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Ziehl Neelsen Staining - Principle, Procedure and Interpretations - HowMed
Ziehl Neelsen Staining - Principle, Procedure and Interpretations - HowMed
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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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