Acid-Fast Staining

Acid-fast stain is a differential stain. More so, it is a physical property of certain bacteria, particularly a bacteria’s resistance to decolorization by certain acids during staining procedures. Most bacteria are decolorized by the use of acid-alcohol. However, the families of Mycobacteriaceae, Nocardiceae, Gordoniaceae, Dietziaceae, and Tsukamurellacea are acid fast. In 1882 acid fast staining was developed by Paul Ehrlich and was later successfully modified by Ziehl And Neelsen. Ehrlich first discovered that Mycobacterium tuberculosis, which is the main causing agent of tuberculosis, retained the primary stain given even after washing it with an acid-alcohol mixture. Acid fastness in the property of certain bacteria (mycobacterium), their structures (spores) or protozoa forms; resist the decolorization by weak mineral acids following staining by the use of an intense dye. The most commonly known acid fast staining technique is the Ziehl-Neelsen technique. The cell walls of acid-fast organisms contain a wax like lipid called mycolic acid. As a result the cell wall is relatively impermeable to ordinary staining techniques. They can be stained by aniline dyes using drastic measures such as application of heat and phenol. The heat softens the wax in the cell wall, allowing the stain to enter, which is a basic fuchsin. The fuchsin dye is more soluble in phenol than in water or alcohol. Phenol in turn is more soluble in lipids or waxes, thus the dye-phenol mixture enters the cell. Once stained, it resists decolorization by weak mineral acid. This is a result of the phenol-dye mixture being more soluble in waxes of the mycobacterium than the acid of alcohol. This way phenol acts as a mordant. While the mycobacterium retain the primary stain, which is pink, the background material gets decolorized and takes up the counter stain which is methylene blue. The methylene blue is used as a counter stain in order for a person to observe the non-acid-fast organisms. In the Kinyoun modification, which is known as a cold stain, the concentrations of phenol and carbolfuchsin are increased so heating isn’t necessary. There is one major problem that could come about when using this technique. Acid-fast organisms are difficult to characterize using standard microbiological techniques, for example the Gram stain. If an acid-fast bacillus were to be gram-stained, the result would be an abnormal gram-positive organism, which would indicate the need for further testing. Thus the type of staining done to the bacteria is very important and the results can vary drastically.