ACID FAST STAINING or ZIEHL NEELSEN STAINING

 Acid fast staining was discovered by Paul Ehrlich and was then modified by Ziehl and Neelsen, which is in common use now.

Acid fast stain helps us to identify Acid-fast bacteria's - bacteria that resist decolourisation with acids due to presence of Mycolic acid in it's cell wall e.g : Mycobacterium tuberculosis.

REAGENTS:

Primary stain - 1%  Carbol fuchsin.

Decolourizer - Sulfuric acid.

Counter stain - Methylene blue.

SMEAR PREPARATION:

• Take a new clean grease free scratch free slide and mark a area of 2x3 cm size using glass cutter.
• Make smear from the yellow purulent portion of the sputum (smear should not be too thick or too thin).  
• Air dry the smear for 15-30 mins and heat fix the smear by passing over bunsen burner for 3-5 times.

STAINING PROCEDURE:

Primary stain

Flood the slide with 1% carbol fuchsin for 5 mins with intermittent heating until vapor rises (helps in penetration of stain) and then rinse with tap water.

Decolourization

Add 25% sulfuric acid over the slide and allow it to stand for 2-4 mins and wash with tap water.

Counter stain

Add 0.1% Methylene blue for 30 secs.

 Now observe under oil immersion(100x).

OBSERVATION:

Tubercle bacilli - appears long, slender, straight or slightly curved Red colored bacilli.

Other non-acid fast bacteria's, pus cells, epithelial cells - appear in Blue color.

REFERENCES:

1. Apurba sastry essentials of medical microbiology - 3rd edn.

2. Koneman's color atlas & textbook of diagnostic microbiology - 7th edn.

3. Ananthanarayan and Paniker's textbook of microbiology.