Improved contrast by a simplified post-staining procedure for ultrathin sections of resin-embedded bacterial cells: Application of ruthenium red
✍ Scribed by Beate Vogt; Ruth Berker; Prof. Dr. Frank Mayer
- Book ID
- 102911637
- Publisher
- John Wiley and Sons
- Year
- 1995
- Tongue
- English
- Weight
- 542 KB
- Volume
- 35
- Category
- Article
- ISSN
- 0233-111X
No coin nor oath required. For personal study only.
✦ Synopsis
High contrast can be obtained in ultrathin sections of bacterial cells embedded in the low-temperature resin Lowicryl by post-staining of the sections on an aqueous ruthenium red solution. Post-staining with lead citrate can be omitted. Combination with post-staining with uranyl acetate results in further improvement of contrast. This is shown for Gram-positive, Gram-negative, and methanogenic bacteria. Improved visibility is demonstrated for wall layers including slime and capsule, cytoplasmic membrane, nucleoid, envelope of PHB inclusion bodies, polyphosphate, and intracellular defective bacteriophages. The procedure is suited as post-staining for immunocytochemical analyses.
Sufficient contrast in investigations of ultrathin sections of resin-embedded bacterial cells by transmission electron microscopy is achieved by several steps of staining : block staining with osmium tetroxide and uranyl acetate ; post-staining, i.e. staining of ultrathin sections, with lead citrate and uranyl acetate (ROBINSON et al. 1987). Drawbacks of conventional post-staining procedures in routine application may be the occurrence of precipitates such as lead carbonate and grainy uranyl acetate particles covering the surface of the sections. These precipitates can be avoided by careful execution of the necessary steps. With the onset of application of low temperature resins such as Lowicryl (ROTH et al. 1981) it turned out that this kind of resin preserves structural integrity of the cells and antigenicity (important for immunocytochemistry). Especially when used for bacterial cells, however, the contrast may not be satisfying. It can only be improved by more intense application of conventional solutions of post-staining chemicals, resulting in a higher probability of formation of precipitates, and more intensive washing steps. The latter is not very welcome in immunocytochemistry because it causes a loss of labelling efficiency. Therefore, an electron microscopic procedure is desirable which avoids these drawbacks. demonstrated that ruthenium red (RR) can be used as a staining agent for extracellular polyanionic polysaccharides. However, when applied as preembedding contrasting solution, the interior cell components were not stained. RR does not penetrate through the cytoplasmic membrane. Later, GUTIERREZ-GONZALVEZ et al. (1984) showed that also other polyanionic polymers such as nucleic acids can be contrasted with RR. Several communications (LINSS et al. , NAGATO et al. 1985) ) dealt with application of RR as a stain for post-staining of sections. Resins such as methacrylate and epoxide compounds did turn out not to be suited because of a very low degree of penetration of an aqueous RR solution into the resin. LINSS et al. (1979) achieved better results with embedding in Durcupan ACM; the sections were treated, prior to application of a combination of RR with osmium tetroxide, with diluted soldium methylate. Contrast was enhanced, but structural preservation suffered. GUTIERREZ-GANZALVEZ et al. (1984) prepared semi-thin sections of Epon 8 12-embedded cells and obtained reasonable contrast for light and electron microscopy by post-staining with a solution of RR in borate buffer, pH 9.2, andor RR dissolved in 15% ethanol. NAGATO et al.