Capsule Staining Technique by Hiss method
- Capsule is the rigid, slimy and gummy covering of the bacterial cell which lies external to the cell wall.
- The cell that posses the capsule are called as capsulated cell and those who lack the capsule are called as the non-capsulated bacteria.
- Capsule is mainly made up of organic compound like Polysaccharides or polypeptides.
- Micro-capsule – Its size is less than 0.2 µ.
- Macro-capsule – Its size is more than 0.2 µ.
- When we stain the cell with normal stain like methylene blue or saffranin it stains the cell as well as capsule as capsule get stained easily.
- So if we want to stain a capsule we need a special capsule staining technique.
- Positive staining method – In positive staining method only capsule is stained.
- Negative staining method – Here capsule is not stained but it is made visible by colourless area of capsule in between coloured cells and coloured background.
So here we are going to study the capsule staining technique and its details.
The two methods which we are going to study are…
- Positive staining method – Hiss Staining method.
- Negative staining method – Maneval’s method.
Hiss Staining method – Positive staining method.
To stain the capsule of bacterial cell by Hiss method.
- Capsulated cell culture.
- 1 % Crystal violet.
- 20 % Copper sulfate.
- A smear is prepared on a clean grease free slide.
- The smear should not be heat fix it should be just air dried.
- After air drying the smear is flooded with 1 % Crystal violet for about 4 – 5 minutes.
- After 5 minutes the smear is rinse with 20 % copper sulfate solution.
- The slide is air dried and observed under oil immersion objective.
Flow chart of Hiss staining technique
- As we know capsule is non-ionic in nature so it doesn,t get stain by a acidic stain but when we apply a basic stain it stains the cell as well as the capsule.
- So further here we treat the smear with hypertonic solution that is that is copper sulphate solution.
- When we apply a copper sulphate solution a ionic difference is created due to which it causes diffusion of stain towards outer surface of cell.
- Further after drying of slide we can observe that the stain which is not passed from the capsular layer during diffusion retains in the capsular layer.
- Finally we can observe a dark violet colour cell and light violet colour capsule.
After staining we can observe that the cells appear dark violet colour and capsule appear light violet colour.