Stain and Staining Procedures

Stain and staining procedures


Micro-organism can be identified by microscopic examination.These micro-organisms are very small, transparent, and invisible to our naked eyes so it is very difficult to observe these micro-organisms.

These microbial cells are colourless so when these cells are suspended in the aqueous medium the refractive index of the cell and aqueous medium is very low and same and due to lack of contrast observation of such unstained organism is very difficult. So staining of micro-organism in microbiology is an essential step for bacteriological studies.

Purpose of staining

  • Staining procedure helps in the production of contrast between micro-organism an aqueous medium.
  • It is useful to study morphological of bacteria.
  • Staining helps in studying internal as well as external characters of bacteria.


1. Dye – Dye is an organic compound made up of auxochrome and chromophore group these groups are linked to a benzene ring. Dye is used for colouring non-biological objects.


a)  Chromophore group– It imparts colour to the compound it is called as chromogen.

b)  Auxochrome group– It has salt formation property with dye.

In simple words Chromophore group is a colour imparting compound and auxochrome group is that binds to chromophore group to a molecule for colouring that molecule.

2. Stain– It is an organic compound containing both auxochrome and chromophore group.

  • Stains are basically used for colouring biological agents.
  • Chromophore group is a charged molecule these molecules get bind to charged cellular compounds like proteins and nucleic acid.
  • Some stains have high affinity towards cellular components as well as some stains very low or no affinity towards cellular components.
  • Such stains having low or no affinity are called as mordant.
  • Mordant is a substance that increases the affinity of stain and molecule to be a stain.

Types of stains

Stains are of three types.

1. Acidic stain- Acidic stains are those which after dissociation or ionization impart a negative charge on chromophore group.Examples are Eosin, Congored etc.

2. Basic stain- Basic stain are those stain which after dissociation or ionization impart a positive charge on chromophore group.Example- Methylene blue.

3.Neutral stain- Neutral stains are those stains which after dissociation or ionization imparts both positive and negative charge on chromophore group.Examples- Leishman’s stain.

There are different types of staining procedures.

  1. Simple staining
  2. Negative staining
  3. Differential staining
  4. Gram staining
  5. Cell wall staining
  6. Acid fast staining
  7. Capsule staining by Maneval’s method
  8. Capsule staining by Hiss method
  9. Endospore staining by Bartholomew and Mittwer’s Method
  10. Endospore Staining by Dorner’s Method
  11. Volutin granule staining





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