ACID FAST STAINING

                                      Acid Fast Stain (Ziehl- Neelsen Method)

Aim:

            To perform acid fast staining to identify Mycobacterium tuberculosis and Mycobacterium leprae.

Introduction :

            The Ziehl Neelsen Method is used for staining Mycobacterium sp. in clinical specimens. The thick outer waxy covering (mycolic acid) of the Mycobacterium cell walls act as a barrier  and does not allow all the stains to enter into the cell. In order to visualize these cells higher concentrations of the staining solution is needed and once this stain enters the cell, it is too difficult to remove the stain using a decolorizer. When the clinical specimen is stained with basic dyes such as carbol fuchsin (primary stain) with the continuous application of heat, softens the waxy lipid outer covering of the cell wall and the stain readily enters the cell and stains the cell cytoplasm. When decolorizing agents such as acid-alcohol is added over the primary stain, some bacterial cells cannot be easily decolorized and such bacterial cells are called as acid fast bacteria. The bacteria with high concentration of lipid are easily decolorized by the decolorizing agent and are said to be non-acid fast bacteria. Finally, the addition of the counter stain, Methylene blue, dyes the colorless non acid fast cells as blue thus differentiating them from the pink acid fast bacteria which are unaffected by the Methylene blue.

 Materials Required:

1.      A clean grease free slide.

2.      A bacterial cell suspension.

3.      Staining agent- carbol fucshin.

4.      Boiling water bath.

5.      Decolourising agent – Acid alcohol

6.      Counter stain – 1% Methylene blue

Procedure :

1. Smear of organisms were prepared on clean glass slide
2. The slide was allowed to air dry and heat fixed..
3. The slide was flooded with carbol fucshin stain and placed on a boiling water bath for steaming for about 3-5 minutes.
4. During steaming the stain is repeatedly added on the slide to avoid drying of smear.
5. Further the slide was delcolourised with acid alcohol until the stain disappear in washing.
6. After decolourisation the slide was given a water wash treatment.
7. Further the smear was flooded with the counter stain that is 1% Methylene blue for about one minute.
8. The slide was then washed with water, air dried and observed under oil immersion objective.

Result: 

On microscopic observation, the acid fast bacterium appeared as pink coloured cells, non-acid fast cells appeared blue.

Discussion:

            Acid fast stain is due to relative solubility of carbol fuchsin and impermeability of call wall. Fuchsin is more soluble on carbolic acid than in water and carbolic acid soluble more easily in lipids than in acid alcohol. Therefore carbol fuchsin has higher  affinity for lipids than acid alcohol and will remain within the cell wall when washed with decolouriser.