ZN Staining


This method is a modification of Ehrlich’s (1882) original method for the differential staining of tubercle bacilli and other acid-fast bacilli with aniline gentian violet followed by strong nitric acid. It incorporates improvements suggested successively by Ziehl and Neelsen.



The ordinary aniline dye solutions do not readily penetrate the substance of the tubercle bacilli and therefore unsuitable for staining it. However by the use of a powerful staining solution that contains phenol, and the application of heat, which act as a mordant and the dye can be made to penetrate the bacillus. Once stained the tubercle bacillus will withstand the action of powerful decolorizing agents for a considerable time and thus still retains the stain when everything else in the microscopic preparation has been decolorized.



  1. Make a smear on clean slide, dry and fix by flaming.
  2. Cover the slide with filtered basic carbol fuchsin.
  3. Heat the slide until steam rises, but without boiling. Allow the preparation to stain for 5 minutes, applying the heat at intervals to keep the stain hot. Do not allow the stain to dry on the slide, if necessary pour on more carbol fuchsin to cover it.
  4. Wash both sides of the slide with water from the tap.
  5. Cover the slide with 25 % sulphuric acid. The red color of preparation is changed to yellow brown. After 3 minute, wash the slide with water, and pour on fresh acid. Repeat this procedure for total 3 times. Decolorization generally requires contact with sulphuric acid for a total time of at least 10 minutes. When it is complete, the film, after washing is only very faintly pink.
  6. Wash the slide well with water to remove all traces of acid.
  7. Counterstain with Loeffler’s methylene blue for 15-20 seconds.
  8. Wash, blot with clean paper, dry and mount.



ZN Stain for Tuberculosis

Acid-fast bacilli are stained bright red, while other organisms, tissue cells and debris are stained blue.


Acid Fastness:

  1. Acid fast bacteria are rich in lipids, fatty acids & higher alcohol. Acid fastness is mainly due to the presence of unsaponifiable wax of the nature of hydroxyl acid known as mycolic acid.
  2. Acid fastness is also depends on the structural integrity of the cell because when the cell is ruptured by mechanical means or autolysis, its acid fastness is lost.
  3. Acid fastness is lost due to exposure to INH-drug.



ZN Stain for Leprae
ZN Stain for Leprae
  1. ZN staining for M. leprae: M. leprae is less acid-fast than M. tuberculosis. In this modification for M. leprae procedure is same except 5% sulphuric acid is used as a decolorizer instead of 25 % used in Z-N stain for M. tuberculosis.
  2. Kinyoun stain(cold staining): This modification of Z-N stain is used for staining of parasitic cysts from stool specimens e.g. Cryptosporidium and Cylclospora. Here heat is not applied while 0.5 – 1% sulphuric acid is used.



Acid-fast Organisms:

  • Mycobacterium tuberculosis
  • Mycobacterium leprae
  • Atypical mycobacteria
  • Nocardia
  • Spores of certain parasites. Like:
    • Cryptosporidium,
    • Cyclospora,
    • Isospora


Sputum Grading: (RNTCP)

Bacilli Result Grading No. of fields examined
>  10 AFB / OIF Positive 3+ 20
1-10 AFB / OIF Positive 2+ 50
10-99 AFB / OIF Positive 1+ 100
1-9 AFB / OIF Scanty Record actual number 100
No AFB / OIF Negative 0 100

OIF = Oil Immersion Field