A gelatinous layer of polysaccharide or polypeptide is called Glycocalyx. These polysaccharides may be composed of a single type of sugars (homopolysaccharide) or several types of sugars (heteropolysaccharides) and lies outside the cell envelope of bacteria. If glycocalyx is firmly attached to the cell wall it is referred to as a capsule. It is 0.2 µm thick layer attached to the cell wall of some bacteria.
The capsule differs from the slime layer that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall and a slime layer is loosely attached. The capsule stain employs an acidic stain and a basic stain to detect capsule production.
The capsule can be found in both Gram-negative bacteria and Gram-positive bacteria. Capsules are usually polymers of polysaccharides, although the capsule of Bacillus anthracis is composed of polypeptide (polyglutamic acid). In case of Acetic acid bacteria capsule is composed of homopolysaccharide. In some gram positive bacteria such as leuconostoc capsule is made of cellulose, consisting of glucose or fructose. In Klebsiella pneumoniae: capsule is composed of glucose, galactose,rhamnose etc.
There are two types of capsule.
- Macro-capsule: thickness of 0.2µm or more, visible under light microscope
- Microcapsule: thickness less than 0.2µm, visible under Electron microscope
Some of the functions are discussed below:
• The capsule is considered a virulence factor because of its ability to cause disease.
• capsules help cells adhere to surfaces
• It may prevent the attachment of bacteriophages and most hydrophobic toxic materials such as detergents
• They may inhibit the engulfment by eukaryotic cells, such as macrophages and, therefore, contribute to virulence.
• Capsule contains water which protects the cell from desiccation
• Capsule act as a source of nutrition in case of short nutrient supply
The main purpose of capsule stain is to distinguish capsular material from the bacterial cell Because Capsules are so tightly packed they stain very poorly with chemical used in simple staining. A capsule stain can be, depending on the method, a misnomer because the capsule may or may not be stained.
Negative staining methods contrast a thin specimen with an optically opaque fluid against darker colored background with stained cells but an unstained capsule. The background is formed with india ink or nigrosin or congo red. India ink is difﬁcult to obtain nowadays; however, nigrosin is easily available. A positive capsule stain requires a mordant that precipitates the capsule. A counterstain dye is used like crystal violet or methylene blue to stain bacterial cell. Capsules appear colourless with stained cells against dark background. Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating.
Reagents used for Capsule Staining
Crystal Violet (1%)
Crystal Violet = 1 gm
Distilled Water = 100 ml
Nigrosine, water soluble = 10 gm
Distilled Water = 100 ml
Procedure of Capsule Staining
- Place a single drop of a negative stain (India Ink, Congo Red, Nigrosin, or Eosin) on a clean microscope slide.
- Using sterile technique, take a loopful of bacterial culture and mix it into the drop of negative stain.
- Place the end of another clean slide at an angle to the end of the slide containing the culture and drag the dye-cell mixture into a thin film along the first slide and let it stand for 5-7 minutes.
- Allow the film to air dry (do not heat fix)
- Flood the smear with crystal violet stain for 1 minute. Drain the excess of crystal violet by tilting the slide.
- Rinse the slide gently with water
- Allow the slide to air dry
- Examine the slider under the microscope (100X) for the presence of capsules.
Result of Capsule Staining
Capsule: Clear halo zone around bacterial cells against the dark background
No Capsule: No Clear halo zone
Examples of Capsule Positive and Negative
Negative: The capsule is found most commonly among gram negative bacteria:
Escherichia coli (in some strains), Neisseria meningitides, Klebsiella pneumonia, Haemophilus influenza, Pseudomonas aeruginosa, Salmonella
Positive: Bacillus megaterium, Streptococcus pneumonia Streptococcus pyogenes, Staphylococcus epidermidis etc.
Quality control of Capsule Staining
Positive control: Klebsiella pneumoniae (ATCC 13883)
Negative control: Alacilgenes denitrificans (ATCC 15173)