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Negative Staining of Thinly Spread Biological Particulates

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 117))

Abstract

The negative staining of virus particles for TEM study was introduced in the late 1950s, following the establishment of a standardized procedure by Brenner and Horne in 1959 (1). Rapidly, this staining technique was applied to other biological particulates, usually when a purified or semipurified aqueous suspension of freely suspended (i.e., nonaggregated) material was available. In addition to viruses, this material ranged from purified enzymes and other soluble protein molecules and components of molecular mass in the range of 200 kDa up to several MDa (such as the molluscan hemocyanins and ribosomes), to isolated cellular organelles, membrane fractions, bacterial cell walls and membranes, and filamenous protein structures of many types, also liposomal and reconstituted membrane systems, and even synthetic polymers.

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References

  1. Brenner, S. and Horne, R. W. (1959) Anegative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34, 60–71.

    Article  Google Scholar 

  2. Harris, J. R. (1997) Negative staining and cryoelectron microscopy, RMS Microscopy Handbook, Number 35, BIOS Scientific Publishers Ltd., Oxford, England.

    Google Scholar 

  3. Harris, J. R. and Horne, R. W. (1994) Negative staining: A brief assessment of current technical benefits, limitations and future possibilities. Micron 26, 5–13.

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  4. Adrian, M., Dubochet, J., Fuller, S. D., and Harris, J. R. (1998) Cryo-negative staining. Micron 29, 145–160.

    Article  PubMed  CAS  Google Scholar 

  5. Harris, J. R., Gebauer, W., and Markl, J. (1995) Keyhole limpet hemocyanin: Negative staining in the presence of trehalose. Micron 26, 25–33.

    Article  CAS  Google Scholar 

  6. Harris, J. R., Gerber, M., Gebauer, W., Wernicke, W., and Markl, J. (1996) Negative stains containing trehalose: Application to tubular and filamentous structures. J. Microsc. Soc. Amer. 2, 43–52.

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  7. Orlova, E. V., Dube, P., Harris, J. R., Beckman, E., Zemlin, F., Markl, J., and van Heel, M. (1997) Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 Å resolution by electron cryomicroscopy and angular reconstitution. J. Mol. Biol. 272, 417–437.

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  8. Horne, R. W. and Pasquali-Ronchetti, I. (1974) A negative staining-carbon film technique for studying viruses in the electron microscope. I. Preparation procedures for examining isocahedral and filamentous viruses. J. Ultrastruct. Res. 47, 361–383.

    Article  PubMed  CAS  Google Scholar 

  9. Harris, J. R. (1996) Immunonegative staining: Epitope localization on macromol-ecules. Methods 10, 234–246.

    Article  PubMed  CAS  Google Scholar 

  10. Hyatt, A. D. (1991) Immunogold labelling techniques, in Electron Microscopy in Biology: A Practical Approach (Harris, J. R., ed.) IRL Press, Oxford, pp. 59–81.

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  11. Harris, J. R., Adrian, M., Bhakdi, S., and Palmer, M. (1998) Cholesterol-strep-tolysin O interation: An EM Study of wild-type and mutant streptolysin O. J. Struct. Biol. 121, 343–355.

    Article  PubMed  CAS  Google Scholar 

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© 1999 Humana Press Inc.

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Robin Harris, J. (1999). Negative Staining of Thinly Spread Biological Particulates. In: Nasser Hajibagheri, M.A. (eds) Electron Microscopy Methods and Protocols. Methods in Molecular Biology™, vol 117. Humana Press. https://doi.org/10.1385/1-59259-201-5:13

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  • DOI: https://doi.org/10.1385/1-59259-201-5:13

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-640-6

  • Online ISBN: 978-1-59259-201-2

  • eBook Packages: Springer Protocols

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