Acid Fast Stain (Kinyoun-Cold Method)- Principle, Procedure and Result Interpretation

Objective of Kinyoun-Cold Method

Identification of acid-fast Mycobacterium spp. and parasites such as Cryptosporidium and Isopora spp.

Principle of Kinyoun-Cold Method

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, carbolfuchsin, is applied to the cells and phenol is used to allow the stain to penetrate into the waxy surface of acid-fast microorganisms. The excess stain is removed with treatment by 1% sulfuric acid. A secondary stain, methylene blue, is then applied to the cells.

Procedure of Kinyoun-Cold Method

1. Prepare and fix the specimen smear prior to staining.
2. Cover the smear with carbolfuchsin for 3 to 5 minutes at room temperature.
3. Gently rinse the slide with water.
4. Run 1% sulfuric acid decolorizer over the slide for approximately 3 minutes.
5. Rinse the slide with water and decolorize again for 1 to 2 minutes until the solution runs clear.
6. Rinse the slide with water.
7. Cover the smear with the secondary or counterstain, methylene blue, for 1 minute.
8. Gently rinse the slide with water.
9. Blot the slide dry with bibulous paper.

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Result Interpretation of Kinyoun-Cold Method

Acid-fast organisms, Mycobacterium spp., will appear pink. Note: Identification of a single acid-fast bacillus in a single sputum is considered diagnostic.
Nonacid-fast organisms will appear blue. In addition, background material should stain blue.

Limitations of Kinyoun-Cold Method

1. May be less sensitive than the Ziehl-Neelsen method.
2. Smears that are too thick may not properlystain.