The Ziehl–Neelsen stain, also known as the acid-fast stain, was first described by two German doctors; Franz Ziehl

Franz Ziehl

Dr. Franz Ziehl was a German bacteriologist. He was a professor in Lübeck. Franz Ziehl introduced the carbolfuchsin stain for the tubercle bacillus in 1882. With pathologist Friedrich Neelsen , he developed the Ziehl-Neelsen stain also known as the acid-fast stain, which is used to identify...

 (1859 to 1926), a bacteriologist and Friedrich Neelsen

Friedrich Neelsen

Friedrich Carl Adolf Neelsen was a German pathologist.-Life:Friedrich C.A. Neelsen was born to Hans Friedrich Neelsen, deacon of the Uetersen vicarage, and his wife Bertha Sophia . He attended school in Uetersen and later in Altona...

 (1854 to 1898), a pathologist. It is a special bacteriological stain used to identify acid-fast

Acid-fast

Acid-fastness is a physical property of certain bacteria, specifically their resistance to decolorization by acids during staining procedures.Acid-fast organisms are difficult to characterize using standard microbiological techniques Acid-fastness is a physical property of certain bacteria,...

 organisms, mainly Mycobacteria. Mycobacterium tuberculosis

Mycobacterium tuberculosis

Mycobacterium tuberculosis is a pathogenic bacterial species in the genus Mycobacterium and the causative agent of most cases of tuberculosis . First discovered in 1882 by Robert Koch, M...

is the most important of this group, as it is responsible for the disease called tuberculosis

Tuberculosis

Tuberculosis, MTB, or TB is a common, and in many cases lethal, infectious disease caused by various strains of mycobacteria, usually Mycobacterium tuberculosis. Tuberculosis usually attacks the lungs but can also affect other parts of the body...

 (TB) along with some others of this genus. It is helpful in diagnosing Mycobacterium tuberculosis since its lipid rich cell wall makes it resistant to Gram stain. It can also be used to stain few other bacteria like Nocardia

Nocardia

Nocardia is a genus of weakly staining Gram-positive, catalase-positive, rod-shaped bacteria. It forms partially acid-fast beaded branching filaments . It has a total of 85 species. Some species are non-pathogenic while others are responsible for nocardiosis. Nocardia are found worldwide in soil...

. The reagents used are Ziehl–Neelsen carbolfuchsin, acid alcohol and methylene blue

Methylene blue

Methylene blue is a heterocyclic aromatic chemical compound with the molecular formula C16H18N3SCl. It has many uses in a range of different fields, such as biology and chemistry. At room temperature it appears as a solid, odorless, dark green powder, that yields a blue solution when dissolved in...

. Acid-fast bacilli will be bright red after staining.

A variation on this staining method is used in mycology

Mycology

Mycology is the branch of biology concerned with the study of fungi, including their genetic and biochemical properties, their taxonomy and their use to humans as a source for tinder, medicinals , food and entheogens, as well as their dangers, such as poisoning or...

 to differentially stain acid-fast incrustations in the cuticular hyphae of certain species of spence

Spence

Spence may refer to:* Spence, Australian Capital Territory, a suburb of Canberra, Australia* Spence, Ontario, a ghost town in Ontario, Canada* Spence, California, an unincorporated community in Monterey County, California, United States...

 in the genus Russula

Russula

Around 750 worldwide species of mycorrhizal mushrooms compose the genus Russula. They are typically common, fairly large, and brightly colored - making them one of the most recognizable genera among mycologists and mushroom collectors...

.

Procedure

• 1. Prepare smear
• 2. Air dry and heat fix it
• 3. Rinse in carbol fuchsin
• 4. Light a cotton swab and hold it underneath until steam appears
• 5. Wash with dilute hydrochloric acid until a faint pink color remains.
• 6. Counterstain with Methylene Blue Chloride for a minute
• 7. Wash with gentle water till violet becomes faint
• 8. Blot dry
• 9. View under oil immersion lens

Studies have shown that an AFB stain without a culture has a poor negative predictive value. An AFB Culture should be performed along with an AFB stain; this has a much higher predictive value.

Modifications

• 5% sulphuric acid is used for destaining Mycobacterium leprae
  Mycobacterium leprae
  Mycobacterium leprae, also known as Hansen’s coccus spirilly, mostly found in warm tropical countries, is a bacterium that causes leprosy . It is an intracellular, pleomorphic, acid-fast bacterium. M. leprae is an aerobic bacillus surrounded by the characteristic waxy coating unique to mycobacteria...
  
  instead of the 20% used for Mycobacterium tuberculosis.
• Kinyoun modification (or cold Ziehl–Neelsen technique) is also available.
• A protocol in which a detergent
  Detergent
  A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...
  
   is substituted for the highly toxic phenol
  Phenol
  Phenol, also known as carbolic acid, phenic acid, is an organic compound with the chemical formula C6H5OH. It is a white crystalline solid. The molecule consists of a phenyl , bonded to a hydroxyl group. It is produced on a large scale as a precursor to many materials and useful compounds...
  
   in the fuchsin staining solution.

Online protocol examples

• Ziehl–Neelsen protocol (PDF
  Portable Document Format
  Portable Document Format is an open standard for document exchange. This file format, created by Adobe Systems in 1993, is used for representing documents in a manner independent of application software, hardware, and operating systems....
  
  format).