Sometimes there is serious contamination of grids caused by "dirt" on the section and/or stain precipitates that render the grid useless. In the case of "dirt" on the sections, which renders micrographs unacceptable for publication, it is necessary to cut new sections and take precautions to prevent the collection of dirt on the sections as outlined by Mollenhauer (11,12). "Dirt" adheres well only to a dry section and "dirt" on most sections comes from the surfaces of staining and wash fluids (11). Keep grids wet throughout the staining process; never blot the grids. Dry the grids after the staining is finished by touching filter paper points to the edge of the grid on the forceps side of the grid. Touching filter paper to the grid opposite the side of forceps results in pulling any form of "dirt" onto
the grid. Capillary fluid often collects between the tips of the forceps as grids are being held; remove this fluid by placing a filter paper point between the tips of the forceps before placing the grid on a piece of filter paper or in a grid box.
Another source of section contamination is bacteria, which can grow on 0.22-pm filters or in wash bottles used with deionized water. This problem often happens with water used in the boat of the knife as well as with water used for the final wash after staining. Using freshly boiled water and replacing the filters on a monthly basis can help to eliminate this potential problem.
Uranyl acetate precipitate often is recognized as needle-like objects on the section and can result from not filtering the stain properly immediately before use or from evaporation during prolonged staining. Many problems with poor staining are associated with the use of old solutions of uranyl acetate. Freshly prepared uranyl acetate stains more intensely and can eliminate prolonged staining times, a factor contributing to uranyl acetate precipitate. Alcoholic uranyl acetate stains are used to improve the contrast of some difficult specimens. Care
should be taken to wash with the same concentration of alcohol that the stain is dissolved in and going through a decreasing series of alcohol concentrations down to water helps to prevent rapid drying and possible precipitate formation.
A common, unrecognized cause of lead precipitates and poor staining intensity with lead citrate is incorrect pH. A pH lower than 12 causes precipitation of lead carbonate which appears as electron dense particles of various sizes and shapes. As the pH approaches 14, staining is severely reduced to the point that there is loss of contrast (2). The use of carbonate free, commercially prepared 1 N NaOH solutions is a simple, practical way to achieve the correct pH when preparing lead citrate solutions. Another possible source of problems in preparing lead citrate is the quality of the deionized or distilled water. Proper maintenance of the deionized or distilled water source is essential.
In the event that there is severe stain precipitation that cannot be worked around, there are methods which can be used to salvage grids with important ultrastructural information. Avery and Ellis (13) proposed the use of oxalic acid, a chelator, dissolved in methanol for removal of uranyl acetate precipitate in en bloc stained sections and on sections stained with hot methanolic uranyl acetate. Oxalic acid removes only uranyl acetate precipitate. Several methods (14-16) have been developed to destain sections with both uranyl acetate and lead precipitates. Treatment of sections with freshly prepared 2% (w/v) aqueous uranyl acetate for 6 to 8 min or 10% (v/v) acetic acid for 1 min followed by several rinses in distilled water can remove both uranyl acetate and lead precipitates. The dilute acetic acid treatment can be used not only for precipitate removal, but also for removing the original stain from sections. These salvage techniques are not always successful with sections that have been exposed extensively to the electron beam. It is better to remove grids from the beam and destain, wash and restain as quickly as possible.
Sometimes a fine, electron dense, "peppery" precipitate appears in sections after post staining; specimens which exhibit this fine osmium related precipitate should be recut and picked up on nickel grids or Marinozzi rings. These sections can then be oxidized with 1% to 2% (w/v) freshly prepared periodic acid for 5 to 10 min at room temperature, washed with deionized water and stained in the conventional manner with uranyl acetate and lead citrate (17,18). Dilute hydrogen peroxide (1-3% v/v) may be used as an oxidizing agent but is much harsher on the sections; periodic acid is the preferred oxidizer.
1. Prepare small volumes (5 mL) of stain on a weekly (more often if needed) basis for more consistent results. This also eliminates problems of disposing of large volumes of old uranyl acetate stain. Check with your local environmental health and safety office for appropriate local regulations for disposal of uranyl acetate and other chemical wastes generated in the laboratory.
2. The correct pH for Reynolds's lead citrate is 12. The cause of incorrect pH in preparing lead citrate is the use of NaOH pellets to prepare a 1 N NaOH solution instead of using a commercially produced, carbonate free 1 N NaOH solution. It is not possible to weigh NaOH pellets accurately due to their size and hygroscopic nature.
3. Lead citrate reacts with CO2 in the atmosphere to form lead carbonate precipitate, which appears as electron dense deposits on the grid. Every effort should be made to reduce the formation of lead carbonate by avoiding CO2 contamination. Spreading fresh pellets of NaOH around the Parafilm in the Petri dish helps to absorb CO2. Because NaOH is very hygroscopic, it is better to buy small bottles (100 g/bottle) of NaOH to insure that the reagent is dry.
4. Grids should be kept wet throughout the staining process and only allowed to dry after all staining is complete.
1. Spurr, A. R. (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43.
2. Reynolds, E. S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212.
3. Marinozzi, V. (1961) Silver impregnation of ultrathin sections for electron microscopy. J. Biophys. Biochem. Cytol. 9, 121-133.
4. Hayat, M. A. (1975) Positive Staining for Electron Microscopy, Van Nostrand Reinhold Co., New York.
5. Luft, J. H. (1961) Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409-414.
6. Mollenhauer, H. H. (1964) Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111-114.
7. Daddow, L. Y. M. (1983) A double lead stain method for enhancing contrast of ultrathin sections in electron microscopy: a modified multiple staining technique. J. Microsc. (Oxford) 129, 147-153.
8. Bell, M. (1988) Artifacts in staining procedures, in Artifacts in Biological Electron Microscopy (Crang, R. F. E. and Klomparens, K. L. eds.), Plenum Press, New York, pp. 81-106.
9. Maunsbach, A. B. and Afzelius, B. A. (1999) Biomedical Electron Microscopy: Illustrated Methods and Interpretations, Academic Press, New York.
10. Chien, K., Van de Velde, R. L., Heusser, R. C., Shiroishi, H., and Cohen, A. H. (1994) A rapid phosphotungstic acid staining method on ultra-thin sections. Proc. Ann. MSA Meeting 52, 318-319.
11. Mollenhauer, H. H. (1974) Poststaining sections for electron microscopy. Stain Technol. 49, 305-308.
12. Mollenhauer, H. H. (1975) Poststaining sections for electron microscopy: an alternate procedure. Stain Technol. 50, 292.
13. Avery, S. W. and Ellis, E. A. (1978) Methods for removing uranyl acetate precipitate from ultrathin sections. Stain Technol. 53, 137-140.
14. Kuo, J. (1980) A simple method for removing stain precipitates from biological sections for transmission electron microscopy. J. Microsc. (Oxford) 120,221-224.
15. Kuo, J. and Husca, G. L. (1980) Removing stain precipitates from biological ultrathin sections. Micron 11, 501-502.
16. Kuo, J., Husca, G. L., and Lucas, L. N. D. (1981) Forming and removing stain precipitates on ultrathin sections. Stain Technol. 56, 199-204.
17. Mollenhauer, H. H. and Morre, D. J. (1978) Contamination on thin sections, cause and elimination. Proc. Ninth Internat. Congress on Electron Microscopy, vol. II, 78-79. Toronto.
18. Ellis, E. A. and Anthony, D. W. (1979) A method for removing precipitate from ultrathin sections resulting from glutaraldehyde-osmium tetroxide fixation. Stain Technol. 54, 282-285.
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