Z.N.STAIN {ZIEHL NEELSEN STAIN},PROCEDURE AND RESULT

1. introduction of z.n.stain
2. principle of z.n. stain

Introduction of z.n.stain

Mycobacterium Tuberculosis was first introduced by a scientist Robert Koch. This Acid-fast nature was first described by Scientist Ehrlich. The stain for AFB was first introduced by scientist Ziehl and modified by Neelsen. Hensen has discovered the disease Leprosy.
Aim:- To differentiate the Acid Fast Bacilli by z.n.stain.

PRINCIPLE OF Z.N.STAIN

The cell wall of Mycobacteria contains various type of lipids and wax, so it is very thick therefore it is difficult to stain, so in Ziehl Neelsen stain, we use two strong moderates.

1. Chemical Mordant:- Carbolic Acid and Phenol
2. Physical Mordant:- Heat which helps to fix the primary stain into the bacterial cell.

1. When a heat-fix bacteria smear is treated with hot carbolfuchsin the whole bacteria take a
2. pink color. In the subsequent process of decolorization with 20% H, SO, and 95% ethyl alcohol. The same bacteria retain this primary colour and don’t decolorize with strong acids and alcohol. These are known as AFB.
3. The bacteria are decolorized with 20% H, SO, and 95% Ethyl alcohol and take up a blue color with counter-stain methylene blue. This is known as Non-AFB.

Reagents

• Carbol Fuchsin,
• 20% Sulphuric Acid.,
• 95% Ethyl alcohol,
• 1% Methylene Blue.

Procedure

1. Prepare a smear, air dry and fix it by heat.
2. Boil 2-3 ml of carbolfuchsin in a test tube and pour it on the smear wait for 5-7 minutes. Pour more carbolfuchsin after boiling when the steam stops rising from the slide. 3. Drain off the carbolfuchsin and decolorise it with 20% sulphuric acid for 2-3 minutes.
3. Wash the slide with tap water.
4. Decolourise with 95% ethyl alcohol for 2 minutes
5. Wash with tap water
6. Counterstain with methylene blue for 2 minutes
7. Wash with tap water and air dry it
8. Examine under the oil immersion objective of the microscope.

Result:-

Mycobacteria – stain pink in colour, Background material and other bacteria take blue color.