ACID FAST STAINING

Acid fast staining is a differential staining technique that differentiates between acid
fast and non-acid fast bacteria and serve as an important tool in the diagnosis of
tuberculosis. Certain bacterial genera (such as Mycobacterium and Nocardia) have a
gram positive cell wall structure but in addition contain a waxy layer of glycolipids
and fatty acids (mycolic acid) in their cell walls.

Because of these lipid contents such bacteria are difficult to Gram stain as
ordinary dye solutions do not penetrate the cell wall. For staining of acid fast bacteria
a solution containing carbol fuchsin in combination with phenol is used. For
penetration of dye into the cell wall, heating of the flooded smear is necessary. After
penetration of the primary stain, acid fast bacteria resist decolourization with acid or
acid alcohol due to a complex formation between carbol fuchsin and mycolic acid.
Non-acid fast bacteria thus take the colour of counter stain as they are decolourized
by acid or acid alcohol.

REQUIREMENTS

1. Glass slide.
2. Wire loop.
3. Saline (0.85%).
4. Ziehl-Neelsen carbol fuchsin (Z.N.C.F) (primary stain).
5. Acid alcohol [conc. hydrochloric acid = 3 ml; ethanol (95%) = 97 ml].
6. Methylene blue or crystal violet or malachite green (secondary stain).
7. Spirit lamp.
8. Ceedar wood oil.
9. Bacterial culture of Mycobacterium tuberculosis or sputum of a patient
suffering from tuberculosis.

PROCEDURE (Ziehl-Neelsen Method)

1. Prepare a smear on a clean glass slide, dry it in air and fix it by heat.
2. Flood the slide with ZNCF stain and heat constantly for at least 5 minutes
over a spirit lamp. Do not boil and do not allow the stain to dry over the slide.
Pour a little more of ZNCF if needed.
3. Throw the stain and wash the slide with water.
4. Decolourize the smear with acid alcohol for 15 30 seconds and then wash
the slide.
5. Apply the counter stain for 1 2 minutes.
6. Wash and air-dry the smear and observe under oil-immersion lens of the
microscope. M. tuberculosis will appear as thin red bacilli while non-acid fast
bacteria will appear according to the colour of counter stain.
REPORT
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