Negative staining, also known as negative contrast staining, is a staining technique discovered by Hall, as opposed to conventional staining (i.e., positive staining). Its principle lies in using heavy metal salts to surround low-electron-density samples, enhancing the electron density around the specimen and creating differences in mass-thickness between fine structures. This enhances the scattering absorption contrast, making the sample appear as bright structures against a dark background. Negative staining solutions include phosphotungstic acid, ammonium molybdate, India ink, etc., with 1–3% phosphotungstic acid being the most commonly used.
Phosphotungstic Acid Negative Staining Solution (3%) is suitable for visualizing macromolecules, bacteria, viruses, protozoa, bacteriophages, organelles, nucleic acid macromolecules, protein crystals, and other macromolecular materials. After staining, the sample appears as transparent bright structures, while the background appears black.
Instructions for Use
1. Droplet Staining Method
- Concentrate the sample by low-speed centrifugation (2000g, 10 min) or other methods to prepare a suspension with appropriate concentration and purity.
- Directly drop the sample suspension onto a grid with a support film and let it stand for 3–5 minutes.
- Use filter paper strips to absorb excess liquid from the edge of the droplet and allow it to dry slightly.
- Add a drop of negative staining solution and let it stand for 2–3 minutes.
- Absorb the excess staining solution, allow it to air-dry naturally, and proceed with microscopic observation.
2. Floating Method
- Concentrate the sample by low-speed centrifugation (2000g, 10 min) or other methods to prepare a suspension with appropriate concentration and purity.
- Float the grid with a support film on a droplet of the sample suspension to pick up the sample.
- Float the grid on the negative staining solution for 1–2 minutes.
- Absorb the excess staining solution, allow it to air-dry naturally, and proceed with microscopic observation.
Precautions
The target sample should be as fresh as possible.
The sample should be a homogeneous suspension with appropriate purity and concentration; otherwise, a specific and clear binding reaction with the staining agent may not occur.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Staining Results
Sample
| Transparent bright structures
|
Background
| Black |
