Acid-Fast Staining : Principle, Procedure, Interpretation and Examples.

                                Acid-Fast Staining

It is the differential staining techniques which was first developed by Ziehl and later on modified by Neelsen. So this method is also called Ziehl- Neelsen staining techniques. Neelsen in 1883 used Ziehl's carbon-fushsin and heat then Decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl-Neelsen staining techniques was developed.

The main aim of this staining is to differentiate bacteria into acid fast group and non acid fast groups.

This method is used for those microorganisms which are not stained by simple or Gram Staining method, particularly the the member of genus Mycobacterium, are resistant and can only be visualised by acid fast staining.

Principle of Acid-Fast Stain:

Acid-fast mycobacteria contain mycolic acid in their outer membrane, making the cells waxy and resistant to staining with aqueous based stains such as the Gram stain. The primary stain, carbol-fushsin is applied to the cells, and heat and phenol are used to allow the stain to penetrate into the waxy surface of acid-fast microorganisms. The excess stain is removed with treatment by acid alcohol (ethanol and hydrochloric acid). A secondary stain, methylene blue, is then applied to the cells.

Components of Ziehl-Neelsen stain:

• Primary stain: Strong Carbol-fushsin ( consists of basic fuchsin and carbolic acid)
• Decolorizer: 20% sulfuric acid (H2SO4) or acid alcohol
• Counter stain: Loeffler's methylene blue or Malachite green

Procedure

1. Prepare and fix the specimen smear prior to staining.
2. Place a small strip of blotting or filter paper over the top of the specimen, and place the slide over a boiling hot water bath on a mesh surface.
3. Cover the filter paper with the primary stain, carbol-fushsin. Leave the slide on the water bath for 3 to 5 minutes. Continue to apply stain if the filter paper begins to dry.
4. Remove the filter paper and rinse the slide with water until the solution runs clear.
5. Run acid-alcohol decolorizer over the slide for approximately 10 to 15 seconds.
6. Rinse the slide with water.
7. Cover the smear with the secondary or counter stain, methylene blue, for 1 minute.
8. Gently rinse the slide with water.
9. Air dry, blot dry and observe under microscope.

Result Interpretation: 

Acid fast: Bright red to intensive purple, Red , straight, or slightly curved rods, occuring singly or in small groups, may appear beaded.

Non- Acid fast: Blue color, in addition, background material should stain blue.

Examples:

Acid-fast: Mycobacterium tuberculosis, Mycobacterium smegmatis.

Non- Mycobacterial bacteria: Nocardia Coccidian Parasites: Cryptosporidium