like processes around the cell circumference, but the cell surface now possessed many short, fine fibrils and small globule-like particles (Figures 2A2C) . Extending the fixation time from 4 to 22 hr did not appear to produce any changes in the surface topography (Figure 2C). Fixation in the presence of ruthenium red (Figure 2D) had an effect similar to that seen in the 4-hr fixation in the presence of alcian blue (see Figures 1C and 1D) and lysine (Figure 2C), in that spike-like fibrils were seen extending between the cell margin and the substratum. These spikes appeared thicker than those seen when alcian blue or lysine was added, and the surface topography appeared rugose, with a few fibrils present between adjacent folds (Figure 2D).
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Discussion |
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Our results confirm and extend what others have done using cationic dyes at the TEM level, and supports the finding that aldehyde fixation alone is inadequate for visualization of the bacterial glycocalyx (Fassel et al. 1993). Our study is unique because we have demonstrated that visualization of different components of the glycocalyx is dependent on the duration of fixation and also on the choice of cationic probe. As shown in Table 1, the cationic probes we employed (alcian blue, safranin O, ruthenium red, and lysine) vary considerably in both charge and shape. The biochemical composition of the microbial glycocalyx is known to contain many anionic mono- and polysaccharides together with proteins, nucleic acids, and lipids (Cooksey 1992
; Nielsen et al. 1997
; Flemming and Wingender 2001
; Starkey et al. 2004
). It has been proposed that preservation of the different elements in the glycocalyx might be related to a charge-coupling effect (Fassel and Edmiston 1999
). Thus, cationic probes such as ruthenium red and lysine are quite small (
1 nm) compared with the larger planar molecules (
4 nm) of alcian blue and safranin O, and charge density varies considerably in these probes.
The variability seen in the phenotypic retention of the glycocalyx for both K. pneumoniae and E. faecalis is probably a result of the selectivity of each probe interacting with different anionic species as a function of probe charge density, and the ability of each probe to penetrate into this highly charged hydrophilic layer. Lysine also has the ability to form polymers with glutaraldehyde, which can be diminished by adding paraformaldehyde to the fixative, but high concentrations of lysine should be avoided to minimize the potential for polymerization of the fixative (Fassel et al. 1997; Fassel and Edmiston 1999
). Fine branches on surface fibrils were seen in Figures 2A2C, and therefore short fibrils on filaments may be a "decorative artifact" resulting from lysine polymerization. Because of the cytochemical variation in the visualization of glycocalyx phenotype with different cationic probes, it was difficult to determine which one represented the correct topography or whether the actual appearance would be a composite of all methods. The spike-like extensions surrounding the cell circumference were first detected by Springer and Roth (1973)
, but the increased presence of small fibrils linking adjacent rugae (Figures 1E and 1F), or the fine fibrillar material visualized with lysine (Figures 2A2C), has not been described. Likewise, the dramatic visualization of glycocalyx in E. faecalis biofilms seen with alcian blue is quite distinct from that seen with safranin O, ruthenium red, or lysine (cfompare Figures 3C and 3D to Figures 3E and 3F and Figures 4A and 4B). Direct comparisons of visualization of the glycocalyx by alcian blue or ruthenium red have suggested that alcian blue stabilizes a more extensive glycocalyx than does ruthenium red (Fassel et al. 1992
). Attempts to improve visualization of microbial glycocalyx by constructing cocktails of different dyes, including alcian blue and ruthenium red, alcian blue and safranin O, as well as alcian blue and lysine, were less successful than the use of individual cationic probes (data not shown). Complex mechanisms related to charge coupling and diffusion in highly charged hydrophilic films, together with other components of the glycocalyx, may be involved in protecting the dyeEPS interaction against forces (surface tension) that may collapse the glycocalyx during dehydration or critical point drying.
Our results suggest that the design of fixations containing cationic dyes may require a trial-and-error approach. Because very little information exists about the type(s) and number of polyanionic subunits in the glycocalyx (Schmitt and Flemming 1999; Starkey et al. 2004
; Sutherland 2001
), it will be necessary to vary time and dye concentrations for application to other microorganisms. On the basis of our experience with cationic dyes to visualize polyanionic substances in the glycocalyx, we predict that anionic dyes (ponceau 2R, orange G, or biebrick scarlet) may be more useful in detecting and enhancing polycationic cell surfaces as found in some strains of Staphlylococcus epidermidis (Starkey et al. 2004
).
In summary, our study shows that low-voltage SEM can provide high-resolution imaging of the bacterial glycocalyx and allow visualization of the filaments that compose it. We also observed that cationic dyes can dramatically increase the visualization of glycocalyx structure, and found that different cationic dyes may selectively permit visualization of different components in the glycocalyx. The actual determination of the "native" structure of the glycocalyx may require the application of cryo-immobilization methods. Instead of using cationic probes to preserve components of the glycocalyx, the cryo-immobilization and cryo-sublimation would avoid typical processing artifacts (such as fixation or dehydration), and water would be sublimated at cryo-temperature and vacuum conditions designed to prevent molecular collapse (Erlandsen et al. 2001). This approach has been successfully used to discern the glycocalyx of the bacterium Proteus mirabilus, and this study revealed that the glycocalyx consisted of a fine meshwork of anastomotic fibrils (Erlandsen et al. 2003
). Our current knowledge of the bacterial glycocalyx has been based on experiments with cationic dyes in TEM. This study demonstrates the potential of field emission SEM for enhancing and visualizing the bacterial glycocalyx and, together with cryo-SEM approaches, may help to clarify the role of the glycocalyx in bacterial adhesion and biofilm formation.
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Acknowledgments |
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We wish to thank Rob Garni and Lisa Erickson for their excellent technical assistance. We also thank Chris Frethem (Electron Microscopy Laboratory in the Characterization Facility at the University of Minnesota) for excellent technical assistance.
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Footnotes |
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Literature Cited |
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