Negative Staining

Structural details of particulate specimens (virus, bacteria, subcellular components) are best revealed by the use of negative staining. This process surrounds the particles with electron-dense materials and reveals the surface by the contrast between the stain (dark) and the specimen (light). Since this is the opposite of the normal positive staining effect, it is called negative staining.

A common negative stain is a solution of 1% phosphotungstic acid (PTA) in water, adjusted to pH with 1N NaOH.

1. Place a drop of the diluted material on a formvar and carbon covered grid.

2. After 30-60 seconds, blot off the excess liquid with filter paper.

3. Add a drop of PTA solution.

4. After 1 minute, blot off the excess with filter paper.

5. Allow the grid to air dry.

6. Examine in the transmission electron microscope.

You might try mixing the sample and the PTA together and the adding one drop, or other combinations. Avoid too dense a concentration of materials, and avoid the use of buffers or compounds that might "cook" in the electron beam (sucrose, for example).

Alternate stains include: uranyl acetate, uranyl formate, and ammonium molybdate.

If the specimen requires a buffer, ammonium acetate is recommended because it does not react with most negative stains.