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.

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, PhD

Dr. Sagar Aryal is a microbiologist, researcher, and science communicator from Nepal. He is also co-founder and Research Head of the Department of Natural Products at the Kathmandu Research Institute for Biological Sciences (KRIBS), where he leads research on natural product discovery and microbial biodiversity. Dr. Aryal earned his Ph.D. in Microbiology from Tribhuvan University, Kathmandu, Nepal, in 2025, supported by a DAAD Research Grant and a University Grants Commission (UGC) Nepal Ph.D. Fellowship. His research interests include Actinomycetes, Myxobacteria, microbial taxonomy, and natural products, particularly the discovery of bioactive compounds with potential pharmaceutical applications. He isolated and identified Nocardia nepalensis, a novel bacterial species from Nepal, contributing to understanding microbial diversity in the region. Dr. Aryal has published more than 16 research articles in international journals and contributed three book chapters to established academic publications. He has presented scientific research findings and developed international research collaborations through visits to more than 18 countries across Asia, Europe, North America, and Oceania. From 2023 to 2025, he served as the American Society for Microbiology (ASM) Young Ambassador to Nepal. Since December 2025, he has served as an Editorial Board Member of Scientific Reports, a Nature Portfolio journal. In recognition of his scientific contributions, Dr. Aryal received the NAST Science and Technology Youth Award 2025 from the Nepal Academy of Science and Technology. He previously received the Frs. Locke and Stiller Research Award (LSRA) for the 2017-2018 cycle from St. Xavier's College, Kathmandu, and a Nepal Academy of Science and Technology (NAST) Research Grant for 2017-2018. Alongside his scientific career, Dr. Aryal has more than 15 years of experience in blogging, content writing, and search engine optimization (SEO).