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Acid Fast Stain

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The most common acid fast staining method is Ziehl-Neelsen Staining method, it was described by two German doctors Franz Ziehl, a bacteriologist and Friedrich Neelsen, a pathologist. this method is used to identify acid fast organisms like Mycobacterium.

Principle :- The lipoid capsule of the acid fast organism are of high molecular weight and remain waxy in room temperature so normal aqueous stain are unable to stain the cell. So in this method Carbolfuchsin are heat steamed and so that the lipoid capsule of the organism takes up the carbolfuchsin.

Technique :-1. Prepare a smears of organisms to be stained.
                    2. Heat fix the smears.
                    3. Cut a absorbant paper to fit the slide leaving one end for handling. Do not allow the paper to               protude beyond the slide,but smears must be covered.
                    4. place the slide on wire gauze on a ring stand.
                    5. saturate the paper with carbolfuschin.
                    6. heat the slide for 5 minutes by passing the slide through the flame of a gas burner.
                    7. Allow the slide to cool and was with water by removing the paper.
                    8. Drain the slide.
                    9. Decolorize with acid alcohol for 30 sec.
                    10. Rinse , Drain and counterstain with methylene blue for 45 sec - 1 min.
                    11. Rinse , blot and examine under microscope.
                    12. Acid Fast microorganism will appear RED and Non-Acid-Fast will be BLUE. 
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