Acid Fast Staining Procedure Steps

In this acid fast staining procedure steps post we have briefly explained about acid fast staining principle, objectives, requirements, acid fast staining method, uses and limitations.

Acid Fast Staining Method

It was Ziehl who originally devised differential Acid fast staining procedures, which were later refined by Neelsen. As a result, Ziehl-Neelsen staining techniques are sometimes known as Ziehl-Neelsen staining procedures. In 1883, Neelsen utilised Ziehl’s carbol-fuchsin, which he heated before decolorizing with acid alcohol and counterstaining with methylene blue.

The acid fast staining method is a differential stain used to identify acid-fast organisms, such as Mycobacterium species. Acid-fast organisms have waxy, almost impenetrable cell walls and contain huge amounts of fatty acids, waxes, and complex lipids, as well as mycolic acid. Disinfectants and dry environments are particularly resistant to acid-fast organisms.


Acid fast staining method organisms require a specific staining approach due to the cell wall’s resistance to most chemicals. Carbolfuchsin, the principal stain used in acid-fast staining, is lipid-soluble and contains phenol, which aids penetration of the cell wall.

This is aided even more by the presence of heat. The smear is then rinsed with a very strong decolorizer, which removes the stain from all non-acid-fast cells while leaving acid-fast organisms’ cell walls intact. The counterstain is then taken up by the decolorized non-acid-fast cells.


Acid Fast Staining Method Steps

Slide Preparation

To remove any fingerprints, clean the slide with a paper towel and alcohol. On the bottom of the slide, draw two circles with your Marker.

Fill each circle with two little drips of water using your inoculation loop. Remove a little amount of bacteria from the culture tube using aseptic technique. Before and after you enter, make sure to burn the tube.

Smear the bacteria on your slide with a drop of water. Allow the slide to dry completely in the open air. Run the slide through the flame 3-4 times with the ‘smear’ side up to heat-fix it. Allow the slide to cool completely before staining it.

Acid Fast Staining Method Steps

Within the slide’s border, cover the smears with a piece of paper towel. Place the slide over a heating beaker of water. Make sure the beaker doesn’t boil dry.

Allow the slide to steam for 3-5 minutes after soaking the paper towel in carbolfuchsin. Remove the stained paper towel gently.

Rinse the slide gently with water to remove any stray paper towel pieces, and then dry thoroughly. 15-30 seconds of acid-alcohol application.

Rinse well and dry thoroughly. Use methylene blue as a counterstain for 1.5 minutes. Using a towel, wipe dry after rinsing with water. Wipe the area gently with a paper towel. Dry the bottom of the slide before placing it on the microscope stage and viewing it with the oil immersion lens.


Acid fast: Bright red to intensive purple, Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded Non-acid fast: Blue color.


Acid Fast Staining Method, Image Source:


Acid-fast: Mycobacterium tuberculosis, Mycobacterium smegmatis.

Non-Mycobacterial bacteria: Nocardia

Coccidian Parasites: Cryptosporidium

Further Readings