Thursday, 9 June 2011

Ziehl–Neelsen Stain For Acid-Fast Organisms – Method and Tips

The Ziehl–Neelsen (ZN) stain is a common standard stain which is readily performed in a majority of histopathology laboratories around the world. It was first described by Dr. Franz Ziehl and Dr Friedrich Neelsen, a German bacteriologist and a German pathologist respectively. The ZN stain is mostly used to identify acid-fast mycobacteria, the most important of which is Mycobacterium Tuberculosis, the organism responsible for tuberculosis (TB). The ZN stain also stains other organisms such as Nocardia.
As Mycobacterium are unable to be visualised on standard haematoxylin and eosin (H+E) and gram stains, the ZN stain was developed. It is based on the tubercle bacilli having a lipid-rich cell wall that takes up phenol-dye solutions (eg. carbol fuchsin, the main dye used in the ZN stain) and after subsequent differentiation, retains the phenol-dye.

Below is the method used by this author.


Carbol fuchsin – (1g basic fuchsin in 10ml ethanol) + (5g phenol in 100ml distilled water), then filter.

Methylene Blue – 0.2% methylene blue


1. Take sections to water.

2. Cover section with filtered carbol fuchsin for 20 minutes.

3. Wash well in tap water.

4. Differentiate in 1% acid alcohol until section is a very pale pink.

5. Wash well in tap water.

6. Stain with methylene blue for 1 minute.

7. Dehydrate, clear and mount.


- Before covering section with carbol fuchsin try covering the section with a little filter paper to reduce precipitate on the slide.

- Some methods still say to the slide to steaming temperature after covering it with carbol fuchsin. This author has found this of no use and is an unnecessary extra step, plus removes the hazard of using a naked flame.

- Before differentiation with acid alcohol wash slide with 70% alcohol for about 1 minute to remove a majority of the stain. This will reduce your differentiation time.

- Blot dry your slide after washing in water after the methylene blue counterstain. This will reduce your dehydration time and therefore result in less leaching of the methylene blue counterstain from the section.

- Some tap water contaminants have been described that stain with carbol fuchsin and are resistant to differentiation. These appear on a different focal plane to true acid-fast organisms within the section.

I welcome any other tips and comments.

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