Negative Staining
Backgroung reference
Hayat, Vol. 2, Chapter 3, p. 101
Negative staining is a technique used to enhance the visualization of small cells, viruses and macromolecules. With this technique samples are allowed to settle onto the surface of a supporting film then the film is flooded with a suspension of heavy metal. the stain dries along the edge of the sample and in surface irregularities. The result is a layer of electron opaque material defining the features of interest in the sample.
Materials
- Carbon-coated formvar grids that have been "glow discharged"
- Bacteria or other materials to be stained
- Filter paper
- Locking forceps
- 2% Phosphotungstic Acid (pH 7.0 with KOH) KPTA
- 2% Uranyl Acetate, pH 4.5Background
Method
- Grasp carbon-coated formvar grid with forceps and slide "O" ring to secure grid. Place secured grid over filter paper.
- Place small drop of bacteria (or other marterial) on grid.
- Let settle for 1 minute.
- Touch torn filter paper to forcep end of drop and remove most of liquid.
- Add drop of stain.
- Remove drop of stain as in step 4. Grid should have an even, shiny appearance.
- Let air dry and observe in TEM.
Notes
- Bacteria should sometimes be washed once in PBS or other suitable buffer. Centrifuge bacteria and resuspend in buffer. Try first without washing.
- Start with 2% KPTA. You may have to dilute it with distilled water to as little as 0.25% to get good "staining".Important that bacteria and/or stain get only on one side of grid.