Negative Staining

Negative staining is used for examination of surface details of small particulate biological species. Also, it is advantageous for the study of biomolecules. The method consists of staining in a layer of dense material so that the specimen is viewed as a light object against a dark background. Whereas positive staining involves direct interaction of stains with the cellular proteins, negative staining does not involve protein-stain reaction. A good negative stain must possess some inherent characteristics: 

  • It should be fairly soluble and should not crystallise during drying; panerai replica watches
  • It should withstand electron bombardment under microscope and thus must not volatilize;
  • It should have high physical density, boiling and melting point.


For viewing of negatively stained specimens under EM, one must use a small objective aperture. For maximal contrast from negatively stained material, TEM at a low accelerating voltage is used.


7.1      Preparation of particulate specimens for negative staining

Phosphotungstic acid (PTA), ammonium molybdate or uranyl acetate (1-3% solution) is routinely used as negative stains for particulate biological specimens.

7.2      Staining Procedure

1   Method I

  • Prepare 2% PTA in double distilled water or in 1% ammonium acetate. Adjust pH (6.8-7.0) with 1N KOH. It is the resultant K-phosphotungstate that forms an electron dense background when dried.
  • Place a drop of the suspension of the particulate specimen on a coated grid and drain off the excess. Do not allow drying. Add a drop of PTA and allow standing for 2 min. Drain off the excess.
  • Dry and observe under a TEM. The minute details of the specimen are seen as unstained, electron-lucent (light) structures in an electron dense (dark) background. 

2     Method II 

  • To an aliquot of a suspension of particulate specimens, add PTA solution. Mix thoroughly.
  • Place a drop of the mixture on to the coated grid and drain off the excess.
  • Dry and observe under a TEM.