Written by MicroDok

Ziehl-Neelsen staining is a type of Acid-fast stain, first introduced by Paul Ehrlich. Ziehl–Neelsen staining is a bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. It is named for two German doctors who modified the stain: the bacteriologist Franz Ziehl (1859–1926) and the pathologist Friedrich Neelsen (1854–1898). The Ziehl-Neelsen stain (ZN stain), also called the hot method of acid fast bacilli (AFB) staining, is a type of differential bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. Acid fast organisms are those which are capable of retaining the primary stain when treated with an acid (fast=holding capacity).

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 Ziehl-Neelsen method of staining for TB is known as the “hot plate” method which means the Ziehl-Neelsen method uses bright field microscopy. To begin the staining process, a bacterial smear must be done. The smear should be evenly spread across the center of the slide. The smear is covered in a piece of bibulous paper and the smear is stained with carbol fuchsin. The slide is heated for five minutes while keeping the bibulous paper moist with the carbol fuchsin. The bibulous paper is removed and the slide is rinsed with distilled water. Acid-alcohol is used to decolorize the slide until the runoff is clear. The decolorizer removes the stain from non-acid-fast cells. The slide is rinsed with distilled water to insure all of the decolorizer is off of the slide. The slide is stained with the counter stain of methylene blue for one minute. Rinse the slide with distilled water. Blot, do not rub, the slide dry in a tablet of bibulous paper. When the slide is dry observe the bacteria under a microscope with oil immersion.


The genus Mycobacterium is a slow growing bacteria, made up of small rods that are slightly curved or straight, and are considered to be gram positive. Some types of Mycobacteria form branches or filaments. Some mycobacteria are free-living saprophytes, but many are pathogens that cause disease in animals and humans. Mycobacterium bovis causes tuberculosis in cattle. Since tuberculosis can be spread to humans, milk is pasteurized to kill any of the bacteria. Some Mycobacteria species that cause disease in humans include Mycobacterium leprae, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium bovis, Mycobacterium africanum and members of the Mycobacterium avium complex. Mycobacterium tuberculosis is a species of Mycobacterium that causes tuberculosis (TB). Mycobacterium tuberculosis is an airborne bacterium that typically infects the human lungs. Symptoms of TB include a bad cough, chest pain, fatigue, weight loss, no appetite, chills, fever and night sweats. The typical regimen for treating a Latent TB infection includes the use of isoniazid, rifapentine, and rifampin. The regimen is changed for those who have developed a drug resistant strain of TB. Testing for TB includes blood testing, skin tests, and chest x-rays. When looking at the smears for TB, it is stained using and acid-fast stain. These 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. 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.


  1. Make a thin smear of the material for study and heat fix by passing the slide 3-4 times through the flame of a Bunsen burner or use a slide warmer at 65-75 C. Do not overheat.
  2. Place the slide on staining rack and pour carbol fuschin over smear and heat gently underside of the slide by passing a flame under the rack until fumes appear (without boiling!). Do not overheat and allow it to stand for 5 minutes.
  3. Rinse smears with water until no color appears in the effluent.
  4. Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until the slide appears light pink in color (15-20 sec).
  5. Wash well with clean water.
  6. Cover the smear with methylene blue or malachite green stain for 1–2 minutes.
  7. Wash off the stain with clean water.
  8. Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot dry).
  9. Examine the smear microscopically, using the 100x oil immersion objective.


  • Acid Fast Bacilli : Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded
  • Cells : Green (malachite green) or Blue (methylene blue)
  • Background material : Green (malachite green) or Blue (methylene blue)


  • Mycobacterium spp: Acid Fast
  • Cyst of Cryptosporidium: Acid Fast
  • Cyst of Isospora: Acid Fast
  • Nocardia spp: Partial Acid Fast
  • Rhodococcus spp: Partial Acid Fast
  • Legionella micdadei: Partially acid fast in tissue


Sandman, Kathleen, Joanne Willey, and Dorothy Wood. Prescott’s Microbiology. 11th ed. New York, NY: McGraw-Hill Higher Education, 2020. Print. p. 541.

Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. 2nd Cambridge University Press, UK. Pp. 178-187.

Willey J.M, Sherwood L.M and Woolverton C.J (2008). Harley and Klein’s Microbiology. 7th ed. McGraw-Hill Higher Education, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

World Health Organization (1993). Laboratory Biosafety Manual, 2nd edn. Geneva: WHO.

World Health Organization (2003). Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens.  WHO/EMC/97.3. Geneva: WHO.

About the author


Leave a Comment