Ziehl–Neelsen stain

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Mycobacterium tuberculosis visualization using the Ziehl–Neelsen stain
Ziehl–Neelsen (acid-fast) stain – diagram of basic steps

Acid-fast stain, first introduced by Paul Ehrlich, also known as the Ziehl–Neelsen staining, is a bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. It is named for two German doctors who modified it: the bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–1898).

Mycobacterium tuberculosis is the most important of this group because it is responsible for tuberculosis (TB). Other important Mycobacterium species involved in human disease are Mycobacterium leprae, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium bovis, Mycobacterium africanum and members of the Mycobacterium avium complex. Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stain.[1] It can also be used to stain a few other bacteria, such as Nocardia. The reagents used for Ziehl–Neelsen staining are – carbol fuchsin, acid alcohol, and methylene blue. Acid-fast bacilli are bright red after staining.

A variation on this staining method is used in mycology to differentially stain acid-fast incrustations in the cuticular hyphae of certain species of fungi in the genus Russula.[2][3] It is also useful in the identification of some protozoa, namely Cryptosporidium and Isospora. The Ziehl–Neelsen stain can also hinder diagnosis in the case of paragonimiasis because the eggs in sputum sample for ovum and parasite (O&P) can be dissolved by the stain, and is often used in this clinical setting because signs and symptoms of paragonimiasis closely resemble those of TB.


A typical AFB stain procedure involves dropping the cells in suspension onto a slide, then air drying the liquid and heat fixing the cells. The slide is flooded with carbol fuchsin, which is then heated to dry and rinsed off in tap water. The slide is then flooded with a 1% solution of hydrochloric acid in isopropyl alcohol (or methanol) to remove the carbol fuchsin, thus removing the stain from cells that are unprotected by a waxy lipid layer. Thereafter, the cells are stained in methylene blue and viewed under a microscope under oil immersion.

Summary of acid-fast stain (Ziehl–Neelsen stain)[4]
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/malachite green Red Blue

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 negative predictive value.

Mechanism of acid-fast staining in acid-fast cells and non-acid-fast cell.[5][6][7]

Mechanism explanation[edit]

Initially, carbol fuchsin stains every cell. When they are de-stained with acid-alcohol, only non-acid-fast bacteria get de-stained since they do not have a thick, waxy lipid layer like acid-fast bacteria. When counter stain is applied, non-acid-fast bacteria pick it up and become blue (methylene blue) or green (malachite green) when viewed under the microscope. Acid-fast bacteria retain carbol fuchsin so they appear red.


See also[edit]


  • "Microbiology with Diseases by Body System", Robert W. Bauman, 2009, Pearson Education, Inc.
  • Morello, Josephine A., Paul A. Granato, Marion E. Wilson, and Verna Morton. Laboratory Manual and Workbook in Microbiology: Applications to Patient Car. 10th ed. Boston: McGraw-Hill Higher Education, 2006. Print.

Online protocol examples[edit]


  1. ^ Morello, Josephine A., Paul A. Granato, Marion E. Wilson, and Verna Morton. Laboratory Manual and Workbook in Microbiology: Applications to Patient Care. 10th ed. Boston: McGraw-Hill Higher Education, 2006. Print.
  2. ^ Romagnesi, H. (1967). Les Russules d'Europe et d'Afrique du Nord. Bordas. ISBN 0-934454-87-6.
  3. ^ Largent, D; D Johnson; R Watling (1977). How to identify fungi to genus III: microscopic features. Mad River Press. ISBN 0-916422-09-7. p 25.
  4. ^ Acid-Fast Stain- Principle, Procedure, Interpretation and Examples. May 8, 2015 by Sagar Aryal
  5. ^ "Online Microbiology Notes". Online Microbiology Notes. Retrieved 2017-11-29.
  6. ^ "Home – microbeonline". microbeonline.com. Retrieved 2017-11-29.
  7. ^ Kumar, Surinder (2012). Textbook of Microbiology. p. 315.
  8. ^ Ellis, RC; LA Zabrowarny. (1993). "Safer staining method for acid fast bacilli". Journal of Clinical Pathology. 46 (6): 559–560. doi:10.1136/jcp.46.6.559. PMC 501296. PMID 7687254.

External links[edit]