Abstract
Negative staining is widely applicable to isolated viruses, protein molecules, macromolecular assemblies and fibrils, subcellular membrane fractions, liposomes and artificial membranes, synthetic DNA arrays, and also to polymer solutions. In this chapter, techniques are provided for the preparation of the necessary support films (continuous carbon and holey/perforated carbon). The range of suitable negative stains is presented, with some emphasis on the benefit of using ammonium molybdate and of negative staintrehalose combinations. Protocols are provided for the single-droplet negative staining technique (on continuous and holey carbon support films), the negative staining-carbon film technique, for randomly dispersed fragile molecules, 2D crystallization of proteins, and for cleavage of cells and organelles. The newly developed cryonegative staining procedure also is included. Immunonegative staining and negative staining of affinity labeled complexes (e.g., biotin-streptavidin) are discussed in some detail. The formation of immune complexes in solution for droplet negative staining is presented, as is the use of carbon-plastic support films as an adsorption surface on which to perform immunolabeling or affinity experiments, before negative staining. Dynamic biological systems can be investigated by negative staining, where the time period is in excess of a few minutes, but there are possibilities to greatly reduce the time by rapid stabilization of molecular systems with uranyl acetate or tannic acid.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Brenner, S. and Horne, R. W. (1959) A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34, 60–71.
Harris, J. R. (1997) Negative Staining and Cryoelectron Microscopy: the Thin Film Techniques. RMS Microscopy Handbook Number 35, BIOS Scientific Publishers Ltd., Oxford.
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.
Adrian, M., Dubochet, J., Fuller, S. D., and Harris, J. R. (1998) Cryo-negative staining. Micron 29, 145–160.
Harris, J. R., Gebauer, W., and Markl, J. (1995) Keyhole limpet hemocyanin: negative staining in the presence of trehalose. Micron 26, 25–33.
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. Am. 2, 43–52.
Harris, J. R. and Scheffler, D. (2002) Routine preparation of air-dried negatively stained and unstained specimens on holey carbon support films: a review of applications. Micron 33, 461–480.
Orlova, E. V., Dube, P., Harris, J. R., et al. (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.
Horne, R. W. and Parquali-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.
Harris, J. R. (1991) Negative staining-carbon film technique: New cellular and molecular applications. J. Electron Microsc. Techn. 18, 269–276.
Roberts, I. M. (1986) Immunoelectron micrisocopy of extracts of virus-infected plants, in Electron Microscopy of Proteins, vol. 5, Viral Structure (Harris, J. R. and Horne, R. W., eds.), Academic Press, London, 293–357.
Harris, J. R. (1996) Immunonegative staining: epitope localization on macromolecules. Methods 10, 234–246.
Petry, F. and Harris, J. R. (1999) Structure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites. Int. J. Parasitol., 29, 1249–1260.
Hyatt, A. D. (1991) Immunogold labelling techniques, in Electron Microscopy in Biology: A Practical Approach (Harris, J. R., ed.), IRL Press, Oxford, 59–81.
Zhao, F.-Q. and Craig, R. (2003) Capturing time-resolved changes in molecular structure by negative staining. J. Struct. Biol. 141, 43–52.
Mitchell, J. C., Harris, J. R., Malo, J., Bath, J., and Turberfield, A. J. (2004) Self-assembly of chiral DNA nanotubes. J. Am. Chem. Soc. 126, 16342–16343.
Harris, J. R., Bhakdi, S., Meissner, U., et al. (2002) Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol esters, and cholesterol derivatives: a TEM study. J. Struct. Biol. 139, 122–135.
Ohi, M., Li, Y., and Walz, T. (2004) Negative staining and image classification—Powerful tools in modern electron microscopy. Biol. Proced. Online 6, 23–34.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Harris, J.R. (2007). Negative Staining of Thinly Spread Biological Samples. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology™, vol 369. Humana Press. https://doi.org/10.1007/978-1-59745-294-6_7
Download citation
DOI: https://doi.org/10.1007/978-1-59745-294-6_7
Publisher Name: Humana Press
Print ISBN: 978-1-58829-573-6
Online ISBN: 978-1-59745-294-6
eBook Packages: Springer Protocols