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Acid-fast stain (Ziehl-Neelsen stain) Principle, Procedure and Results MSc. Sarah Ahmed

Acid-fast stain (Ziehl-Neelsen stain)

Members of the genus mycobacterium are resistant to simple and gram staining procedure due to their thick waxy (fatty) cell wall which makes the penetration of dyes extremely difficult and requires the application of heat. But once the dye has penetrated, it can not be removed even with the vigorous use of acid alcohol as decolourizing agent, due to this property, these organisms are called acid fast.

Principle

**The stain binds to the mycobacterial cell wall. ** After staining, an acid decolorizing solution is applied. This removes the red dye from the background cells, tissue fibres, and any organisms in the smear except mycobacteria which keep the dye and are therefore referred to as acid fast bacilli (AFB) ** Following decolorization, sputum smear is counterstained with malachite green, or methylene blue which stains the background material, providing a contrast colour against the red AFB which can be seen.

Acid-fast stain procedure

Prepare a smear of the microorganism on a clean slide. Allow the smear to air-dry then fix it by flaming. Place the slide over a steam bath and flood the smear with strong carbol fuchsin and keep it for 5 mins. caution :don,t allow stain to dry; replenish stain as needed. also, prevent stain from boiling by adjusting the temperature applied. Allow to cool and then wash with tap water. Decolourize with acid alcohol, till the effluent runs almost clear with a slight red tinge.

Acid-fast stain procedure

Wash with tap water Counterstain with methylene blue for 2 mins. Wash smear with tap water. Blot dry and examine with a microscope.
Acid fast bacteria appear red while non acid fast bacteria appear blue blue.

Reagent

Acid Fast
Non-Acid Fast
Carbol Fuchsin with heat
Red (Hot Pink)
Red (Hot Pink)
Acid Alcohol
Red
Colorless
Methylene Blue/Malachite Green
Red
Blue/Green
Results

THE END