Kinyoun stain

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The Kinyoun method, or Kinyoun stain (cold method), developed by Robert Koch in 1882 and later modified by other scientist, is an acid-fast procedure used to stain any species of the genus Mycobacterium , Nocardia [1] and Cryptosporidium species. Certain species of bacteria have a waxy lipid called mycolic acid, in their cell walls which allow them to be stained with Acid-Fast better then a Gram-Stain. The unique ability of mycobacteria to resist decolorization byacid-alcohol is why they are termed acid-fast. [2] It involves the application of a primary stain (basic fuchsin), a decolorizer (acid-alcohol), and a counterstain (methylene blue). [3] Unlike the Ziehl-Neelsen stain (Z-N stain), the Kinyoun method of staining does not require heating. [4] [5] In the Ziehl-Neelsen stain, heat acts as a physical mordant while phenol (carbol of carbol fuschin) acts as the chemical mordant. Since the Kinyoun stain is a cold method (no heat applied), the concentration of carbol fuschin used is increased. [6]

<i>Mycobacterium</i> genus of bacteria

Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. Over 190 species are recognized in this genus. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis and leprosy in humans. The Greek prefix myco- means "fungus," alluding to the way mycobacteria have been observed to grow in a mold-like fashion on the surface of cultures. It is acid fast and cannot be stained by the Gram stain procedure.

<i>Nocardia</i> genus of Gram-positive bacteria

Nocardia is a genus of weakly staining Gram-positive, catalase-positive, rod-shaped bacteria. It forms partially acid-fast beaded branching filaments. It contains a total of 85 species. Some species are nonpathogenic, while others are responsible for nocardiosis. Nocardia species are found worldwide in soil rich in organic matter. In addition, they are oral microflora found in healthy gingiva, as well as periodontal pockets. Most Nocardia infections are acquired by inhalation of the bacteria or through traumatic introduction.

<i>Cryptosporidium</i> genus of apicomplexan parasitic alveolate

Cryptosporidium is a genus of apicomplexan parasitic alveolates that can cause a respiratory and gastrointestinal illness (cryptosporidiosis) that primarily involves watery diarrhea with or without a persistent cough in both immunocompetent and immunodeficient humans.


Summary of acid-fast stain (Kinyoun stain)
Application ofReagentCell Colour
Acid FastNon-acid fast
Primary dyeCarbol fuchsinRedRed
DecolorizerAcid alcoholRedColorless
Counter StainMethylene blueRedBlue

Staining Procedure

Kinyoun stain on organism Mycobacterium smegmatis Kinyoun Stain, organism Mycobacterium smegmatis.jpg
Kinyoun stain on organism Mycobacterium smegmatis

Make an Acid-Fast Stain [7]

  • Slide with organism smear
  • Carbol Fuchsin
  • Acid-Alcohol
  • Methylene blue
  • bibulous paper
  • Microscope
  1. Make smear on a slide with organisms
    • Clean slide, wax label slide, spread organism, air dry for 10 minutes, heat fix
  2. Dip slide into Carbol Fuchsin for 20 minutes.
  3. Rinse slide
  4. Dip slide into Acid-alcohol for 3-5 seconds.
  5. Rinse Slide
  6. Dip slide into Methylene blue for 30 seconds.
  7. Rinse Slide
  8. Blot slide dry with bibulous paper.
  9. Observe under Microscope.


The Kinyoun method can be modified as a weak acid fast stain, which uses 5% sulfuric acid instead of hydrochloric acid. The weak acid fast stain in addition to staining mycobacteria will stain organisms that are not able to maintain the carbol fuchsin after decolorizing with HCl. [8]

See also

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Auramine–rhodamine stain

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Fuchsine magenta dye, chemical

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  1. Geo F. B et al, Jawetz, Melnick and Adelberg's Medical Microbiology, 25th Edition, Lange Medical,2004, page 182
  2. .dalynn (October 2014). "KINYOUN CARBOL FUCHSIN STAIN" (PDF).
  3. Hussey, A. M., Zayaitz, A., "Acid-Fast Stain Protocols" Archived 2011-10-01 at the Wayback Machine , American Society for Microbiology , 8 September 2008. Retrieved on 1 November 2014.
  4. Murray PR, Baron E, Pfaller M, Tenover F, Yolken, Eds. Manual of clinical microbiology. 7th ed. Washington, DC: ASM, 1999.
  5. Baron EJ, Finegold SM. Bailey and Scott's diagnostic microbiology. 8th ed. St. Louis: Mosby, 1990.
  6. Ananthanarayan and Panicker's Textbook of Microbiology, 9th Edition, Universities Press (India), 2013, page 353
  8. "Kinyoun Stain - an overview | ScienceDirect Topics". Retrieved 2019-05-22.