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LAB 4-M 265

Acid Fast Stain

(Ziehl-Neelsen Stain)

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The acid-fast stain is a differential stain used to identify acid-fast organismssuch as members of the genus Mycobacterium .

Acid-fast organisms are characterized by wax-like, nearly impermeable cell

walls; they contain mycolic acid and large amounts of fatty acids, waxes,and complex lipids. Acid-fast organisms are highly resistant to disinfectantsand dry conditions .

Because the cell wall is so resistant to most compounds, acid-fastorganisms require a special staining technique. The primary stain used inacid-fast staining, carbolfuchsin, is lipid-soluble and contains phenol, whichhelps the stain penetrate the cell wall. This is further assisted by theaddition of heat. The smear is then rinsed with a very strong decolorizer,which strips the stain from all non-acid-fast cells but does not permeate thecell wall of acid-fast organisms. The decolorized non-acid-fast cells thentake up the counterstain .

A few genera ,particularly the members ofMycobacterium(M.tuberculosis & M.leprae) and Nocardia sp.only are acid fastbacteria

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The reagents for the Ziehl-Neelsen method:

1.Primary stain: Carbol Fuchsin 2.Decolorizing agent:Acid Alcohol

(3% hcl + 95%Ethanol)

3.Counter stain :Methylene Blue

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1. Prepare smears of organisms to be stained.

2. Heat fix the smears.

3. Cut or tear absorbent paper (bibulous paper) to fit the slide leaving one endfor handling. Do not allow the paper to protrude beyond the slide, but thesmears must be covered.

4. Place the slide on a sink rack. 5. Saturate the paper with carbolfuschin.

6. Heat the slides with a hand-held bunsen burner until steam can be seenrising from the surface. Alternately remove the burner and reheat the slide tomaintain steaming for 3-5 minutes. As the paper begins to dry during thestaining process add a drop or two of carbolfuschin to keep the slidemoist. Adding too much stain will cool the slide (and drip on thebench). Overheating the slide or letting it dry will distort the cells. Underheating the slide will fail to stain acid-fast cells.

7.At the end of staining remove the paper with tweezers and wash the slidethoroughly.

8.Drain the slide.

9.Decolorize with acid-alcohol for 30 seconds.

10. Rinse, drain, and counter stain with methylene blue for1 minute.

11. Rinse, blot, and examine. First observe each organism on its separate

smear. Then examine the mixed smear. 12.Acid-fast organisms will appear red and non-acid-fast organisms will be

blue.

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To the right, a mixed culture has been acid-fast stained.

A. Non Acid-fast bacteria

B. Acid-fast bacteria (red cells)