Phospotungstic Acid
Instructions for Phospotungstic Acid
Application
Phosphotungstic Acid is used as a negative stain for the examination of small isolated subcellular
tissue components, virus particles, bacterial flagella and pili and protein molecules, etc. This
technique makes use of the principle of surrounding the particles with electron dense material leaving
them electron transparent so a negative image of the structural geometry can be seen. This reverse contrast
effect was first reported by FARRANT (1954) when examining ferritin molecules and by HALL (1955) using virus
particles.
The advantages of negative staining are threefold:
1) The technique is simple and rapid.
2) Very high electron contrast and good structural preservation is obtained.
3) Only very small amounts of sample are needed.
Procedure
There are two methods normally used to prepare negatively stained samples for electron microscopy.
1) BRENNER et. al. (1959). A 1 or 2% solution of stain is prepared and its pH adjusted,
to match that sample, with either NaOH or HCI.
A small quantity of the sample is mixed with an equal volume of the stain and a small drop of
the mixture is either placed on a carbon or plastic coated E.M. grid, using a Pasteur pipette,
or sprayed onto the coated grid using - a glass nebulizer. Excess liquid is removed with edge of
a filter paper and the grid allowed to dry.
2) A drop of the sample is placed on a coated E.M. before it dries one or two drops of stain from
pippette are washed over it. Again, the excess removed with a filter paper and the grid dried.-
In each of these methods it is necessary to experiment with the sample concentration in
order to obtain a satisfactory
distribution of particles in the image.
For further more specific details of the technique, see KAY (1965) and HAYAT (1972).
References
FARRANT J.L. (1954) An Electron Microscopic, Study of Ferritin.
Biochem, biophys. Acta 13, 569.
HALL C.E. Particles.(1955) Electron Densitometry of Stained Virus Particles. J. Biochem, biophys. Cytol 1, 1.
BRENNER S. and R. W. HORNE (1959) A Negative Staining Method, for High Resolution Electron Microscopy of Viruses. Biochem,
biophys. Acta 34, 103.
KAY D.H. (1965) ed. Techniques for Electron Microscopy (Chapter 11 by R.W. Horne)' Blackwell Scientific Press.
HAYAT M.A. Microscopy.(1972) ed. Principles and Techniques of Electron Biological Applications. Van Nostranol Princeton.
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