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Ziehl Neelsen Staining -Principle, Procedure And Interpretations
in Microbiology
Principle
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.
Reagents
1. Carbol fuschin (basic dye)
2. Mordant (heat)
3. 20% sulphuric acid (decolorizer)
4. Methylene blue (counter stain) or Malachite
green
Procedure
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.
Result
Acid fast bacilli stain pink, straight or slightly curved rods, at times having beaded appearance. The
background appears blue due to methylene blue.
Interpretations
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.
Modifications
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
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Hot ZN stain is the usual method in which we heat the smear to enhance the dye penetration.
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January 3, 2013 at 7:43 pm

Stephen Thyaka
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.
Reply
October 2, 2013 at 10:23 am
mukite denis
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
Reply
October 24, 2013 at 9:01 am
Moses Hakizimfura
Thank you very much for ur contribution in healthy . From Dr . Moses
Reply
October 24, 2013 at 9:03 am
Moses Hakizimfura
6/11/2014 Ziehl Neelsen Staining -Principle, Procedure and Interpretations | howMed
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October 24, 2013 at 9:03 am
Moses Hakizimfura
As a medical student .
Reply
December 12, 2013 at 11:54 am
charles kuwin
GRAM STAIN PROCEDURE
Reply
December 26, 2013 at 1:23 pm
A.C.Ayyappan.,DMLT.,
Thank you sir , that is very very useful for all lab technician.Kindly inform to anothar microbiological
techniq..
Reply
January 28, 2014 at 12:07 pm
Aboagye Mensah
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
Reply
May 5, 2014 at 9:56 am
andy moses
It is terrific, makes learning easier.
Reply
May 5, 2014 at 10:01 am
andy moses
Note.. for mycobacteria Tuberculosis, 3percent acid alcohol is used, while for mycobacteria leprea,
1percent acid alcohol is used.
Reply
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6/11/2014 Ziehl Neelsen Staining -Principle, Procedure and Interpretations | howMed
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