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Acid-Fast Staining Tutorial Send Print

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Created: Friday, 03 May 2002
Last update: Wednesday, 25 August 2010
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The acid fast stain is a technique that differentiates Mycobacterium species from other bacteria. Nocardia species may also fall under the category of "acid-fast."

Acid fast organisms generally do not stain well with dyes, including the Gram stain dyes. Prolonged application of more concentrated dyes, facilitated by heat (Ziehl-Neelson stain) or lipid solvent (Kinyoun) treatment, are used to stain the mycobacteria. Once stained, acid fast bacteria are able to retain stains that are more easily removed in other bacteria using acidic ethanol. This trait gives them their designation as "acid fast." The high lipid content of the mycobacterial cell wall is probably responsible for the weak initial staining and the subsequent strong retention following prolonged staining.

In the classic Ziehl-Neelson technique of acid-fast staining, the smear is flooded with carbol-fuchsin dye, heated, and then decolorized. The non-acid fast organisms are then counterstained with methylene blue. Acid-fast organism will appear red due to the retention of the carbol-fuchsin while the background and any non-acid-fast organisms will appear blue due to the methylene blue counterstain. The Kinyoun method is similar but heat treatment is not necessary because a more concentrated fuchsin dye is used. Both methods are seen in the animation.

A third type of acid-fast stain is the fluorochrome stain which utilizes auramine, a fluorescent dye. This stain uses the same principle as the above stains, but visualization is via fluorescent microscopy.

This "animated" (with appropriate and/or inappropriate sounds) PowerPoint presentation can be utilized in the classroom to provide:
1. An introduction (brief history and structural basis) to the Acid-Fast Stain.
2. Stepwise instructions for the techniques.
3. "Cartoons" of how the cells appear at each step.
4. Views of typical Acid-Fast morphologies.

Please note: The tutorial above is optimized for Microsoft Internet Explorer and will not work in Netscape Navigator. In IE, upon clicking on the link to the tutorial above, you will see frames containing the outline and the slide show. You may use the outline on the left-hand side to scroll through the slide show. In the lower right-hand corner of your screen there is a "Full slide show" icon. If you click on this, you will be able to view the full slide show with sound effects. Use the space bar to advance the slides, use the ESC key to exit the slide show at any time.
For Netscape Navigator users: Please use this alternate link to view a non-optimized version of the tutorial. Please be aware that you will not be able to see the tutorial as full slide show with animations, but you will be able to click through the slides using the list on the left-hand side of your screen.
Acid-Fast Tutorial for Netscape

Legend written by:
Kristen Catlin
American Society for Microbiology
Washington D.C. 20036

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