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The use of the negative staining method for the electron-microscopic study of subcellular particles from animal tissues

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Summary

  1. 1.

    The negative staining (negative contrast) method for investigating the structure of particulate biological material (viruses, bacteria) in the electron microscope has been found to be applicable to subcellular fractions of guinea pig brain and other animal tissue preparations.

  2. 2.

    The method involves the transfer to specimen grids of fine droplets of the particulate material mixed with potassium phosphotungstate at pH 7.4. Under suitable conditions the droplets leave, on drying, a glass of electron-dense phosphotungstate in which the subcellular particles show up as areas of relatively low electron-density. The phosphotungstate appears to protect the particles from excessive distortion; it has the property of penetrating into minute pores and clefts and shows up fine structure with a potentially much higher resolution than is obtainable in thin sections.

  3. 3.

    The potentialities of the method have been illustrated by work with myelin fragments, mitochondria and particles derived from nerve endings.

  4. 4.

    The myelin period has been estimated to be 140–170 Å, the mean value of which is somewhat below the diffraction results of Finean.

  5. 5.

    The results with mitochondria are consistent with the Robertson rather than the Sjöstrand model. Long sausage-shaped mitochondria are frequently seen which would appear in thin sections as multiple oval profiles, thus confirming the deductions of Wessel.

  6. 6.

    The results with nerve ending particles clearly demonstrate the existence, as discrete cytoplasmic organelles, of synaptic vesicles. They are seen to have a flattened, disc-like shape rather than a spherical, and remain firmly stuck together even when the outer wall of the particle is broken.

  7. 7.

    Other structures seen in brain fractions include nuclei, microsomes and sheets of endoplasmic reticulum studded with ribosomes.

  8. 8.

    The method appear to be highly suitable for work with subcellular fractions and to be capable of giving valuable information complementary to that obtainable with thin sections.

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Authors and Affiliations

  1. Electron Microscope Laboratory and the Biochemistry Department, Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge, England

    R. W. Horne & V. P. Whittaker

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  1. R. W. Horne
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  2. V. P. Whittaker
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Horne, R.W., Whittaker, V.P. The use of the negative staining method for the electron-microscopic study of subcellular particles from animal tissues. Z.Zellforsch 58, 1–16 (1962). https://doi.org/10.1007/BF00406937

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  • DOI: https://doi.org/10.1007/BF00406937

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