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.