Modified acid-fast staining principles are, that when mycobacteria and Nocardia species stain with carbol fuchsin, they stained and resist the decolorizing acid. But other bacteria decolorized by the acid. Therefore Mycobacteria and Nocardia species are called ACID FAST bacteria (AFB).

Ziehl-Neelsen stain procedure

Following are the 4 steps in acid-fast staining.

  1. First, cover the smear on the slide with Carbol fuchsin and start heating until the steam rises. Allow the smear + stain for 5 minutes. After 5 minutes wash the slide.
  2. Cover the slide with 20% Sulphuric acid (H2SO4). The red color of the smear changed into a yellowish-brown.
  3. Wash the slide. Repeat step 2. Until the film is only faintly pink in color.
  4. Counterstain with methylene blue for 20 to 25 seconds. Wash the slide, dry in the air.

Why ziehl-neelsen stain (ZN staining) is called acid fast?

In the ziehl Neilsen stain, we use strong acids such as sulphuric acid and Ethanol as the decolorizing agents. When bacteria retain their color even after reacting with sulphuric acid, Such bacteria are called ACID-FAST bacteria and the stain is called an ACID-FAST stain.

Mycobacterium Leprae is called a Less acid-fast bacteria because it is decolorized by the 5% concentration of sulphuric acid (H2S04). Similarly Legionella species are also less acid-fast bacteria as they decolorized by 1% concentration of acid.

What stain is used for acid-fast bacteria?

Modified acid-fast stain or ziehl-Nielsen stain use for acid-fast bacteria like mycobacteria, Nocardia. These acid-fast bacteria retain their color even after reacting with decolorization with Sulphuric acid.

What color indicates a positive result in the acid-fast stain?

  1. Acid-fast bacteria appear in RED color as a positive result.
  2. Background in blue color.
  3. Other cells’ color is blue or green.

Acid-fast (AFB) stain results

It is better to report AFB quantitatively when acid-fast bacteria are found.

what is the Bacteria Index in AFB

1 to 2 bacteria seen on complete smearDoubtful repeat the Test
3 to 9 AFB seen on the entire smearRare (+1)
=10b AFB seen on the entire smearfew (+2)
=1 AFB per oil immersion Numerous (+3)
ziehl-neelsen stain

Ziehl-neelsen stain control

Two sputum smears having high and low Acid-fast bacilli should be stained with the test batch. In this way can check and interpret the result correctly.

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