Procedure used to stain certain acid-fast species of bacteria
The Kinyoun method or Kinyoun stain (cold method), developed by
Joseph J. Kinyoun, is a procedure used to
stainacid-fast species of the bacterial genus Mycobacterium.[1] It is a variation of a method developed by
Robert Koch in 1882. 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 than a Gram-Stain. The unique ability of mycobacteria to resist decolorization by acid-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 fuchsin) 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]
Clean slide, wax label slide, spread organism, air dry for 10 minutes, heat fix
Dip slide into Carbol Fuchsin for 20 minutes.
Rinse slide
Dip slide into Acid-alcohol for 3–5 seconds.
Rinse Slide
Dip slide into Methylene blue for 30 seconds.
Rinse Slide
Blot slide dry with bibulous paper.
Observe under Microscope.
Modification
The Kinyoun method can be modified as a weak acid fast stain, which uses 0.5–1.0%
sulfuric acid instead of
hydrochloric acid. The weak acid fast stain, in addition to staining Mycobacteria, will also stain organisms that are not able to maintain the carbol fuchsin after decolorizing with HCl, such as Nocardia species and Cryptosporidium.