Ziehl–Neelsen stain
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The Ziehl-Neelsen stain, also known as the acid-fast stain, is a bacteriological
staining Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology (microscopic study of biological tissues), in cytology (microscopic study of cells), and in the ...
technique used in cytopathology and
microbiology Microbiology () is the scientific study of microorganisms, those being unicellular (single cell), multicellular (cell colony), or acellular (lacking cells). Microbiology encompasses numerous sub-disciplines including virology, bacteriology, prot ...
to identify acid-fast bacteria under
microscopy Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of micr ...
, particularly members of the ''
Mycobacterium ''Mycobacterium'' is a genus of over 190 species in the phylum Actinomycetota, assigned its own family, Mycobacteriaceae. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis ('' M. tuberculosis'') and ...
'' genus. This staining method was initially introduced by Paul Ehrlich (1854–1915) and subsequently modified by the German bacteriologists
Franz Ziehl Franz Ziehl (13 April 1857 in Wismar – 7 April 1926) was a German bacteriologist. He was a professor in Lübeck. Franz Ziehl introduced the carbol fuchsin stain for the tubercle bacillus in 1882. With pathologist Friedrich Neelsen (1854–1898 ...
(1859–1926) and
Friedrich Neelsen Friedrich Carl Adolf Neelsen (March 29, 1854, Uetersen – April 11, 1894, Dresden) was a German pathologist. Life Friedrich C.A. Neelsen was born to Hans Friedrich Neelsen, deacon of the Uetersen vicarage, and his wife Bertha Sophia (née Lueders ...
(1854–1898) during the late 19th century. The acid-fast staining method, in conjunction with auramine phenol staining, serves as the standard diagnostic tool and is widely accessible for rapidly diagnosing tuberculosis (caused by ''
Mycobacterium tuberculosis ''Mycobacterium tuberculosis'' (M. tb) is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. First discovered in 1882 by Robert Koch, ''M. tuberculosis'' has an unusual, waxy coating on its c ...
'') and other diseases caused by atypical mycobacteria, such as leprosy (caused by '' Mycobacterium leprae'') and ''Mycobacterium avium-intracellulare'' infection (caused by ''Mycobacterium avium'' complex) in samples like
sputum Sputum is mucus that is coughed up from the lower airways (the trachea and bronchi). In medicine, sputum samples are usually used for a naked eye examination, microbiological investigation of respiratory infections and cytological investigations ...
, gastric washing fluid, and
bronchoalveolar lavage fluid Bronchoalveolar lavage (BAL) (also known as bronchoalveolar washing) is a diagnostic method of the lower respiratory system in which a bronchoscope is passed through the mouth or nose into an appropriate airway in the lungs, with a measured amoun ...
. These acid-fast bacteria possess a waxy lipid-rich outer layer that contains high concentrations of mycolic acid, rendering them resistant to conventional staining techniques like the Gram stain. After the Ziehl-Neelsen staining procedure using carbol fuchsin, acid-fast bacteria are observable as vivid red or pink rods set against a blue or green background, depending on the specific
counterstain A counterstain is a stain with colour contrasting to the principal stain, making the stained structure easily visible using a microscope. Examples include the malachite green counterstain to the fuchsine stain in the Gimenez staining technique a ...
used, such as
methylene blue Methylthioninium chloride, commonly called methylene blue, is a salt used as a dye and as a medication. Methylene blue is a thiazine dye. As a medication, it is mainly used to treat methemoglobinemia by converting the ferric iron in hemoglobin ...
or malachite green, respectively. Non-acid-fast bacteria and other cellular structures will be colored by the counterstain, allowing for clear differentiation.


Mycobacteria

In anatomic pathology specimens,
immunohistochemistry Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to an ...
and modifications of Ziehl–Neelsen staining (such as Fite-Faraco staining) have comparable diagnostic utility in identifying ''Mycobacterium''. Both of them are superior to traditional Ziehl–Neelsen stain. ''Mycobacterium'' are slow-growing rod-shaped bacilli that are slightly curved or straight, and are considered to be
Gram positive In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall. Gram-positive bact ...
. Some mycobacteria are free-living saprophytes, but many are pathogens that cause disease in animals and humans. ''
Mycobacterium bovis ''Mycobacterium bovis'' is a slow-growing (16- to 20-hour generation time) aerobic bacterium and the causative agent of tuberculosis in cattle (known as bovine TB). It is related to ''Mycobacterium tuberculosis'', the bacterium which causes tube ...
'' causes tuberculosis in cattle. Since tuberculosis can be spread to humans, milk is pasteurized to kill any of the bacteria. ''
Mycobacterium tuberculosis ''Mycobacterium tuberculosis'' (M. tb) is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. First discovered in 1882 by Robert Koch, ''M. tuberculosis'' has an unusual, waxy coating on its c ...
'' that causes tuberculosis (TB) in humans is an
airborne Airborne or Airborn may refer to: Arts, entertainment, and media Films * ''Airborne'' (1962 film), a 1962 American film directed by James Landis * ''Airborne'' (1993 film), a comedy–drama film * ''Airborne'' (1998 film), an action film sta ...
bacterium that typically infects the human lungs. Testing for TB includes blood testing, skin tests, and chest X-rays. When looking at the smears for TB, it is stained using an acid-fast stain. These acid-fast organisms like ''Mycobacterium'' contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stain. It can also be used to stain a few other bacteria, such as ''
Nocardia ''Nocardia'' is a genus of weakly staining Gram-positive, catalase-positive, rod-shaped bacteria. It forms partially acid-fast beaded branching filaments (acting as fungi, but being truly bacteria). It contains a total of 85 species. Some spec ...
''. The reagents used for Ziehl–Neelsen staining are carbol fuchsin, acid alcohol, and
methylene blue Methylthioninium chloride, commonly called methylene blue, is a salt used as a dye and as a medication. Methylene blue is a thiazine dye. As a medication, it is mainly used to treat methemoglobinemia by converting the ferric iron in hemoglobin ...
. Acid-fast bacilli are bright red after staining.


Fungi

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. The results of Ziehl–Neelsen staining is variable because many fungal cell walls are not acid fast. An example of a common type of acid-fast fungus that is usually stained with Ziehl–Neelsen staining is called '' Histoplasma'' (HP). ''Histoplasma'' is found in soil and the feces of birds and bats. Humans can contract histoplasmosis by inhalation of the fungal spores. ''Histoplasma'' enters the body and goes to the lungs where the spores turn into yeast. 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. Another variation on this staining method is used in mycology to differentially stain acid-fast incrustations in the cuticular
hyphae A hypha (; ) is a long, branching, filamentous structure of a fungus, oomycete, or actinobacterium. In most fungi, hyphae are the main mode of vegetative growth, and are collectively called a mycelium. Structure A hypha consists of one or ...
of certain species of fungi in the genus '' Russula''. Some free endospores can be confused with small yeasts, so staining is used to identify the unknown fungi. It is also useful in the identification of some protozoa, namely ''
Cryptosporidium ''Cryptosporidium'', sometimes informally called crypto, is a genus of apicomplexan parasitic alveolates that can cause a respiratory and gastrointestinal illness (cryptosporidiosis) that primarily involves watery diarrhea (intestinal cryptosp ...
'' and '' Isospora''. The Ziehl–Neelsen stain can also hinder diagnosis in the case of paragonimiasis because the eggs in sputum sample for ovum and parasite (O&P) can be dissolved by the stain


History

In 1882
Robert Koch Heinrich Hermann Robert Koch ( , ; 11 December 1843 – 27 May 1910) was a German physician and microbiologist. As the discoverer of the specific causative agents of deadly infectious diseases including tuberculosis, cholera (though the Vibrio ...
discovered the etiology of tuberculosis. Soon after Koch's discovery, Paul Ehrlich developed a stain for mycobacterium tuberculosis, called the alum hematoxylin stain. 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 by
Joseph Kinyoun Joseph James Kinyoun (November 25, 1860 – February 14, 1919) was an American physician and the founder of the United States' Hygienic Laboratory, the predecessor of the National Institutes of Health. Biography Early life Joseph James "Jo ...
by using the Ziehl–Neelsen staining technique but removing the heating step from the procedure. This new stain from Kinyoun was named the Kinyoun stain.


Procedure

A typical AFB stain procedure involves dropping the cells in suspension onto a slide, then air drying the liquid and heat fixing the cells. 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.


Mechanism explanation

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 the
cell walls A cell wall is a structural layer surrounding some types of cells, just outside the cell membrane. It can be tough, flexible, and sometimes rigid. It provides the cell with both structural support and protection, and also acts as a filtering mech ...
of the bacteria. The acidity of the dyes causes them to bind more strongly to the cell walls of the bacteria than to other cells or tissues. This results in the selective staining of only those cells that have a high density of cell wall material, such as acid-fast bacteria. The Ziehl-Neelsen stain is a two step staining 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 basic
fuchsin Fuchsine (sometimes spelled fuchsin) or rosaniline hydrochloride is a magenta dye with chemical formula C20H19N3·HCl.
with the cell wall components of bacteria creates a new molecule that is responsible for the color.


Modifications

* 1% sulfuric acid alcohol for actinomycetes,
nocardia ''Nocardia'' is a genus of weakly staining Gram-positive, catalase-positive, rod-shaped bacteria. It forms partially acid-fast beaded branching filaments (acting as fungi, but being truly bacteria). It contains a total of 85 species. Some spec ...
. * 0.5–1% sulfuric acid alcohol for oocysts of isospora,
cyclospora ''Cyclospora'' is a genus of apicomplexan parasites. It includes the species ''Cyclospora cayetanensis'', the causative agent of cyclosporiasis. Members of ''Cyclospora'' are characterized as having oocysts with two Apicomplexan life cycle, spor ...
. * 0.25–0.5% sulfuric acid alcohol for bacterial endospores. * Differential staining – glacial acetic acid used, no heat applied, secondary stain is Loeffler's methylene blue. * Kinyoun modification (or cold Ziehl–Neelsen technique) is also available. * A protocol in which a
detergent A detergent is a surfactant or a mixture of surfactants with cleansing properties when in dilute solutions. There are a large variety of detergents, a common family being the alkylbenzene sulfonates, which are soap-like compounds that are more ...
is substituted for the highly toxic phenol in the fuchsin staining solution.


See also

* Lowenstein–Jensen medium * Gram stain * Kinyoun stain * Acid-fastness *
Franz Ziehl Franz Ziehl (13 April 1857 in Wismar – 7 April 1926) was a German bacteriologist. He was a professor in Lübeck. Franz Ziehl introduced the carbol fuchsin stain for the tubercle bacillus in 1882. With pathologist Friedrich Neelsen (1854–1898 ...
*
Friedrich Neelsen Friedrich Carl Adolf Neelsen (March 29, 1854, Uetersen – April 11, 1894, Dresden) was a German pathologist. Life Friedrich C.A. Neelsen was born to Hans Friedrich Neelsen, deacon of the Uetersen vicarage, and his wife Bertha Sophia (née Lueders ...


References


Bibliography

* "Microbiology with Diseases by Body System", Robert W. Bauman, 2009, Pearson Education, Inc.


External links

*
Ziehl–Neelsen
protocol (
PDF Portable Document Format (PDF), standardized as ISO 32000, is a file format developed by Adobe in 1992 to present documents, including text formatting and images, in a manner independent of application software, hardware, and operating systems. ...
format). {{DEFAULTSORT:Ziehl-Neelsen stain Microscopy Microbiology techniques Laboratory techniques Histopathology Histotechnology Staining dyes Staining Bacteriology Acid-fast bacilli