TheZiehl-Neelsen stain, also known as theacid-fast stain, is a bacteriologicalstaining technique used incytopathology andmicrobiology to identifyacid-fast bacteria undermicroscopy, particularly members of theMycobacterium genus. This staining method was initially introduced byPaul Ehrlich (1854–1915) and subsequently modified by the German bacteriologistsFranz Ziehl (1859–1926) andFriedrich Neelsen (1854–1898) during the late 19th century.
The acid-fast staining method, in conjunction withauramine phenol staining, serves as the standard diagnostic tool and is widely accessible for rapidly diagnosingtuberculosis (caused byMycobacterium tuberculosis) and other diseases caused byatypical mycobacteria, such asleprosy (caused byMycobacterium leprae) andMycobacterium avium-intracellulare infection (caused byMycobacterium avium complex) in samples likesputum, gastric washing fluid, andbronchoalveolar lavage fluid. These acid-fast bacteria possess a waxy lipid-rich outer layer that contains high concentrations ofmycolic acid, rendering them resistant to conventional staining techniques like theGram stain.[1][2]
After the Ziehl-Neelsen staining procedure usingcarbol fuchsin, acid-fast bacteria are observable as vivid red or pink rods set against a blue or green background, depending on the specificcounterstain used, such asmethylene blue ormalachite green, respectively. Non-acid-fast bacteria and other cellular structures will be colored by the counterstain, allowing for clear differentiation.[3]
In anatomic pathology specimens,immunohistochemistry and modifications of Ziehl–Neelsen staining (such asFite-Faraco staining) have comparable diagnostic utility in identifyingMycobacterium. Both of them are superior to traditional Ziehl–Neelsen stain.[4]
Mycobacterium are slow-growing rod-shapedbacilli that are slightly curved or straight, and are considered to beGram positive. Some mycobacteria are free-livingsaprophytes, but many arepathogens that cause disease in animals and humans.Mycobacterium bovis causes tuberculosis in cattle. Since tuberculosis can be spread to humans, milk is pasteurized to kill any of the bacteria.[5]Mycobacterium tuberculosis that causestuberculosis (TB) in humans is anairborne bacterium that typically infects the human lungs.[6][7] Testing for TB includes blood testing, skin tests, and chest X-rays.[8] When looking at the smears for TB, it is stained using an acid-fast stain. These acid-fast organisms likeMycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as aGram stain.[9] It can also be used to stain a few other bacteria, such asNocardia. The reagents used for Ziehl–Neelsen staining arecarbol fuchsin, acid alcohol, andmethylene blue. Acid-fast bacilli are bright red after staining.[citation needed]
Ziehl–Neelsen staining is a type of narrow spectrum fungal stain. Narrow spectrum fungal stains are selective, and they can help differentiate and identify fungi.[10] The results of Ziehl–Neelsen staining is variable because many fungal cell walls are not acid fast.[11] An example of a common type of acid-fast fungus that is usually stained with Ziehl–Neelsen staining is calledHistoplasma (HP).[12]Histoplasma is found in soil and the feces of birds and bats.[13] Humans can contracthistoplasmosis by inhalation of the fungal spores.Histoplasma enters the body and goes to the lungs where the spores turn into yeast.[14] The yeast gets into the blood stream and affects lymph nodes and other parts of the body. Usually people do not get sick from inhaling the spores, but if they do they usually have flu like symptoms.[15] Another variation on this staining method is used inmycology to differentially stain acid-fast incrustations in the cuticularhyphae of certain species offungi in the genusRussula.[16][17] Some free endospores can be confused with small yeasts, so staining is used to identify the unknown fungi.[18] It is also useful in the identification of some protozoa, namelyCryptosporidium andIsospora. The Ziehl–Neelsen stain can also hinder diagnosis in the case ofparagonimiasis because the eggs in sputum sample for ovum and parasite (O&P) can be dissolved by the stain[citation needed]
In 1882Robert Koch discovered the etiology of tuberculosis.[19] Soon after Koch's discovery, Paul Ehrlich developed a stain for mycobacterium tuberculosis, called the alum hematoxylin stain.[20] Franz Ziehl then altered Ehrlich's staining technique by using carbolic acid as the mordant. Friedrich Neelsen kept Ziehl's choice of mordant but changed the primary stain to carbol fuchsin. Ziehl and Neelsen's modifications together have developed the Ziehl–Neelsen stain. Another acid-fast stain was developed byJoseph Kinyoun by using the Ziehl–Neelsen staining technique but removing the heating step from the procedure. This new stain from Kinyoun was named theKinyoun stain.[21]
A typical AFB stain procedure involves dropping the cells in suspension onto a slide, then air drying the liquid and heat fixing the cells.[22]
| Summary of acid-fast stain (Ziehl–Neelsen stain)[23] | |||||
|---|---|---|---|---|---|
| Application of | Reagent | Cell colour | |||
| Acid fast | Non-acid fast | ||||
| Primary dye | Carbol fuchsin | Red | Red | ||
| Decolorizer | Acid alcohol | Red | Colorless | ||
| Counter stain | Methylene blue/malachite green | Red | Blue | ||
Studies have shown that an AFB stain without a culture has a poor negative predictive value. An AFB culture should be performed along with an AFB stain; this has a much higher negative predictive value.[citation needed]
The mechanism of action of the Ziehl-Neelsen stain is not completely understood, but it is thought to involve a chemical reaction between the acidic dyes and thecell walls of thebacteria. The acidity of the dyes causes them to bind more strongly to the cell walls of the bacteria than to other cells ortissues. This results in the selective staining of only those cells that have a high density of cell wall material, such as acid-fast bacteria.[27]
The Ziehl-Neelsen stain is a two stepstaining process. In the first step, the tissue is stained with a basic fuchsin solution, which stains all cells pink. In the second step, the tissue is incubated in an acid alcohol solution, which decolorizes all cells except for acid-fast cells, which retain the color and appeared as red. The mechanisms by which this color is produced are not well understood, but it is thought that the interaction of the basicfuchsin with the cell wall components of bacteria creates a new molecule that is responsible for the color.[28]
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