What Does Acid-Fast Stain?
Acid-fast stain (AF) is an important special staining technique used in the histology lab. This is a differential stain used to identify acid-fast bacterial organisms, such as members of the genus Mycobacterium and Nocardia.
The discovery of TB
German scientist and physician, Robert Koch, was a Nobel Laureate in Medicine and a founder of the science of bacteriology. In 1882, his discovery of Mycobacterium tuberculosis, (the bacterial agent responsible for tuberculosis) was a major event in the history of medicine. At this time, he described the appearance of the bacterium as a result of a complex staining procedure which became the original AF stain.
No, not zinc, Ziehl and Neelson…
Although there are various types of AF staining techniques, the Ziehl-Neelson (ZN) stain is used most widely. This technique was first described in the 1800s by two German doctors: bacteriologist Frank Ziehl, and pathologist Friedrich Neelson, both of whom modified Koch’s staining technique in order to further improve on it. It was Ziehl who first used carbolic acid (phenol) as the mordant. Neelsen kept Ziehl’s mordant, but changed the primary stain to the basic fuchsin, and in the mid-1890s, this technique was named the ZN method.
General Principles of Acid-Fast Stain
Acid-fast bacteria have a cell wall consisting of mycolic acid, fatty acids, waxes, and complex lipids. This produces a waxy cell wall that is almost impermeable, and therefore resistant to most compounds- including the routine bacterial special stains, such as the Gram stain. A special staining technique is therefore needed to identify these organisms.
This is where the ZN method comes in:
Primary Stain: Carbol fuchsin is the primary stain in this technique. This is lipid-soluble and contains phenol to help drive the primary stain into the waxy cell walls of these bacteria. Heat may also be used (‘hot staining’) at this stage to soften the cell wall and further help the stain permeate.
Decolorizer: The slide is now rinsed with a strong acid-alcohol decolorizer. This strips the stain from all non-acid-fast cells. However, the acid cannot penetrate the cell wall of acid-fast bacteria (hence the term ‘acid-fast’).
Counterstain: After decolorization, the slide is counterstained with methylene blue. The decolorised non-acid-fast cells are able to take up the methylene blue counterstain, and therefore stain blue. The rod-shaped acid-fast bacteria that resisted decolorisation, however, will stain red-pink, the color of the initial carbol fuchsin stain.
Who Uses AF Staining Methods?
The ZN method continues to remain a reliable and effective way to demonstrate the acid-fast bacteria, and it is used for both research and diagnostic purposes.
Researchers studying acid-fast organisms, such as members of the genus Mycobacterium and Nocardia, will use this staining technique routinely to examine sections to evaluate the bacteria in certain tissues.
The AF stain is also used diagnostically by pathologists to identify these organisms, and is an especially important technique in the diagnosis of tuberculosis. It is also used in the diagnosis of other mycobacterial diseases, including leprosy due to infection with Mycobacterium leprae. Other mycobacterial organisms, the so-called ‘atypical species’, can also cause tuberculosis-like infections, especially in immunosuppressed patients. Mycobacterium avium-intracellulare complex, for example, often causes systemic infections in people with HIV/AIDS.
How is AF used in your lab?
Want to know more about histology? Visit the Bitesize Bio Histology Hub for tips and trick for all your histology experiments.