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History

In 1882 Robert Koch reported the discovery of the tubercle bacillus (4) and described the appearance of the bacilli resulting from a complex staining procedure.  During  the same time period several other researchers (Ehrlich, Ziehl, Rindfleisch, and Neelsen), intending to improve on Koch’s method, introduced modifications to the reagents and the procedure.  Franz Ziehl was the first to use carbolic acid (phenol) as the mordant.  Friedrich Neelsen kept Ziehl’s mordant, but changed the primary stain to the basic fuchsin (first used by Ehrlich in 1882).  This method became known as the Ziehl-Neelsen method in the early to mid 1890s.  In this method heat is used to help drive the primary stain into the waxy cell walls of these difficult-to-stain cells.  The use of heat in this method has been the reason that this technique is called the “hot staining” method.

The Ziehl-Neelsen method has endured as a reliable and effective way to demonstrate the acid-fast bacteria. 

In 1915, Kinyoun published a method that has become known as the “cold staining” method because the heating step was removed in favor of using a higher concentration of the carbolfuchsin primary stain. 

Purpose 

The acid-fast stain is performed on samples to demonstrate the characteristic of  acid fastness in certain bacteria and the cysts of Cryptosporidium and Isospora.  Clinically, the most important application is to detect Mycobacterium tuberculosis in sputum samples to confirm or rule out a diagnosis of tuberculosis in patients.

Theory 

There are three common acid-fast staining methods, Ziehl-Neelsen (hot), Kinyoun (cold), and Auramine-Rhodamine Fluorochrome (Truant method).  The emphasis in this Atlas-Protocol project will be on the Ziehl-Neelsen and the Kinyoun methods because the slides produced by these methods can be visualized using a standard bright-field microscope.  The fluorochrome method is used by large laboratories that have a fluorescent (ultraviolet) microscope.  For comparison purposes, the recipe for the reagents and the protocol for all three methods are included below, but images for the fluorochrome method will not be a part of the Atlas at this time. 

Many bacterial cells are easily stained with simple stains or using the Gram stain.  A few types of bacteria, such as the mycobacteria and Nocardia species, do not stain using these techniques or, if stained, they produce a variable reaction because their walls are not permeable to the rosaniline dyes in common staining regimens (12).  The cell walls of the mycobacteria contain mycolic acids giving the cell walls a high lipid content.  This characteristic is thought to be the reason (5, 10) these bacteria are difficult to stain.  To view these cells in samples staining requires higher concentrations of the dye solution and/or a heating period (4). However, once a stain is introduced into the cell wall, removing it with a decolorizer is even more difficult. The expression “acid fast” is derived from the observation that even with the addition of hydrochloric acid to the alcohol decolorizer, some of the stained cells retain the primary stain (carbolfuchsin).  Cells that release the primary stain (carbolfuchsin) with decolorizing will be visible after the counterstaining step is complete.  Bacteria described as acid fast will appear red when examining specimens using bright-field microscopy.  Non-acid-fast cells and field debris will appear blue.   

Acid fastness is a characteristic that is shared by just a few organisms, so staining to determine if organisms possess this trait is useful in microbial identification schemes.