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

by

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.

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.

Read Also:

Limitations of Kinyoun-Cold Method

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

About Author

Photo of author

Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He is doing his Ph.D. at the Central Department of Microbiology, Tribhuvan University, Kathmandu, Nepal. He was awarded the DAAD Research Grant to conduct part of his Ph.D. research work for two years (2019-2021) at Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. Sagar is interested in research on actinobacteria, myxobacteria, and natural products. He is the Research Head of the Department of Natural Products, Kathmandu Research Institute for Biological Sciences (KRIBS), Lalitpur, Nepal. Sagar has more than ten years of experience in blogging, content writing, and SEO. Sagar was awarded the SfAM Communications Award 2015: Professional Communicator Category from the Society for Applied Microbiology (Now: Applied Microbiology International), Cambridge, United Kingdom (UK).

2 thoughts on “Acid Fast Stain (Kinyoun-Cold Method)- Principle, Procedure and Result Interpretation”

  1. ”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”, so these cells contains waxy layer , is this the reason why we heat the slide after primary staining (AFB) so that the stain can penetrate inside the cell.

    Reply

Leave a Comment

This site uses Akismet to reduce spam. Learn how your comment data is processed.