The Objective of capsule staining is to observe bacterial capsule by distinguishing capsular material from the bacteria cell.In some bacteria, the cell wall is surrounded by a viscous cell envelope called ‘capsule’. It is made of polysaccharide, glycoprotein or polypeptide.When the capsule is too thin to be observed under light microscope, it is called ‘microcapsule’ and when it is so thick that many cells are embedded in the capsular matrix; it is called ‘slime’.
Bacteria can be classified into the following two group, based on the presence or absence of capsule.
- Capsulated bacteria: A bacteria, which possesses capsule is a capsulated bacteria.
- Non-capsulated Bacteria: A bacteria, which does not possess capsule.
In capsule staining, a smear of bacteria is made at the center of a slide. It is not heat-fixed, as the cell shrinkage caused by heating may create a clear zone around the cell, which may be mistaken as capsule.
Moreover, as the capsular material is water-soluble and may be washed away with vigorous washing, only two reagents are used in staining without any in-between water-washing. One of the two reagents is the primary stain, crystal violet. It imparts dark purple-blue color to both, the cell as well as the capsule.
However, it is ‘absorbed’ into the cell due to the ionic nature of the cellular components, whereas it simply ‘adheres’ to the capsule, because of the non-ionic nature of the capsular material. To prevent dislodging of the capsular material, the smear is not water-washed.
The second reagent, copper sulphate acts both as a decolorizing agent and a counter-stain. It decolorizes by removing excess crystal violet from around the cells as well as the crystal violet adhering to the capsule. It also counter- stains the capsule and imparts a light blue color to it. The capsule now appears light blue, whereas the cell appears deep purple-blue.
Method 1: Copper sulphate solution (20%)
Reagents And Material Required
- Microscopic Slide
- Immersion oil
- Microscope with 100X objective lens
- Crystal violet stain
- Stain solutions: Depending on the type of method used (crystal violet, India ink, Nigrosin, Copper Sulfate, Basic carbol fuschin solution, Methylene blue solution etc.
- In this case, Copper sulphate solution (20%)
- Gas burner
- Clean the slide properly under water such that water does not remain as drops on its surface.
- The adhering water is wiped out with bibulous paper and the slide is air-dried.
- A smear of bacteria is prepared at the center of the slide in two methods as follows.(a)If bacteria grown on agar plate or agar slant are to be observed, a drop of water is put at the center of the slide and a loop of bacteria from the plate or slant is transferred to it by a loop sterilized over flame. Then, by slow rotation of the loop in the drop, a bacteria suspension is made and it is spread till a smear is obtained. (b) If bacteria grown in liquid broth are to be observed, a drop of the bacteria suspension is directly placed at the center of the slide by a flame-sterilized loop and a smear is made by spreading.
- The smear is air-dried. No heat-fixation is done as the resultant cell shrinkage may create a clear zone around the bacteria, which may be mistaken as capsule.
- The smear is flooded with the primary stain, crystal violet, for 5-7 minutes.
- The smear is washed with 20% copper sulphate solution. To prevent dislodging of the capsular material, the smear is not water-washed.
- The slide is blotted dry with bibulous paper.
- The slide is clipped to the stage of the microscope and the smear observed under low power and high dry objectives.
- A drop of immersion oil is put on the smear.
- The smear is observed under oil-immersion objective.
1. Colour of the area surrounding the cells:
Light blue: Capsule present (capsulated bacteria)
Dark purple–blue: Capsule absent (non-capsulated bacteria)
2. Colour of the cells
3. If capsulated, size of the capsules:
Small, moderate or large.
4. Relative size of cells and capsules
By eye estimation, make drawing of the field under the oil-immersion objective, indicating the relative size of the cells and the capsules.
Method 2: India Ink Method
In this method, two dyes, crystal violet, and India ink are used. The capsule is seen as a clear halo around the microorganism against the black background. This method is used for demonstrating Cryptococcus.
- The background will be dark (color of India ink).
- The bacterial cells will be stained purple (bacterial cells take crystal violet-basic dyes as they are negatively charged).
- The capsule (if present) will appear clear against the dark background (capsule does not take any stain).
- Place a single drop of India ink on a clean microscope slide, adjacent to the frosted edge.
- Using a flamed loop and sterile technique, remove some Klebsiella pneumoniae from culture tube or plate and mix it into the drop of India ink. Be sure there are no large clumps of organism, but try to avoid spreading the drop.
- Place the end of another clean microscope slide at an angle to the end of the slide containing the organism. Spread out the drop out into a film. This is done by contacting the drop of India ink with the clean microscope slide and using the capillary action of the dye/ slide to spread the India ink across the smear.
- Allow the film to air dry (will take 5-7 minutes). DO NOT heat or blot dry! Heat will melt the capsule!
- Saturate the slide with crystal violet for 1 minute and rinse slightly & very gently with water. Be cautious water may remove the capsule from the cell.
- Let the slide air dry for a few minutes. DO NOT blot the slide! Blotting will remove the bacteria from the slide and/or distort the capsule.
- Observe the slide under oil immersion.
Look for purple cells surrounded by a clear halo on a dark background. The halo is the capsule. You may need to decrease the amount of light in order to make the capsule easier to see.