To perform acid fast staining

 Experiment

Aim: 

To perform acid fast staining.

Requirements:

Glassware: glass slide, glass rod, beaker,dropper,cover slip etc.

Chemical/Reagent: Carbol fuchsin solution, Acid alcohol solution (3% HCl in alcohol),sulphuric acid solution (20-25% v/v in water), Methylene blue counter stain (0.3% w/v aq).

Theory:

The ZN staining technique is based on the work of five people. Koch, Ehrlich, Ziehl, Rindfleisch and Neelsen, but it is popularly known as the ‘Ziehl-Neelsen’ method.

The technique was developed by Paul Ehrlich (1882) and was modified later by Ziehl- Neelsen and therefore also known as Ziehl Neelsen staining. This is a differential staining used to identify mainly the members of mycobacterium tuberculosis and leprae. These organism are difficult to stain by ordinary staining methods due to presence of high lipid content in their cell wall.

Bacteria are classified as:-

1. ACID FAST: if they retain the primary stain after the application of strong acid and appeared red.

2. NON ACID FAST: if they do not retain the primary stain and are counterstained by methylene blue.

Reagents: 1. Carbol fuchsin solution, Acid alcohol solution (3% Hcl in alcohol)

Alternatively: sulphuric acid solution (20-25% v/v in water) Methylene blue counter stain (0.3% w/v aq).

Image Source: laboratory info . com

Procedure:

1. Prepare a smear of purulent portion of the specimen on a clean glass slide

2. Air dry and heat fix the smear

3. Flood the smear with freshly filtered carbol fuchsin. Heat gently until steam rises

4. Continue to heat for 5 min so that steam is seen but without boiling. Do not allow the side to dry and add more stain from time to time to prevent this drying.

5. Cool and wash the stain of the slide with water.

6. Cover the slide with acid alcohol solution for 3 min. wash with running water and drain. Repeat de colorization process until smear becomes faint pink in color.

7. Cover the slide with methylene blue stain and leave it for 2 min

8. Wash with tap water , blot dry air the slide under the oil immersion object

Results: 

Acid fast organism will appear bright red on a blue background while non acid fast organism will appear dark blue in color.