Acid Fast Staining Technique and its Details
- In nature, there is a variety of micro-organism each micro-organism have some special characters.
- Most of the microorganisms are easily stained by simple staining procedures.
- But there is some micro-organism that is not easily stained by this technique because they have a waxy covering on its surface. If anyhow 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 Ziehl-Neelsen staining method is used. It is also called as Acid-fast staining method.
- 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 an acid-fast organism.
- Non-acid-fast organism- The organism that easily gets stained by a staining procedure as well as decolourises easily by a strong acid are a non-acid fast organism.
- A clean grease free slide.
- A bacterial cell suspension.
- Staining agent- Ziehl Neelsen carbol fuchsin.
- Boiling water bath.
- Decolourising agent – Acid alcohol.
- Counter stain – 1% Malachite green or 0.3 % Methylene blue.
- Take a clean grease free slide and prepare a smear using nichrome wire loop.
- Air dry and heat fix the slide.
- The slide is flooded with ZNCF stain and placed on a boiling water bath for steaming for about 3-5 minutes.
- During steaming the stain is repeatedly added on the slide to avoid drying of smear.
- Further, the slide is treated to the decolourising agent that is acid alcohol until the stain disappears in washing.
- After decolourisation, the slide is given a water wash treatment.
- Further, the smear is flooded with the counterstain that is 1% Malachite green or 0.3 % Methylene blue for about 2 minutes.
- After 2 minutes the slide is washed with water, air dried and observed under oil immersion objective.
Flowchart of the Acid Fast staining procedure
Functions of reagents used in acid-fast staining technique
1. ZNCF ( Ziehl Neelsen carbol fuchsin)
- It is a primary stain.
- Many acid-fast bacteria are not stained with the common stain like carbol fuchsin because they are prepared in aqueous solution.
- These acid fast bacteria require a stain that is prepared in phenolic stain and ZNCF stain is prepared in phenolic solution.
- As these acid-fast bacteria have a waxy covering on their surface and phenolic stain solubilizes waxy covering and stain the cell.
- The cells stained with ZNCF appear pink in colour.
2. Acid alcohol
- It is the decolourising agent.
- It is prepared from the 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 counterstain.
- It stains the decolourised cell and these cells appear green or blue in colour.
- Acid-fast bacteria have a waxy covering on its surface or we can say it has high lipid content in the cell wall.
- The cell wall of acid-fast bacteria is made up of lipids like Mycolic acid and Glycolipids.
- Due to these high lipid content in the cell wall, these cell wall has less permeability.
- So first it is necessary to increase the permeability of the cell wall so the stain can easily penetrate in the cell.
- The permeability of the cell wall is increased by using phenolic solution and steaming.
- After the permeability of the cell increases the cells get the stain.
- Now once the acid-fast bacteria get stained it doesn’t decolourise even by the strong decolourising agent and appear pink in colour.
- But the non-acid fast bacteria get decolourise easily and get stained by counterstain and appear green or blue in colour.
- If we use Malachite green stain cells get the stain and appear green in colour and if we use Methylene blue stain cells get the stain and appear blue in colour.
- Acid-fast bacteria appear pink in colour.
- Non-acid fast bacteria appear green or blue in colour.
Some important points
- 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.
- For increasing the permeability we use heat steaming because steaming softens the waxy material and allow easy penetration of stain.
- Acid-fast staining is useful in the diagnosis of Tuberculosis and leprosy.