Microbiology | Microbe World

Acid Fast Staining Technique and its Details

Introduction

• In nature there are variety of micro-organism each micro-organism have some special characters.
• Most of the micro-organisms are easily stained by simple staining procedures.
• But there are some micro-organism that are not easily stained by these technique because they have a waxy covering on its surface.If any how they get stained they are don’t get decolourise even by strong acid.
• Such organism require a special staining technique.
• Acid fast staining technique is a differential staining technique in bacteriology.
• This staining technique was discovered by scientist Paul Ehrlich in 1883.
• Acid fast staining technique helps us to differentiate the organism as acid fast and non-acid fast organisms.
• For staining such organism Zeihl-Neelsen’s staining method is used.It is also called as Acid fast staining method.

Definition

1. Acid fast organism- The organism that get stained by acid fast staining technique but don’t get decolourised even by strong acid are called as acid fast organism.
2. Non acid fast organism- The organism that easily get stained by a staining procedure as well as decolourise easily by a strong acid are non-acid fast organism.

Requirement

1. A clean grease free slide.
2. A bacterial cell suspension.
3. Staining agent- Zeihl Neelsen carbol fucshin.
4. Boiling water bath.
5. Decolourising agent – Acid alcohol.
6. Counter stain – 1% Malachite green or 0.3 % Methylene blue.

Procedure

1. Take a clean grease free slide and prepare a smear using nichrome wireloop.
2. Air dry and heat fix the slide.
3. The slide is flooded with ZNCF 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 is treated to the decolourisng agent that is acid alcohol ntil the stain disappear in washing.
6. After decolourisation the slide is given a water wash treatment.
7. Further the smear is flooded with the counter stain that is 1% Malachite green or 0.3 % Methylene blue for about 2 minutes.
8. After 2 minutes the slide is washed with water,air dryied and observe under oil immersion objective.

Flow chart of Acid Fast staining procedure

Functions of reagents used in acid fast staining technique

1. ZNCF ( Zeihl Neelsen carbol fucshin)

• It is a primary stain.
• Many acid fast bacteria are not stained with common stain like carbol fucshin because they are prepared in aqueous solution.
• These acid fast bacteria requires a stain that is prepared in phenolic stain and ZNCF stain is prepared in phenolic soloution.
• As these acid fast bacteria have a waxy covering on there surface and phenolic stain solubilizes waxy covering and stain the cell.
• The cells stained with ZNCF appearpink in colour.

2. Acid alcohol 

• It is the decolourising agent.
• It is prepared from combination of acid that is 3% hydrochloric acid and alcohol that is 95% ethanol.

3.  1% Malachite green or 0.3 % Methylene blue

• It acts as a counter stain.
• It stain the decolourised cell and these cell appear green or blue in colour.

Mechanism

1. Acid fast bacteria have a waxy covering on its surface or we can say it has high lipid content in the cell wall.
2. Cell wall of acid fast bacteria is made up of lipids like Mycolic acid and Glycolipids.
3. Due to these high lipid content in the cell wall these cell wall has less permeability.
4. So first it is necessary to increase permeability of cell wall so the stain can easily penetrate in the cell.
5. Permeability of the cell wall is increased by using phenolic solution and steaming.
6. After the permeability of cell increases the cells get stain.
7. Now once the acid fast bacteria get stained it doesn’t decolourise even by strong decolourisng agent and appear pink in colour.
8. But the non-acid fast bacteria get decolourise easily and get stained by counter stain and appear green or blue in colour.
9. If we use Malachite green stain cells get stain and appear green in colour and if we use Methylene blue stain cells get stain and appear blue in colour.

Observation

1. Acid fast bacteria appear pink in colour.
2. Non acid fast bacteria appear green or blue in colour.

Some important points

1. The permeability of acid fast cell is increased by phenolic stain because phenolic stain have high affinity towards the waxy covering and it is more soluble in waxy covering.
2. For increasing the permeability we use heat steaming because steaming softens the waxy material and allow easy penetration of stain.

Applications

1. Acid fast staining is useful in diagnosis of Tuberculosis and leprosy.