Capsule Staining – Concept, Agents, Method And Result

Capsule staining’s main purpose is to distinguish capsular material from a bacterial cell. So what is a capsule? A capsule is the gelatinous outer layer of a bacterial cell, it surrounds and adheres to the wall of a cell.

Some capsules are composed of polypeptides, but most are comprised of polysaccharides. Most bacterial cells produce a slime layer, a capsule differs in the way that it is a thick, detectable, discrete layer that sits outside of the cell wall.

A bacterial capsule is non ionic which means that what the capsule stain does is that it utilizes a basic stain and an acidic stain to detect capsule production.

The Concept Of Capsule Staining

Capsules stain poorly with agents used in simple staining, capsule stains can be a misnomer depending on the method, this is because the capsule may or may not be stained.

The negative staining methods contain a dark coloured, translucent, background with an unstained capsule but the cells themselves are stained. The background is formed with either congo red, nigrosin or india ink.

Both nigrosin and congo red can be obtained a lot easier than india ink which has become more difficult to obtain in recent years.

The reason for its rarity is due to the demand for it india ink is a permanent opaque black color. It also mixes remarkably easily.

A positive capsule stain differs from the negative staining method as it requires a mordant that precipitates the capsule. A mordant is a substance that is typically an inorganic oxide, it combines with a stain and fixes the stain to a material.

If you use dyes like crystal violet and methylene blue then the bacterial cell will take up the dye. The capsules will appear colorless with stained cells against a dark background.

Capsules are fragile; they can be easily diminished, distorted, desiccated or even destroyed by heating.

A drop of serum can be used during the smearing process, this enhances the size of the capsule and this makes it more easily observed with the usual compound light microscope.

Agents Used For Capsule Staining

The two main agents used for capsule staining are:

Crystal Violet

Crystal violet or gentian violet is a triarylmethane dye used mainly as a histological stain in Gram’s method of classifying bacteria.

Made up of an organic salt chloride, it is typically used in creams as a tropical treatment of bacterial and fungal infections. As well as this it has also been used as a histological stain to treat dying wood, silk and also paper.

The dye is made up of two components, the dye content and one hundred milliliters of distilled water.


Nigrosin is a mixture of black synthetic dyes that are made by heating a mixture of agents such as nitrobenzene, hydrochloric acid and aniline in the presence of copper or iron.

Whilst mainly used as a colorant for lacquers and varnishes as well as being the ink in marker pens it is used as an agent for the negative staining of a bacteria.

Negative staining with nigrosin can reveal some microorganisms that cannot be stained via regular methods. The ink will only enter dead cells making it more viable for testing as living cells will exclude the dye.

The dye is made up of nigrosine water soluble and much like crystal violet it is also made up of one hundred milliliters of distilled water.

The Method Of Capsule Staining

Points To Remember

  • Make sure to clean your microscope with lens cleaner, this removes all oils from the lenses
  • Be cautious when handling the slide as the organisms have not been killed.

So how do we go about staining a capsule well to start with

  1. Choose the negative dye you wish to use. This can be india ink, congo red, nigrosin or eosin. Once you have selected the dye, place a small drop on the slide.
  2. Using the sterile technique, add a loopful of bacterial culture to the slide you dropped the ink on and then smear the bacteria in the dye.
  3. Drag the mixture of ink and cells into a thin film using another slide ensuring the mixture runs along the first slide and then leave it to stand for around 5 to 7 minutes.
  4. Allow the slide to air dry. It is vital you do not heat fix the mixture.
  5. Once dry you are going to use the crystal violet stain and flood the smear for around sixty seconds. This will stain the cells, not the capsules. After you have left it to mix, simply drain the crystal violet by tilting the slide at a 45 degree angle, this will allow it to drain off until it air dries.
  6. Finally, examine the smear under a microscope and look for the presence of any encapsulated cells, these will be indicated due to the clear zone surrounding the cells.

The Result Of Capsule Staining

How do you know if you have successfully stained any capsules well? Any stained capsules will have a noticeable clear zone up against a dark background. If you do not notice any clear zones then none of the bacteria has been captured.

Some Examples Of Capsule Positive And Negative


Bacillus anthracis, Streptococcus pneumonia, Rhizobium spp, Klebsiella pneumoniae, Neisseria meningitidis, Clostridium spp.


Neisseria gonorrhoreae

Quality Control Of Capsule Staining

  • Positive control: Klebsiella pneumoniae.
  • Negative control: Alacilgenes denitrificans
Jennifer Dawkins