Romanowsky stain
Encyclopedia
Romanowsky staining is a prototypical staining
technique that was the forerunner of several distinct but similar methods, including Giemsa
, Jenner
, Wright
, Field, and Leishman
stains, which are used to differentiate cell
s in pathologic
specimens.
had used mixtures of acidic and basic dyes for this purpose in 1879: e.g. Fuchsine
(acid dye) and methylene blue (basic dye). In 1891 Ernst Malachowski and Dmitry Leonidovich Romanowsky independently developed techniques using a mixture of Eosin
Y and modified Methylene blue
that produced a surprising hue
unattributable to either staining component: a beautiful, distinctive shade of purple
. Requirement for the occurrence of the Malachowski-Romanowsky-Giemsa effect are: I A cationic dye: The best dye is azure B and, though azure A gives the nuclear purple colour, the cytoplasmic blue is inferior. No other cationic dye such as methylene blue is suitable. 2 An anionic dye: Most commonly eosin Y is used. Because the aqueous dye solutions were unstable, methanol
was introduced as a solvent, and William Boog Leishman
and James Homer Wright
advocated use of methanol as a fixative prior to staining. Gustav Giemsa
improved this technique by standardizing the dye solutions and adding glycerol
to increase stability.
The demethylation
of Methylene Blue in aqueous solution using heat and alkali
produces a mixture of Azure A
, Azure B, Methylene Violet and Methylene Blue. Eosin Y is then added to produce a "neutral" dye. The precipitate is then dissolved in a mixture of methanol and glycerol to form a stock solution; this is diluted with water or an aqueous buffer to form a 'working' solution that is used in the staining of pathology specimens. The 'working' solution is stable for 3 hours.
Immunochromatographic capture procedures (Rapid Diagnostic Tests
) are nonmicroscopic diagnostic options for the laboratory that may not have appropriate microscopy expertise available.
Staining (biology)
Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes...
technique that was the forerunner of several distinct but similar methods, including Giemsa
Giemsa stain
Giemsa stain, named after Gustav Giemsa, an early German microbiologist, is used in cytogenetics and for the histopathological diagnosis of malaria and other parasites.-Uses:...
, Jenner
Jenner's stain
Jenner's Stain is used in microscopy for staining blood smears....
, Wright
Wright's stain
Wright's stain is a histologic stain that facilitates the differentiation of blood cell types. It is used primarily to stain peripheral blood smears and bone marrow aspirates which are examined under a light microscope...
, Field, and Leishman
Leishman stain
Leishman's stain, also Leishman stain, is used in microscopy for staining blood smears. It provides excellent stain quality. It is generally used to differentiate and identify leucocytes, malaria parasites, and trypanosomas. It is based on a mixture of methylene blue and eosin.Leishman stain uses a...
stains, which are used to differentiate cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
s in pathologic
Pathology
Pathology is the precise study and diagnosis of disease. The word pathology is from Ancient Greek , pathos, "feeling, suffering"; and , -logia, "the study of". Pathologization, to pathologize, refers to the process of defining a condition or behavior as pathological, e.g. pathological gambling....
specimens.
Microscopic examination of stained blood films
Paul EhrlichPaul Ehrlich
Paul Ehrlich was a German scientist in the fields of hematology, immunology, and chemotherapy, and Nobel laureate. He is noted for curing syphilis and for his research in autoimmunity, calling it "horror autotoxicus"...
had used mixtures of acidic and basic dyes for this purpose in 1879: e.g. Fuchsine
Fuchsine
Fuchsine or rosaniline hydrochloride is a magenta dye with chemical formula C20H19N3·HCl. There are other similar chemical formulations of products sold as fuchsine, and several dozen other synonyms of this molecule....
(acid dye) and methylene blue (basic dye). In 1891 Ernst Malachowski and Dmitry Leonidovich Romanowsky independently developed techniques using a mixture of Eosin
Eosin
Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. It can be used to stain cytoplasm, collagen and muscle fibers for examination under the microscope. Structures that stain readily with eosin are termed eosinophilic....
Y and modified Methylene blue
Methylene blue
Methylene blue is a heterocyclic aromatic chemical compound with the molecular formula C16H18N3SCl. It has many uses in a range of different fields, such as biology and chemistry. At room temperature it appears as a solid, odorless, dark green powder, that yields a blue solution when dissolved in...
that produced a surprising hue
Hue
Hue is one of the main properties of a color, defined technically , as "the degree to which a stimulus can be describedas similar to or different from stimuli that are described as red, green, blue, and yellow,"...
unattributable to either staining component: a beautiful, distinctive shade of purple
Purple
Purple is a range of hues of color occurring between red and blue, and is classified as a secondary color as the colors are required to create the shade....
. Requirement for the occurrence of the Malachowski-Romanowsky-Giemsa effect are: I A cationic dye: The best dye is azure B and, though azure A gives the nuclear purple colour, the cytoplasmic blue is inferior. No other cationic dye such as methylene blue is suitable. 2 An anionic dye: Most commonly eosin Y is used. Because the aqueous dye solutions were unstable, methanol
Methanol
Methanol, also known as methyl alcohol, wood alcohol, wood naphtha or wood spirits, is a chemical with the formula CH3OH . It is the simplest alcohol, and is a light, volatile, colorless, flammable liquid with a distinctive odor very similar to, but slightly sweeter than, ethanol...
was introduced as a solvent, and William Boog Leishman
William Boog Leishman
Lieutenant-General Sir William Boog Leishman FRS was a Scottish pathologist and British Army medical officer. He was Director-General of Army Medical Services from 1923 to 1926....
and James Homer Wright
James Homer Wright
Dr. James Homer Wright was an early and influential American pathologist, who from 1896 to 1926 was chief of pathology at Massachusetts General Hospital. Wright was born in Pittsburgh, Pennsylvania....
advocated use of methanol as a fixative prior to staining. Gustav Giemsa
Gustav Giemsa
Gustav Giemsa was a German chemist and bacteriologist who was a native of Medar-Blechhammer. He is remembered for creating a dye solution commonly known as "Giemsa stain"...
improved this technique by standardizing the dye solutions and adding glycerol
Glycerol
Glycerol is a simple polyol compound. It is a colorless, odorless, viscous liquid that is widely used in pharmaceutical formulations. Glycerol has three hydroxyl groups that are responsible for its solubility in water and its hygroscopic nature. The glycerol backbone is central to all lipids...
to increase stability.
The demethylation
Demethylation
Demethylation is the chemical process resulting in the removal a of methyl group from a molecule.A common way of demethylation is the replacement of a methyl group by a hydrogen atom, resulting in a net loss of one carbon and two hydrogen atoms....
of Methylene Blue in aqueous solution using heat and alkali
Alkali
In chemistry, an alkali is a basic, ionic salt of an alkali metal or alkaline earth metal element. Some authors also define an alkali as a base that dissolves in water. A solution of a soluble base has a pH greater than 7. The adjective alkaline is commonly used in English as a synonym for base,...
produces a mixture of Azure A
Azure A
' is an organic compound with the chemical formula C14H14ClN3S. It is a green to dark brown dye. It is used as a screening test for mucopolysaccharides....
, Azure B, Methylene Violet and Methylene Blue. Eosin Y is then added to produce a "neutral" dye. The precipitate is then dissolved in a mixture of methanol and glycerol to form a stock solution; this is diluted with water or an aqueous buffer to form a 'working' solution that is used in the staining of pathology specimens. The 'working' solution is stable for 3 hours.
Immunochromatographic capture procedures (Rapid Diagnostic Tests
Malaria antigen detection tests
Malaria antigen detection tests are a group of commercially available tests that allow the rapid diagnosis of malaria by people who are not otherwise skilled in traditional laboratory techniques for diagnosing malaria or in situations where such equipment is not available. There are currently over...
) are nonmicroscopic diagnostic options for the laboratory that may not have appropriate microscopy expertise available.
External links
- Diagnosis and Treatment of Malaria CDCCenters for Disease Control and PreventionThe Centers for Disease Control and Prevention are a United States federal agency under the Department of Health and Human Services headquartered in Druid Hills, unincorporated DeKalb County, Georgia, in Greater Atlanta...
website on malaria diagnosis and treatment - Field stain Staining methods for the identification of malaria parasites
- Laboratory Diagnosis of Malaria This Royal Perth HospitalRoyal Perth HospitalRoyal Perth Hospital is an 855-bed teaching hospital located on the northeastern edge of the CBD of Perth, Western Australia . Royal Perth Hospital also has specialised rehabilitation facilities at Shenton Park.-History:...
(Australia) website provides an excellent overview of malaria diagnostics - UK malaria advisory, diagnostic and reference service for malaria The Malaria Reference Laboratory of the Public Health Laboratory Service in the UK
- Chenzinsky and Romanowsky On the 120th Anniversary of the Discovery of the Romanowsky Effect