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The acid-fast stain was described by Franz Ziehl and Friedrich Neelsen. Thus, known as the Ziehl–Neelsen stain. This test is specially used to identify acid fast organism, mainly mycobacterium species. In routine practice this test is used to diagnose Mycobacterium tuberculosis, causative organism of tuberculosis. These bacteria have high amount of mycolic acids in their cell wall, which resists ordinary staining like grams staining. List of organism used to stain by this method is described in table 1.


Sr. No.



Mycobacteria like M. tuberculosisM. lepraeM. smegmatis and typical Mycobacterium


Actinomyces with mycolic acid in well wall like: Nocardia, Rhodococcus, Gordonia (Actinomycete), Tsukamurella, Dietzia


Head ok sperm




Cell inclusion


Cysts of some coccidian parasites in faecal matter like Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis.


Fungal yeast

Table 1: example of organism stained by acid fast stain


A few species of bacteria having high content of mycolic acid in well wall do not readily stain with simple stains. However, heating them with carbolfuchsin will allow dye penetration. The heat allows the stain into the cells. Once the microorganisms stained with carbolfuchsin, they are not easily decolorized by acid-alcohol, and hence are termed acid-fast. This acid-fastness is due to the high lipid content of mycolic acid in the cell wall of these microorganisms. The Ziehl-Neelsen acid-fast staining procedure was developed by Franz Ziehl, a German biochemist, and Friedrich Neelsen, a German pathologist, in the late 1800s. Acid-fast microorganisms will retain this dye and appear red under microscope. Microorganisms that are not stained termed non-acid-fast and appear blue or brown due to the counterstaining with methylene blue after they have been decolorized by the acid-alcohol.

A modified method developed by Joseph Kinyoun in the early 1900s, known as Kinyoun staining procedure. A heating step was replaced by employing wetting agent (Tergitol No. 7) to make sure dye penetration.


1.       Understand the biochemical basis of the acid-fast stain

2.       Perform an acid-fast stain

3.       Differentiate bacteria into acid-fast and non-acidfast groups


1.       ZIEHL’S carbolfuchsin:

Carbolfuchsin prepared with Tergitol No. 4 (a drop per 30 ml of carbolfuchsin)


Carbolfuchsin prepared with Triton-X (2 drops per 100 ml of carbolfuchsin)

2.       Alkaline methylene blue

3.       Acid-alcohol

4.       Clean glass slides

5.       Inoculating loop

6.       Hot plate

7.       Microscope

8.       Blotting paper

9.       Paper towel

10.   Seder wood oil

11.   Staining racks

12.   1-ml pipettes


1.       Take a loop full of culture on clean slide.

2.       Prepare a smear

3.       Allow the smear to air dry and then heat-fix

4.       Place the slide on a water bath

5.       Cover the slide with paper towel

6.       Flood the slide with Ziehl’s carbolfuchsin stain.

7.       Heat the slide 3 to 5 minute (Do not allow the slide to dry out, and avoid excess flooding)

8.       Remove the slide from water bath, cool down, and rinse with water for 30 seconds

9.       Decolorize by treating with acid-alcohol drop by drop until the slide remains only slightly pink ( it requires 10 to 30 seconds and must be done carefully).

10.   Rinse the slide with water (10 to 30 seconds)

11.   Remove excess water by using blotting paper

12.   Alkaline methylene blue for about 2 minutes to counter stain

13.   Rinse with water (30 seconds)

14.   Remove excess water by using blotting paper

15.   Examine the slide under oil immersion lens