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 Microscope, microscopic level. Stains and dyes are frequently used in histology (microscopic study of biological tissue (biology), tissues), in cytology (microscopic ...
technique used in
cytopathology Cytopathology (from Greek , ''kytos'', "a hollow"; , ''pathos'', "fate, harm"; and , ''-logia'') is a branch of pathology that studies and diagnoses diseases on the cellular level. The discipline was founded by George Nicolas Papanicolaou in ...
and
microbiology Microbiology () is the branches of science, scientific study of microorganisms, those being of unicellular organism, unicellular (single-celled), multicellular organism, multicellular (consisting of complex cells), or non-cellular life, acellula ...
to identify
acid-fast Acid-fastness is a physical property of certain bacterial and eukaryotic cells, as well as some sub-cellular structures, specifically their resistance to decolorization by acids during laboratory staining procedures. Once stained as part of a sa ...
bacteria under
microscopy Microscopy is the technical field of using microscopes to view subjects too small to 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 microscopy: optical mic ...
, 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 (''Mycobacterium tuberculo ...
'' genus. This staining method was initially introduced by
Paul Ehrlich Paul Ehrlich (; 14 March 1854 – 20 August 1915) was a Nobel Prize-winning German physician and scientist who worked in the fields of hematology, immunology and antimicrobial chemotherapy. Among his foremost achievements were finding a cure fo ...
(1854–1915) and subsequently modified by the German bacteriologists Franz Ziehl (1859–1926) and Friedrich Neelsen (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 Tuberculosis (TB), also known colloquially as the "white death", or historically as consumption, is a contagious disease usually caused by ''Mycobacterium tuberculosis'' (MTB) bacteria. Tuberculosis generally affects the lungs, but it can al ...
(caused by ''
Mycobacterium tuberculosis ''Mycobacterium tuberculosis'' (M. tb), also known as Koch's bacillus, 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'' ha ...
'') and other diseases caused by atypical mycobacteria, such as
leprosy Leprosy, also known as Hansen's disease (HD), is a Chronic condition, long-term infection by the bacteria ''Mycobacterium leprae'' or ''Mycobacterium lepromatosis''. Infection can lead to damage of the Peripheral nervous system, nerves, respir ...
(caused by ''
Mycobacterium leprae ''Mycobacterium leprae'' (also known as the leprosy bacillus or Hansen's bacillus) is one of the two species of bacteria that cause Hansen's disease (leprosy), a chronic but curable infectious disease that damages the peripheral nerves and ta ...
'') 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 Cytopathology, cytological ...
, gastric washing fluid, and bronchoalveolar lavage fluid. These acid-fast bacteria possess a waxy lipid-rich outer layer that contains high concentrations of
mycolic acid Mycolic acids are long fatty acids found in the cell walls of Mycobacteriales taxon, a group of bacteria that includes ''Mycobacterium tuberculosis'', the causative agent of the disease tuberculosis. They form the major component of the cell wall ...
, rendering them resistant to conventional staining techniques like the
Gram stain Gram stain (Gram staining or Gram's method), is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. It may also be used to diagnose a fungal infection. The name comes ...
. After the Ziehl-Neelsen staining procedure using
carbol fuchsin Carbol fuchsin, carbol-fuchsin, carbolfuchsin, or Castellani's paint is a mixture of phenol and basic fuchsin that is used in bacterial staining procedures. It is commonly used in the staining of mycobacteria because it has an affinity for the my ...
, acid-fast bacteria are observable as vivid red or pink rods set against a blue or green background, depending on the specific counterstain used, such as
methylene blue Methylthioninium chloride, commonly called methylene blue, is a salt used as a dye and as a medication. As a medication, it is mainly used to treat methemoglobinemia. It has previously been used for treating cyanide poisoning and urinary trac ...
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 is a form of immunostaining. It involves the process of selectively identifying antigens in cells and tissue, by exploiting the principle of Antibody, antibodies binding specifically to antigens in biological tissues. Alber ...
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 Bacilli is a Taxonomy (biology), taxonomic Class (biology), class of bacteria that includes two orders, Bacillales and Lactobacillales, which contain several well-known pathogens such as ''Bacillus anthracis'' (the cause of anthrax). ''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. The Gram stain i ...
. Some mycobacteria are free-living saprophytes, but many are
pathogen In biology, a pathogen (, "suffering", "passion" and , "producer of"), in the oldest and broadest sense, is any organism or agent that can produce disease. A pathogen may also be referred to as an infectious agent, or simply a Germ theory of d ...
s that cause disease in animals and humans. ''
Mycobacterium bovis ''Mycobacterium bovis'' is a slow-growing (16- to 20-hour generation time) Aerobic organism, aerobic bacterium and the causative agent of tuberculosis in cattle (known as bovine TB). It is related to ''Mycobacterium tuberculosis'', the bacterium ...
'' 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), also known as Koch's bacillus, 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'' ha ...
'' that causes
tuberculosis Tuberculosis (TB), also known colloquially as the "white death", or historically as consumption, is a contagious disease usually caused by ''Mycobacterium tuberculosis'' (MTB) bacteria. Tuberculosis generally affects the lungs, but it can al ...
(TB) in humans is an airborne 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 Gram stain (Gram staining or Gram's method), is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. It may also be used to diagnose a fungal infection. The name comes ...
. It can also be used to stain a few other bacteria, such as ''
Nocardia ''Nocardia'' is a genus of weakly staining Gram-positive, catalase, catalase-positive, rod-shaped bacteria. It forms partially acid-fast beaded branching filaments (appearing similar to fungi, but being truly bacteria). It contains a total of 8 ...
''. The reagents used for Ziehl–Neelsen staining are
carbol fuchsin Carbol fuchsin, carbol-fuchsin, carbolfuchsin, or Castellani's paint is a mixture of phenol and basic fuchsin that is used in bacterial staining procedures. It is commonly used in the staining of mycobacteria because it has an affinity for the my ...
, acid alcohol, and
methylene blue Methylthioninium chloride, commonly called methylene blue, is a salt used as a dye and as a medication. As a medication, it is mainly used to treat methemoglobinemia. It has previously been used for treating cyanide poisoning and urinary trac ...
. 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 Mycology is the branch of biology concerned with the study of fungus, fungi, including their Taxonomy (biology), taxonomy, genetics, biochemistry, biochemical properties, and ethnomycology, use by humans. Fungi can be a source of tinder, Edible ...
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 o ...
of certain species of
fungi A fungus (: fungi , , , or ; or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and mold (fungus), molds, as well as the more familiar mushrooms. These organisms are classified as one ...
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 called crypto, is an apicomplexan genus of alveolates which are parasitism, parasites that can cause a respiratory and gastrointestinal illness (cryptosporidiosis) that primarily involves watery diarrhea (inte ...
'' 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 and anthrax, he i ...
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 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 of the
bacteria Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
. 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 The Actinomycetales is an Scientific classification, order of Actinomycetota. A member of the order is often called an actinomycete. Actinomycetales are generally Gram-positive bacteria, gram-positive and anaerobic and have mycelia in a filamento ...
,
nocardia ''Nocardia'' is a genus of weakly staining Gram-positive, catalase, catalase-positive, rod-shaped bacteria. It forms partially acid-fast beaded branching filaments (appearing similar to fungi, but being truly bacteria). It contains a total of 8 ...
. * 0.5–1% sulfuric acid alcohol for oocysts of isospora, cyclospora. * 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 Cleanliness, cleansing properties when in Concentration, dilute Solution (chemistry), solutions. There are a large variety of detergents. A common family is the alkylbenzene sulfonate ...
is substituted for the highly toxic
phenol Phenol (also known as carbolic acid, phenolic acid, or benzenol) is an aromatic organic compound with the molecular formula . It is a white crystalline solid that is volatile and can catch fire. The molecule consists of a phenyl group () ...
in the fuchsin staining solution.


See also

* Lowenstein–Jensen medium *
Gram stain Gram stain (Gram staining or Gram's method), is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. It may also be used to diagnose a fungal infection. The name comes ...
* Kinyoun stain * Acid-fastness * Franz Ziehl * Friedrich Neelsen


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 Inc., Adobe in 1992 to present documents, including text formatting and images, in a manner independent of application software, computer hardware, ...
format). {{DEFAULTSORT:Ziehl-Neelsen stain Histopathology Histotechnology Staining dyes Bacteriology Acid-fast bacilli