Negative Staining

What is Staining?

Staining is a combination of physical and chemical processes.

The physical phenomena include,

  1. Adsorption,
  2. Absorption,
  3. Capillary action and
  4. Osmosis

On the other hand, the affinity of basic dyes for acidic parts and acidic dyes for basic parts indicates that chemical reactions are also involved.

What is Negative Staining?

The major objective of staining is to increase the contrast between the cell and the surroundings.

It can also be achieved by staining the background rather than staining the cells.

This is called negative staining.

The Negative staining is also known as Relief Staining.

This is achieved by using a dye, which does not enter the cells and forms a thin film on the slide.

Principle of Negative Staining

Cells are not heat-fixed. Also, they are not stained, thereby preventing any damage to the cell due to fixatives in heat fixation.

The cells do not shrink because of the lack of staining and therefore can be seen in their natural shape and size. Cells appear larger when stained with negative staining than with monochrome staining.

When stained with Nigrosin or India Ink (Acidic stain) cells appear colorless against a bluish-black background.

Requirements for Negative staining

  1. Bacterial cell suspension
  2. Microincinerator or Bunsen burner
  3. Inoculating loop
  4. Staining tray
  5. Two glass slides
  6. Lens paper
  7. Microscope

Chemicals or Reagents

  • 10% Nigrosin solution
  • 1 % Congo red solution


procedure of Negative staining
  1. Put a drop of nigrosin on one end of a clean grease slide.
  2. With the wire loop, take the culture suspension by using the aseptic transfer technique.
  3. Gently mix that drop of nigrosin and culture suspension thoroughly.
  4. With the help of the second slide spread it out to form a thin film, allow it to dry, and observe under an oil immersion lens.
  5. The cells appear colorless against a bluish-black background.

Note: Do not heat fix that sample slide.

Results of Negative staining

A – vegetative cells 

B- Spores. 

Negative staining of Bacillus spp.

Result of negative staining, Negative staining of Bacillus spp.


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