Acid-Fast Staining Techniques

The purpose of acid-fast staining is to separate or differentiate acid-fast organisms from non-acid-fast organisms. These acid-fast organisms do not respond to dye like regular organisms, and as such can be difficult to observe under a microscope. Acid-fast staining can dye these organisms for observation for medical testing purposes, such as determining the levels of certain bacteria and microorganisms in HIV patients. Mycobacterium and nocardia are two specific examples of acid-fast organisms that are generally tested for in acid-fast staining tests.

Procedures

• Most bacteria are stained with a dye called gram stain dye or ethanol, in order to be observed under a microscope. Acid-fast cells, such as mycobacterial cells, resist staining with contention dye due to the lipid content in the cell wall. As a result, a special procedure must be done which dyes these difficult-to-stain cells. There are three different techniques that apply stain to these acid-fast bacteria which are actually able to change the pigment of the cells, despite the resistance to dye. These three procedures are called Ziehl-Neelson staining, Kinyoun staining and fluorochrome staining. These methods of staining create a more permanent pigmentation, and the acid-fast bacteria remain dyed for a longer period of time than cells dyed with traditional dye, once the procedure has been completed.

Ziehl-Neelson Staining

• Ziehl-Neelson staining is distinct from other forms of staining because it uses heat to stain the acid-fast bacteria. Ziehl-Neelson staining is a two-step technique. A bacterial sample is taken and saturated with a carbol-fuchsin dye in the first step of the technique. This slide, along with the carbol-fuchsin dye, is then heated. During the heating process, the acid-fast bacteria loses its color and takes on the color of the carbol-fuschin dye (usually a red color). Once this is completed, a methylene blue substance is applied. The methylene blue substance does not bond to the acid-fast bacteria because of the lipids in the cell wall, but it does bond to the other organisms on the slide. As a result, two different colors are present: the acid-fast bacteria is stained red and the other organisms blue. The two organisms can now be distinguished by their color when studied under a microscope.

Kinyoun Method

• The Kinyoun method does not utilize heat to stain the acid-fast bacteria. Instead, this technique simply uses a much stronger and more concentrated fusion dye. This concentrated dye bonds only to acid-fast bacteria, while a different-color dye is applied that bonds only to the non-acid-fast organisms. Again, two different colors result, allowing the acid-fast bacteria and organisms to be distinguished from the non-acid-fast organisms under a microscope.

Fluorochrome Stain

• The final method, fluorochrome stain, uses a different technique of reading the results. Two types of stain are again applied under this technique, resulting in different-colored acid-fast and non-acid-fast organisms. However, the dye applied to the acid-fast organisms is a fluorochrome stain, which is fluorescent. A special fluorescent microscope is then used to read the results, which can identify the fluorescent dye that stained only the acid-fast organisms.

When is Acid-Fast Staining Done?

• The acid-fast staining technique is used in testing for diseases caused by mycobacteria. Mycobacteria cause infections which have tuberculosislike symptoms. Typically, these infections appear only in people with weakened immune systems, such as HIV or AIDS patients.