Negative Staining

Negative staining also refers to as “Indirect staining”. This technique helps to visualize various microorganisms by using both light and electron microscopy. In bright field microscopy, the method of indirect staining involves the use of the liquid medium (black coloured dyes) like Nigrosin and India ink that stains the background, leaving the bacteria unstained.

But, in transmission electron microscopy, the negative staining technique makes the use of solid matrix (commonly potassium phosphotungstate). In TEM, the specimen is mixed in water or ammonium acetate along with the solution of phosphotungstate. Then, the suspension is applied to the carbon-coated grid either by spraying and spreading the sample. In this article, we will discuss the use of negative staining to elucidate the organism morphology via light microscopy.

Content: Negative Staining

  1. Definition
  2. Purpose
  3. Negative Stain
  4. Principle
  5. Procedure
  6. Advantages
  7. Disadvantages
  8. Conclusion

Definition of Negative Staining

Negative staining can define as one of the rapid, uncomplicated and qualitative staining techniques, which examines the bacterial morphological characteristics. It is named as negative staining because it uses negative or acidic stains that do not bind to the test specimen.

In negative staining, the specimen appears clear against a dark coloured background because the negatively charged stain does not penetrate into the negatively charged cell surface. Therefore, this technique is just the opposite of simple staining that stains the specimen against the clear background.


It determines the cell shape, size, structure and arrangement. It also helps us to stain the organisms that are too sensitive to be heat fixed. By using this method, we can prepare a sample for the microscopic examination via different microscopy techniques.

Negative Stain

It can define as the acidic dye that readily gives off H+ ion and accepts OH- ion, and hence is negatively charged. As the negative stain carries a negative charge, it sometimes may also refer as “Anionic or Acidic stain”.

The negative stain facilitates the elucidation of colourless bacteria against a coloured background. There will be repulsion between the negatively charged dye and a negatively charged bacterial cell. As a result, the specimen seems clear or transparent, outlined by the stained or dark background.

Examples of negative stain

  • 10% Nigrosin: Dissolve 10 grams of nigrosin, 0.5 ml of formalin in 100 ml of distilled water.
  • 2% Eosin: Dissolve 2 grams of eosin in 100 ml of distilled water.
  • (1% aqueous) Congo red: Dissolve 10 grams of Congo red into 1000 ml of distilled water.


Negative staining makes the use of negative or acidic dyes such as Nigrosin, Congo red, India ink etc. The negative stains carry a negatively charged chromophore group that readily give up the proton ions. As we know the surface of the bacteria carries a negative charge, thus the cell surface will not take up the colour of the negatively charged stain. As a result of repulsion, the bacterial cell surface will appear colourless with the dark or coloured background.

Principle of indirect staining


  1. Take a clean, grease-free, dry glass slide.
  2. Put a minimal drop of nigrosin towards one end of the glass slide via a dropper.
  3. Then, take the inoculum from the culture plates or slant culture via a sterilized inoculating loop and mix it with a drop of nigrosin.
  4. After that take another clean, grease-free, dry glass slide and place the one end of it towards the centre.
  5. Then tilt the glass slide over the stain containing the test organism by making an acute angle.
  6. Slightly draw the tilted slide until it touches the drop of culture organism, and then move it across the edge of glass slide to make an even, broad and thin bacterial smear.
  7. Allow the glass slide to air dry (do not heat fix).
  8. At last, put a drop of oil immersion and observe the glass slide under the microscope for the appearance of colourless bacterial cell with a gray background.

Procedure of negative staining


  • Through negative staining, clear unstained cells are easily observable against the black coloured stained background.
  • A negative staining method does not involve the heat-fixing of the specimen. As a result, the cell will not deform by the heat.
  • It can also stain the heat-sensitive microorganisms like spirochetes, yeasts etc.
  • Negative staining technique also permits the examination of the transparent capsule of various microorganisms like Cryptococcus neoformans.
  • It is quite an easy and rapid method that makes the use of a single acidic stain only.


  • Negative staining does not provide much information about the cell rather than the cell size, shape and arrangement.
  • By using this technique, we cannot examine a particular strain or type of the organism.


Therefore, we can conclude that a negative staining technique is a simple method to examine the microorganism by the use of a single acidic stain. The result of negative staining depends upon the appearance of the unstained cells with a dark coloured background.

In negative staining, there is a repulsion between the negatively charged stain and the specimen, which results in the observation of different shapes and sizes of the organisms as unstained outlines against stained background.

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