kinyoun stain kinyoun stain the kinyoun method or kinyoun stain (cold method), developed by joseph j. kinyoun, is a procedure used to stain acid-fast species of the bacterial genus mycobacterium. 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. it involves the application of a primary stain (basic fuchsin), a decolorizer (acid-alcohol), and a counterstain (methylene blue). unlike the ziehl–neelsen stain (z-n stain), the kinyoun method of staining does not require heating. 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. summary of acid-fast stain (kinyoun stain) application of reagent cell colour acid fast non-acid fast primary dye carbol fuchsin red red decolorizer acid alcohol red colorless counter stain methylene blue red blue staining procedure kinyoun stain on organism mycobacterium smegmatis materials slide with organism smear carbol fuchsin acid-alcohol methylene blue bibulous paper microscope instructions make smear on a slide with organisms 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.