Home » Microbiology » Ziehl Neelsen Staining -Principle, Procedure and Interpretations

Ziehl Neelsen Staining -Principle, Procedure and Interpretations


This procedure is used to stain mycobacterium tuberculosis and mycobacterium leprae. These bacteria are also called acid fast bacilli. They stain with carbol fuschin, which is a red dye. They retain the dye when treated with acid, which is because of the presence of mycolic acid in their cell wall.

ziel neelson stainingReagents
  1. Carbol fuschin (basic dye)
  2. Mordant (heat)
  3. 20% sulphuric acid (decolorizer)
  4. Methylene blue (counter stain) or Malachite green
  1. Fix the smear of the specimen over the glass slide, either by heating or alcohol fixation.
  2. Pour carbol fuschin over smear and heat gently until fumes appear. Do not overheat and allow it to stand for 5 minutes, then wash it off with water.
  3. Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until the slide appears light pink in color. Wash off with water.
  4. Pour methylene blue, wait for two minutes, again wash with water
  5. Allow it to air dry and examine under oil immersion lens.

Acid fast bacilli stain pink, straight or slightly curved rods, at times having beaded appearance. The background appears blue due to methylene blue.


If definite bacilli are seen, report as AFB positive. However, it is better to report the result quantitatively as follows:

  • > 10 AFB/high power field                 –>+++
  • 1-10 AFB/high power field                 –> ++
  • 10-100 AFB/100 high power fields  –> +
  • 1-9 AFB/100 high power fields         –> exact number

However if no AFB is seen, write the result as ‘no AFB seen’ and never write negative.


a.      Mycobacterium leprae

Same method is used with 5% sulphuric acid due to less mycolic acid in cell wall.

b.      Nocardia and Legionella

1% sulphuric acid is used

c.       Stool Specimen

We use 3% acid alcohol instead of sulphuric acid so as to stain Cryptosporidium parvum.

Hot and Cold Ziehl Neelsen Staining

Hot ZN stain is the usual method in which we heat the smear to enhance the dye penetration.

Whereas in cold ZN staining, instead of heating, we increase the concentration of the basic dye and phenol and incorporate a wetting agent chemical.

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  1. Thanks i salute you guys. Here there is a rising problem , in ZN staining (AFB) . With consideration of factors that are not inevitable by nature like climate change . The variation of climatic change is the one of the most cause of many bias over in ZN staining of AFB. This is leading to many deaths world wide i therefore request a good set up of the stain at both (National)high level and (district) low level.

  2. thanks very much, i really liked the information on the web site, my concern is that, when we are staining the smear suspecting leprae, what is the concentration of the stains, thanks

  3. Moses Hakizimfura

    Thank you very much for ur contribution in healthy . From Dr . Moses

  4. Moses Hakizimfura

    I am doing the research !

  5. Moses Hakizimfura

    As a medical student .

  6. A.C.Ayyappan.,DMLT.,

    Thank you sir , that is very very useful for all lab technician.Kindly inform to anothar microbiological techniq…..

  7. Increasing the carbol fuschin staining time to 25 mins is also used for the Cold ZN staining.
    Can I have the actual concentration of the carbol fuschin and example of the wet chemical agent

  8. It is terrific, makes learning easier.

  9. Note.. for mycobacteria Tuberculosis, 3percent acid alcohol is used, while for mycobacteria leprea, 1percent acid alcohol is used.

  10. Very good information

  11. keerthi balakrishnan

    thank u.. So much fr vry detailed info abt z.n method

  12. thank u so much becoz of this
    my concept is very much clear thank u once again

  13. Please send the all staining metho and interpretation In my mail address.

  14. Thank you so much for this wonderful information. It really made me to understand the details.

  15. Iam a nursing student first yr the results have helped me alot

  16. Thanks for finally talking about > %blog_title% < Loved it!

  17. what about non acid fast?

  18. thanks for the explanation ,it has help me out on my final applied clinical labaratory exam …..thanks again…..

  19. Harunah Ssenyange

    i do appreciate that really

  20. thank you for infermation

  21. Thanks I got clear and cheaper way of understanding.

  22. ibrahim sanusi

    oh!! Thank’s you very murch sir on saering this knowlegw.this is a nice idear

  23. cyprian roman chamwanga

    Thanks for your short understandable lecture

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