Ziehl Neelsen Staining Method in Histopathology

Ziehl neelsen staining method in histopathology post we have briefly explained about Ziehl neelsen stain method’s principle, requirements, ziehl neelsen staining technique procedure, result with Label.

Ziehl Neelsen Staining Technique

It is the differential staining techniques which was first developed by Ziehl and later on modified by Neelsen. So this ziehl neelsen staining technique is also called Ziehl-Neelsen staining techniques. Neelsen in 1883 used Ziehl’s carbol-fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl-Neelsen staining techniques were developed.

The main aim of ziehl neelsen staining technique is to differentiate bacteria into acid fast group and non-acid fast groups. This ziehl neelsen staining technique is used for those microorganisms which are not staining by simple or Gram staining method, particularly the member of genus Mycobacterium, are resistant and can only be visualized by acid-fast staining.


Mycobacterial cell walls contain a waxy substance composed of mycolic acids. These are ß-hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms. The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular species (Lyon H 1991).

Basic fuchsin binds to negatively charged groups in bacteria. The mycolic acid (and other cell wall lipids) presents a barrier to dye entry as well as elusion (washing out with solvent) and this is partly overcome by adding a lipophilic agent to a concentrated aqueous solution of basic fuchsin and partly by heating.


Staining solution

Stock Solution A: Dissolve 0.6 ml of L.O.C. High Suds (Amway) with 100ml of double distilled water.

Stock Solution B: Dissolve 1 gm of Basic fuchsin with 10 ml of Absolute ethyl alcohol. The two solutions can be kept as stock solution and mixed before use.

Working Solution: Mix 50ml of Stock Solution A with 5ml of Stock Solution B, this solution for 1 month.

3% Hydrochloric Acid

3% hydrochloric acid in 95% ethyl alcohol: Mix 95ml Absolute ethyl alcohol,  2 ml Distilled water, 3 ml Concentrated hydrochloric acid. Make up the alcohol solution then add the concentrated acid. Use extreme care when handling concentrated acid.

0.25% Methylene Blue

0.25% methylene blue in 1% acetic acid: Mix 0.25 gm Methylene blue, and 1 ml glacial Acetic acid with 99 ml double distilled water.


1. Place the working solution in a coplin jar and pre-heat for 10 minutes in a 58-60oC water bath. Deparaffinize sections and immerse in water.

2. 15 minutes staining in a pre-heated working solution in a water bath 2 minutes, immerse the coplin jar containing the slides in cold running tap water.

3. Wash the slides in running water for 1 minute after removing them from the coplin jar. Differentiate in 3% hydrochloric acid in 95% ethyl alcohol until no colour runs from the slide.

4. To remove the acid alcohol, wash briefly in water. 15 to 30 seconds, counterstain with 0.25 percent methylene blue in 1 percent acetic acid. Wash, dehydrate, clear, and mount in DPX.


Ziehl Neelsen Staining

Ziehl Neelsen Staining Technique Result

Acid fast bacilli: Red

Nuclei: Blue

Other tissue constituents: Blue


1. Ziehl neelsen staining technique used for examination and identification of Mycobacterium

2. Ziehl neelsen staining technique used to differentiate between acid-fast and non-acid fast bacilli

3. Ziehl neelsen staining technique used for the identification of some fungal species such as Cryptosporidium.

Further Readings