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Staining is a technique used to enhance contrast in samples, generally at the
microscopic The microscopic scale () is the scale of objects and events smaller than those that can easily be seen by the naked eye, requiring a lens (optics), lens or microscope to see them clearly. In physics, the microscopic scale is sometimes regarded a ...
level.
Stain A stain is a discoloration that can be clearly distinguished from the surface, material, or medium it is found upon. They are caused by the chemical or physical interaction of two dissimilar materials. Accidental staining may make materials app ...
s and dyes are frequently used in
histology Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
(microscopic study of biological tissues), in
cytology Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living an ...
(microscopic study of
cell Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...
s), and in the
medical Medicine is the science and practice of caring for a patient, managing the diagnosis, prognosis, prevention, treatment, palliation of their injury or disease, and promoting their health. Medicine encompasses a variety of health care practic ...
fields of
histopathology Histopathology (compound of three Greek words: ''histos'' "tissue", πάθος ''pathos'' "suffering", and -λογία '' -logia'' "study of") refers to the microscopic examination of tissue in order to study the manifestations of disease. Spe ...
,
hematology Hematology ( always spelled haematology in British English) is the branch of medicine concerned with the study of the cause, prognosis, treatment, and prevention of diseases related to blood. It involves treating diseases that affect the produc ...
, and
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 1 ...
that focus on the study and
diagnoses Diagnosis is the identification of the nature and cause of a certain phenomenon. Diagnosis is used in many different disciplines, with variations in the use of logic, analytics, and experience, to determine "cause and effect". In systems enginee ...
of
disease A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that a ...
s at the microscopic level. Stains may be used to define biological tissues (highlighting, for example,
muscle fiber A muscle cell is also known as a myocyte when referring to either a cardiac muscle cell (cardiomyocyte), or a smooth muscle cell as these are both small cells. A skeletal muscle cell is long and threadlike with many nuclei and is called a muscl ...
s or
connective tissue Connective tissue is one of the four primary types of animal tissue, along with epithelial tissue, muscle tissue, and nervous tissue. It develops from the mesenchyme derived from the mesoderm the middle embryonic germ layer. Connective tiss ...
),
cell Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...
populations (classifying different
blood cell A blood cell, also called a hematopoietic cell, hemocyte, or hematocyte, is a cell produced through hematopoiesis and found mainly in the blood. Major types of blood cells include red blood cells (erythrocytes), white blood cells (leukocytes), ...
s), or
organelle In cell biology, an organelle is a specialized subunit, usually within a cell, that has a specific function. The name ''organelle'' comes from the idea that these structures are parts of cells, as organs are to the body, hence ''organelle,'' the ...
s within individual cells. In
biochemistry Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...
, it involves adding a class-specific ( DNA,
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s,
lipid Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...
s,
carbohydrate In organic chemistry, a carbohydrate () is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula (where ''m'' may or ma ...
s) dye to a substrate to qualify or quantify the presence of a specific compound. Staining and
fluorescent tag In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Gener ...
ging can serve similar purposes. Biological staining is also used to mark cells in
flow cytometry Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flo ...
, and to flag
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s or
nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
s in
gel electrophoresis Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
. Light microscopes are used for viewing stained samples at high magnification, typically using bright-field or epi-fluorescence illumination. Staining is not limited to only biological materials, since it can also be used to study the structure of other materials; for example, the
lamellar A ''lamella'' (plural ''lamellae'') is a small plate or flake, from the Latin, and may also be used to refer to collections of fine sheets of material held adjacent to one another, in a gill-shaped structure, often with fluid in between though s ...
structures of
semi-crystalline polymer Crystallization of polymers is a process associated with partial alignment of their molecular chains. These chains fold together and form ordered regions called lamellae, which compose larger spheroidal structures named spherulites. Polymers can ...
s or the domain structures of
block copolymer In polymer chemistry, a copolymer is a polymer derived from more than one species of monomer. The polymerization of monomers into copolymers is called copolymerization. Copolymers obtained from the copolymerization of two monomer species are ...
s.


''In vivo'' vs ''In vitro''

''
In vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
staining'' (also called
vital staining A vital stain in a casual usage may mean a stain that can be applied on living cells without killing them. Vital stains have been useful for diagnostic and surgical techniques in a variety of medical specialties. In supravital staining, living cells ...
or intravital staining) is the process of dyeing living tissues. By causing certain cells or structures to take on contrasting colours, their form (
morphology Morphology, from the Greek and meaning "study of shape", may refer to: Disciplines * Morphology (archaeology), study of the shapes or forms of artifacts * Morphology (astronomy), study of the shape of astronomical objects such as nebulae, galaxies ...
) or position within a cell or tissue can be readily seen and studied. The usual purpose is to reveal cytological details that might otherwise not be apparent; however, staining can also reveal where certain chemicals or specific chemical reactions are taking place within cells or tissues. ''
In vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...
'' staining involves colouring cells or structures that have been removed from their biological context. Certain stains are often combined to reveal more details and features than a single stain alone. Combined with specific protocols for fixation and sample preparation, scientists and physicians can use these standard techniques as consistent, repeatable diagnostic tools. A
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 ...
is stain that makes cells or structures more visible, when not completely visible with the principal stain. * Crystal violet stains both Gram positive and Gram negative organisms. Treatment with alcohol removes the crystal violet colour from gram negative organisms only. Safranin as counterstain is used to colour the gram negative organisms that got decolorised by alcohol. While ex vivo, many cells continue to live and metabolize until they are "fixed". Some staining methods are based on this property. Those stains excluded by the living cells but taken up by the already dead cells are called
vital stain A vital stain in a casual usage may mean a stain that can be applied on living cells without killing them. Vital stains have been useful for diagnostic and surgical techniques in a variety of medical specialties. In supravital staining, living cell ...
s (e.g.
trypan blue Trypan blue is an azo dye. It is a direct dye for cotton textiles. In biosciences, it is used as a vital stain to selectively colour dead tissues or cells blue. Live cells or tissues with intact cell membranes are not coloured. Since cells a ...
or
propidium iodide Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. When in an aqueous solution, PI has a fluo ...
for eukaryotic cells). Those that enter and stain living cells are called
supravital stain Supravital staining is a method of staining used in microscopy to examine living cells that have been removed from an organism. It differs from intravital staining, which is done by injecting or otherwise introducing the stain into the body. Thu ...
s (e.g.
New Methylene Blue is an organic compound of the thiazine class of heterocycles. It is used as a stain and as an antimicrobial agent. It is classified as an azine dye, and the chromophore is a cation, the anion is often unspecified. Applications NMB is a staini ...
and
brilliant cresyl blue Brilliant cresyl blue is a supravital stain used for counting reticulocyte Reticulocytes are immature red blood cells (RBCs). In the process of erythropoiesis (red blood cell formation), reticulocytes develop and mature in the bone marrow a ...
for
reticulocyte Reticulocytes are immature red blood cells (RBCs). In the process of erythropoiesis (red blood cell formation), reticulocytes develop and mature in the bone marrow and then circulatory system, circulate for about a day in the blood stream before ...
staining). However, these stains are eventually toxic to the organism, some more so than others. Partly due to their toxic interaction inside a living cell, when supravital stains enter a living cell, they might produce a characteristic pattern of staining different from the staining of an already fixed cell (e.g. "reticulocyte" look versus diffuse "polychromasia"). To achieve desired effects, the stains are used in very dilute solutions ranging from to (Howey, 2000). Note that many stains may be used in both living and fixed cells.


Preparation

The preparatory steps involved depend on the type of analysis planned. Some or all of the following procedures may be required.
Wet mount A microscope slide is a thin flat piece of glass, typically 75 by 26 mm (3 by 1 inches) and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is mounted (secured) on the slide, and then b ...
s are used to view live organisms and can be made using water and certain stains. The liquid is added to the slide before the addition of the organism and a coverslip is placed over the specimen in the water and stain to help contain it within the
field of view The field of view (FoV) is the extent of the observable world that is seen at any given moment. In the case of optical instruments or sensors it is a solid angle through which a detector is sensitive to electromagnetic radiation. Human ...
. Fixation, which may itself consist of several steps, aims to preserve the shape of the cells or tissue involved as much as possible. Sometimes
heat fixation In the fields of histology, pathology, and cell biology, fixation is the preservation of biological tissues from decay due to Autolysis (biology), autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the ...
is used to kill, adhere, and alter the specimen so it accepts stains. Most chemical fixatives (chemicals causing fixation) generate
chemical bond A chemical bond is a lasting attraction between atoms or ions that enables the formation of molecules and crystals. The bond may result from the electrostatic force between oppositely charged ions as in ionic bonds, or through the sharing of ...
s between
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s and other substances within the sample, increasing their rigidity. Common fixatives include
formaldehyde Formaldehyde ( , ) (systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section F ...
,
ethanol Ethanol (abbr. EtOH; also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound. It is an Alcohol (chemistry), alcohol with the chemical formula . Its formula can be also written as or (an ethyl ...
,
methanol Methanol (also called methyl alcohol and wood spirit, amongst other names) is an organic chemical and the simplest aliphatic alcohol, with the formula C H3 O H (a methyl group linked to a hydroxyl group, often abbreviated as MeOH). It is a ...
, and/or
picric acid Picric acid is an organic compound with the formula (O2N)3C6H2OH. Its IUPAC name is 2,4,6-trinitrophenol (TNP). The name "picric" comes from el, πικρός (''pikros''), meaning "bitter", due to its bitter taste. It is one of the most acidic ...
. Pieces of tissue may be embedded in
paraffin wax Paraffin wax (or petroleum wax) is a soft colorless solid derived from petroleum, coal, or oil shale that consists of a mixture of hydrocarbon molecules containing between 20 and 40 carbon atoms. It is solid at room temperature and begins to ...
to increase their mechanical strength and stability and to make them easier to cut into thin slices.
Mordant A mordant or dye fixative is a substance used to set (i.e. bind) dyes on fabrics by forming a coordination complex with the dye, which then attaches to the fabric (or tissue). It may be used for dyeing fabrics or for intensifying stains in ...
s are chemical agents which have power of making dyes to stain materials which otherwise are unstainable Mordants are classified into two categories: a) Basic mordant: React with acidic dyes e.g. alum, ferrous sulfate, cetylpyridinium chloride etc. b) Acidic mordant : React with basic dyes e.g. picric acid, tannic acid etc. Direct Staining: Carried out without mordant. Indirect Staining: Staining with the aid of a mordant. Permeabilization involves treatment of cells with (usually) a mild
surfactant Surfactants are chemical compounds that decrease the surface tension between two liquids, between a gas and a liquid, or interfacial tension between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming ...
. This treatment dissolves
cell membrane The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...
s, and allows larger dye molecules into the cell's interior. Mounting usually involves attaching the samples to a glass microscope slide for observation and analysis. In some cases, cells may be grown directly on a slide. For samples of loose cells (as with a blood smear or a
pap smear The Papanicolaou test (abbreviated as Pap test, also known as Pap smear (AE), cervical smear (BE), cervical screening (BE), or smear test (BE)) is a method of cervical screening used to detect potentially precancerous and cancerous processes in t ...
) the sample can be directly applied to a slide. For larger pieces of tissue, thin sections (slices) are made using a microtome; these slices can then be mounted and inspected.


Standardization

Most of the dyes commonly used in microscopy are available as BSC-certified stains. This means that samples of the manufacturer's batch have been tested by an independent body, the
Biological Stain Commission The Biological Stain Commission (BSC) is an organization that provides third-party testing and certification of dyes and a few other compounds that are used to enhance contrast in specimens examined in biological and medical laboratories. The BS ...
(BSC), and found to meet or exceed certain standards of purity, dye content and performance in staining techniques ensuring more accurately performed experiments and more reliable results. These standards are published in the commission's journal
Biotechnic & Histochemistry ''Biotechnic & Histochemistry'' is a peer-reviewed scientific journal that covers all aspects of histochemistry and microtechnic in the biological sciences from botany to cell biology to medicine. It is published by Taylor & Francis on behalf of ...
. Many dyes are inconsistent in composition from one supplier to another. The use of BSC-certified stains eliminates a source of unexpected results. Some vendors sell stains "certified" by themselves rather than by the Biological Stain Commission. Such products may or may not be suitable for diagnostic and other applications.


Negative

A simple staining method for bacteria that is usually successful, even when the positive staining methods fail, is to use a
negative stain In microscopy, negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and ...
. This can be achieved by smearing the sample onto the slide and then applying
nigrosin In staining dyes, nigrosin (CI 50415, Solvent black 5) is a mixture of black synthetic dyes made by heating a mixture of nitrobenzene, aniline, and hydrochloric acid in the presence of copper or iron. Related to induline, it is a mixture of phena ...
(a black synthetic dye) or India ink (an aqueous suspension of carbon particles). After drying, the microorganisms may be viewed in bright field microscopy as lighter inclusions well-contrasted against the dark environment surrounding them. Negative staining is able to stain the background instead of the organisms because the cell wall of microorganisms typically has a negative charge which repels the negatively charged stain. The dyes used in negative staining are acidic. Note: negative staining is a mild technique that may not destroy the microorganisms, and is therefore unsuitable for studying pathogens.


Positive

Unlike negative staining, positive staining uses basic dyes to color the specimen against a bright background. While chromophore is used for both negative and positive staining alike, the type of chromophore used in this technique is a positively charged ion instead of a negative one. The negatively charged cell wall of many microorganisms attracts the positively charged chromophore which causes the specimen to absorb the stain giving it the color of the stain being used. Positive staining is more commonly used than negative staining in microbiology. The different types of positive staining are listed below.


Simple versus differential

Simple Staining is a technique that only uses one type of stain on a slide at a time. Because only one stain is being used, the specimens (for positive stains) or background (for negative stains) will be one color. Therefore, simple stains are typically used for viewing only one organism per slide. Differential staining uses multiple stains per slide. Based on the stains being used, organisms with different properties will appear different colors allowing for categorization of multiple specimens. Differential staining can also be used to color different organelles within one organism which can be seen in
endospore staining Endospore staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample. Within bacteria, endospores are protective structures used to survive extreme conditions, including high temperatures making them h ...
.


Types


Techniques


Gram

Gram staining In microbiology and bacteriology, 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. The name comes from the Danish bac ...
is used to determine gram status to classifying bacteria broadly based on the composition of their
cell wall 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 ...
. Gram staining uses
crystal violet Crystal violet or gentian violet, also known as methyl violet 10B or hexamethyl pararosaniline chloride, is a triarylmethane dye used as a histological stain and in Gram's method of classifying bacteria. Crystal violet has antibacterial, antif ...
to stain cell walls,
iodine Iodine is a chemical element with the symbol I and atomic number 53. The heaviest of the stable halogens, it exists as a semi-lustrous, non-metallic solid at standard conditions that melts to form a deep violet liquid at , and boils to a vi ...
(as a mordant), and a
fuchsin Fuchsine (sometimes spelled fuchsin) or rosaniline hydrochloride is a magenta dye with chemical formula C20H19N3·HCl.
or
safranin Safranin (Safranin O or basic red 2) is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endos ...
counterstain to (mark all bacteria). Gram status, helps divide specimens of bacteria into two groups, generally representative of their underlying phylogeny. This characteristic, in combination with other techniques makes it a useful tool in clinical microbiology laboratories, where it can be important in early selection of appropriate
antibiotic An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention of ...
s. On most Gram-stained preparations,
Gram-negative Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall ...
organisms appear red or pink due to their counterstain. Due to the presence of higher lipid content, after alcohol-treatment, the porosity of the cell wall increases, hence the CVI complex (crystal violet – iodine) can pass through. Thus, the primary stain is not retained. In addition, in contrast to most Gram-positive bacteria, Gram-negative bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of lipopolysaccharide.


Endospore

Endospore staining Endospore staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample. Within bacteria, endospores are protective structures used to survive extreme conditions, including high temperatures making them h ...
is used to identify the presence or absence of
endospore An endospore is a dormant, tough, and non-reproductive structure produced by some bacteria in the phylum Bacillota. The name "endospore" is suggestive of a spore or seed-like form (''endo'' means 'within'), but it is not a true spore (i.e., no ...
s, which make bacteria very difficult to kill. Bacterial spores have proven to be difficult to stain as they are not permeable to aqueous dye reagents.  Endospore staining is particularly useful for identifying endospore-forming bacterial
pathogen In biology, a pathogen ( el, πάθος, "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 ...
s such as '' Clostridium difficile''. Prior to the development of more efficient methods, this stain was performed using the Wirtz method with heat fixation and counterstain. Through the use of malachite green and a diluted ratio of carbol fuchsin, fixing bacteria in osmic acid was a great way to ensure no blending of dyes. However, newly revised staining methods have significantly decreased the time it takes to create these stains. This revision included substitution of carbol fuchsin with aqueous Safranin paired with a newly diluted 5% formula of malachite green. This new and improved composition of stains was performed in the same way as before with the use of heat fixation, rinsing, and blotting dry for later examination. Upon examination, all endospore forming bacteria will be stained green accompanied by all other cells appearing red.


Ziehl-Neelsen

A
Ziehl–Neelsen stain Ziehl–Neelsen staining is a type of acid-fast stain, first introduced by Paul Ehrlich. Ziehl–Neelsen staining is a bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. It is named for two German doctors who modi ...
is an acid-fast stain used to stain species of ''
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 do not stain with the standard laboratory staining procedures such as Gram staining. This stain is performed through the use of both red coloured
carbol fuchsin Carbol fuchsin, carbol-fuchsin, carbolfuchsin, or Castellani's paint (CAS ) 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 fo ...
that stains the bacteria and a counter stain 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 ...
.


Haematoxylin and eosin (H&E)

Haematoxylin and eosin stain Hematoxylin and eosin stain (American and British English spelling differences, or haematoxylin and eosin stain or hematoxylin-eosin stain; often abbreviated as H&E stain or HE stain) is one of the principal tissue staining, stains used in hi ...
ing is frequently used in
histology Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
to examine thin tissue sections.
Haematoxylin Haematoxylin or hematoxylin (), also called natural black 1 or C.I. 75290, is a compound extracted from heartwood of the logwood tree (''Haematoxylum campechianum'') with a chemical formula of . This naturally derived dye has been used as a ...
stains cell nuclei blue, while
eosin Eosin is the name of several fluorescent acidic compounds which bind to and form salts with basic, or eosinophilic, compounds like proteins containing amino acid residues such as arginine and lysine, and stains them dark red or pink as a resul ...
stains cytoplasm, connective tissue and other extracellular substances pink or red. Eosin is strongly absorbed by
red blood cell Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...
s, colouring them bright red. In a skillfully made H&E preparation the red blood cells are almost orange, and collagen and cytoplasm (especially muscle) acquire different shades of pink.


Papanicolaou

Papanicolaou stain Papanicolaou stain (also Papanicolaou's stain and Pap stain) is a multichromatic (multicolored) cytological staining technique developed by George Papanicolaou in 1942. The Papanicolaou stain is one of the most widely used stains in cytology, w ...
ing, or PAP staining, was developed to replace fine needle aspiration cytology (FNAC) in hopes of decreasing staining times and cost without compromising quality. This stain is a frequently used method for examining cell samples from a variety of tissue types in various organs. PAP staining has endured several modifications in order to become a “suitable alternative” for FNAC. This transition stemmed from the appreciation of wet fixed smears by scientists preserving the structures of the nuclei opposed to the opaque appearance of air dried Romanowsky smears. This led to the creation of a hybrid stain of wet fixed and air dried known as the ultrafast papanicolaou stain. This modification includes the use of nasal saline to rehydrate cells to increase cell transparency and is paired with the use of alcoholic formalin to enhance colors of the nuclei. The papanicolaou stain is now used in place of cytological staining in all organ types due to its increase in morphological quality, decreased staining time, and decreased cost. It is frequently used to stain
Pap smear The Papanicolaou test (abbreviated as Pap test, also known as Pap smear (AE), cervical smear (BE), cervical screening (BE), or smear test (BE)) is a method of cervical screening used to detect potentially precancerous and cancerous processes in t ...
specimens. It uses a combination of
haematoxylin Haematoxylin or hematoxylin (), also called natural black 1 or C.I. 75290, is a compound extracted from heartwood of the logwood tree (''Haematoxylum campechianum'') with a chemical formula of . This naturally derived dye has been used as a ...
,
Orange G Orange G also called C.I. 16230, Acid Orange 10, or orange gelb is a synthetic azo dye used in histology in many staining formulations. It usually comes as a disodium salt. It has the appearance of orange crystals or powder. Staining Orange G ...
,
eosin Y Eosin Y, also called C.I. 45380 or C.I. Acid Red 87, is a member of the triarylmethane dyes. It is produced from fluorescein by bromination. Use Eosin Y is commonly used as the red dye in red inks. It is commonly used in histology, most nota ...
,
Light Green SF yellowish Light green SF, also called C.I. 42095, light green SF yellowish, is a green triarylmethane dye. Uses Biomedical It is used in histology for staining collagen; for that purpose it is a standard dye in North America. In Masson's trichrome i ...
, and sometimes
Bismarck Brown Y Bismarck brown Y also called C.I. 21000 and C.I. Basic Brown 1, is a diazo dye with the idealized formula H2N)2C6H3N2sub>2C6H4. The dye is a mixture of closely related compounds. It was one of the earliest azo dyes, being described in 1863 by ...
.


PAS

Periodic acid-Schiff Periodicity or periodic may refer to: Mathematics * Bott periodicity theorem, addresses Bott periodicity: a modulo-8 recurrence relation in the homotopy groups of classical groups * Periodic function, a function whose output contains values tha ...
is a histology special stain used to mark
carbohydrate In organic chemistry, a carbohydrate () is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula (where ''m'' may or ma ...
s (
glycogen Glycogen is a multibranched polysaccharide of glucose that serves as a form of energy storage in animals, fungi, and bacteria. The polysaccharide structure represents the main storage form of glucose in the body. Glycogen functions as one o ...
,
glycoprotein Glycoproteins are proteins which contain oligosaccharide chains covalently attached to amino acid side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycos ...
,
proteoglycan Proteoglycans are proteins that are heavily glycosylated. The basic proteoglycan unit consists of a "core protein" with one or more covalently attached glycosaminoglycan (GAG) chain(s). The point of attachment is a serine (Ser) residue to whic ...
s). PAS is commonly used on liver tissue where glycogen deposits are made which is done in efforts to distinguish different types of glycogen storage diseases. PAS is important because it can detect glycogen granules found in tumors of the ovaries and pancreas of the endocrine system, as well as in the bladder and kidneys of the renal system. Basement membranes can also show up in a PAS stain and can be important when diagnosing renal disease. Due to the high volume of carbohydrates within the cell wall of hyphae and yeast forms of fungi, the Periodic acid -Schiff stain can help locate these species inside tissue samples of the human body.


Masson

Masson's trichrome Masson's trichrome is a three-colour staining procedure used in histology. The recipes evolved from Claude L. Pierre Masson's (1880–1959) original formulation have different specific applications, but all are suited for distinguishing cells ...
is (as the name implies) a three-colour staining protocol. The recipe has evolved from Masson's original technique for different specific applications, but all are well-suited to distinguish cells from surrounding
connective tissue Connective tissue is one of the four primary types of animal tissue, along with epithelial tissue, muscle tissue, and nervous tissue. It develops from the mesenchyme derived from the mesoderm the middle embryonic germ layer. Connective tiss ...
. Most recipes produce red
keratin Keratin () is one of a family of structural fibrous proteins also known as ''scleroproteins''. Alpha-keratin (α-keratin) is a type of keratin found in vertebrates. It is the key structural material making up scales, hair, nails, feathers, ho ...
and muscle fibers, blue or green staining of
collagen Collagen () is the main structural protein in the extracellular matrix found in the body's various connective tissues. As the main component of connective tissue, it is the most abundant protein in mammals, making up from 25% to 35% of the whole ...
and
bone A bone is a Stiffness, rigid Organ (biology), organ that constitutes part of the skeleton in most vertebrate animals. Bones protect the various other organs of the body, produce red blood cell, red and white blood cells, store minerals, provid ...
, light red or pink staining of
cytoplasm In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. The ...
, and black
cell nuclei The cell nucleus (pl. nuclei; from Latin or , meaning ''kernel'' or ''seed'') is a membrane-bound organelle found in eukaryotic cells. Eukaryotic cells usually have a single nucleus, but a few cell types, such as mammalian red blood cells, ha ...
.


Romanowsky

The
Romanowsky stain Romanowsky staining, also known as Romanowsky–Giemsa staining, is a prototypical staining technique that was the forerunner of several distinct but similar stains widely used in hematology (the study of blood) and cytopathology (the study of ...
s is considered a polychrome staining effect and is based on a combination of eosin plus (chemically reduced
eosin Eosin is the name of several fluorescent acidic compounds which bind to and form salts with basic, or eosinophilic, compounds like proteins containing amino acid residues such as arginine and lysine, and stains them dark red or pink as a resul ...
) and demethylated
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 ...
(containing its oxidation products
azure A Azure A is an organic compound with the chemical formula C14H14ClN3S. It is a light blue to dark blue dye. It is used as a screening test for mucopolysaccharides. It can also be used to stain lysosome in blood smears, and is often used in Giemsa ...
and
azure B Azure may refer to: Colour * Azure (color), a hue of blue ** Azure (heraldry) ** Shades of azure, shades and variations Arts and media * ''Azure'' (Art Farmer and Fritz Pauer album), 1987 * Azure (Gary Peacock and Marilyn Crispell album), 2013 ...
). This stain develops varying colors for all cell structures (“Romanowsky-Giemsa effect) and thus was used in staining neutrophil polymorphs and cell nuclei. Common variants include
Wright's stain Wright's stain is a hematologic stain that facilitates the differentiation of blood cell types. It is classically a mixture of eosin (red) and methylene blue dyes. It is used primarily to stain peripheral blood smears, urine samples, and bone m ...
,
Jenner's stain Jenner's stain (methylene blue eosinate) is used in microscopy for staining blood smear A blood smear, peripheral blood smear or blood film is a thin layer of blood smeared on a glass microscope slide and then stained in such a way as to allo ...
, May-Grunwald stain,
Leishman stain Leishman stain, also known as Leishman's stain, is used in microscopy for staining blood smears. It is generally used to differentiate between and identify white blood cells, malaria parasites, and trypanosomas. It is based on a methanolic mixture ...
and
Giemsa stain Giemsa stain (), named after German chemist and bacteriologist Gustav Giemsa, is a nucleic acid stain used in cytogenetics and for the histopathological diagnosis of malaria and other parasites. Uses It is specific for the phosphate groups of ...
. All are used to examine
blood Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood in the c ...
or
bone marrow Bone marrow is a semi-solid tissue found within the spongy (also known as cancellous) portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production (or haematopoiesis). It is composed of hematopoietic ce ...
samples. They are preferred over H&E for inspection of blood cells because different types of
leukocytes White blood cells, also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from mult ...
(white blood cells) can be readily distinguished. All are also suited to examination of blood to detect blood-borne parasites such as
malaria Malaria is a mosquito-borne infectious disease that affects humans and other animals. Malaria causes symptoms that typically include fever, tiredness, vomiting, and headaches. In severe cases, it can cause jaundice, seizures, coma, or death. S ...
.


Silver

Silver stain In pathology, silver staining is the use of silver to selectively alter the appearance of a target in microscopy of histological sections; in temperature gradient gel electrophoresis; and in polyacrylamide gels. In traditional stained glass, silv ...
ing is the use of
silver Silver is a chemical element with the Symbol (chemistry), symbol Ag (from the Latin ', derived from the Proto-Indo-European wikt:Reconstruction:Proto-Indo-European/h₂erǵ-, ''h₂erǵ'': "shiny" or "white") and atomic number 47. A soft, whi ...
to stain
histologic section Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
s. This kind of staining is important in the demonstration of
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s (for example type III
collagen Collagen () is the main structural protein in the extracellular matrix found in the body's various connective tissues. As the main component of connective tissue, it is the most abundant protein in mammals, making up from 25% to 35% of the whole ...
) and DNA. It is used to show both substances inside and outside cells. Silver staining is also used in temperature gradient gel electrophoresis. ''Argentaffin cells''
reduce Reduction, reduced, or reduce may refer to: Science and technology Chemistry * Reduction (chemistry), part of a reduction-oxidation (redox) reaction in which atoms have their oxidation state changed. ** Organic redox reaction, a redox react ...
silver solution to metallic silver after
formalin Formaldehyde ( , ) ( systematic name methanal) is a naturally occurring organic compound with the formula and structure . The pure compound is a pungent, colourless gas that polymerises spontaneously into paraformaldehyde (refer to section F ...
fixation. This method was discovered by Italian
Camillo Golgi Camillo Golgi (; 7 July 184321 January 1926) was an Italian biologist and pathologist known for his works on the central nervous system. He studied medicine at the University of Pavia (where he later spent most of his professional career) betwee ...
, by using a reaction between
silver nitrate Silver nitrate is an inorganic compound with chemical formula . It is a versatile precursor to many other silver compounds, such as those used in photography. It is far less sensitive to light than the halides. It was once called ''lunar caustic' ...
and
potassium dichromate Potassium dichromate, , is a common inorganic chemical reagent, most commonly used as an oxidizing agent in various laboratory and industrial applications. As with all hexavalent chromium compounds, it is acutely and chronically harmful to health ...
, thus precipitating silver chromate in some cells (see
Golgi's method Golgi's method is a silver staining technique that is used to visualize nervous tissue under light microscopy. The method was discovered by Camillo Golgi, an Italian physician and scientist, who published the first picture made with the techniqu ...
). A''rgyrophilic cells'' reduce silver solution to metallic silver after being exposed to the stain that contains a
reductant In chemistry, a reducing agent (also known as a reductant, reducer, or electron donor) is a chemical species that "donates" an electron to an (called the , , , or ). Examples of substances that are commonly reducing agents include the Earth meta ...
. An example of this would be
hydroquinone Hydroquinone, also known as benzene-1,4-diol or quinol, is an aromatic organic compound that is a type of phenol, a derivative of benzene, having the chemical formula C6H4(OH)2. It has two hydroxyl groups bonded to a benzene ring in a ''para'' ...
or formalin.


Sudan

Sudan stain Sudan ( or ; ar, السودان, as-Sūdān, officially the Republic of the Sudan ( ar, جمهورية السودان, link=no, Jumhūriyyat as-Sūdān), is a country in Northeast Africa. It shares borders with the Central African Republic t ...
ing utilizes Sudan dyes to stain sudanophilic substances, often including
lipid Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...
s.
Sudan III Sudan III is a lysochrome (fat-soluble dye) diazo dye. It is structurally related to azobenzene. Uses It is used to color nonpolar substances such as oils, fats, waxes, greases, various hydrocarbon products, and acrylic emulsions. Its main use ...
,
Sudan IV Sudan IV (C24H20N4O) is a lysochrome (fat-soluble dye) diazo dye used for the staining of lipids, triglycerides and lipoproteins on frozen paraffin sections. It has the appearance of reddish brown crystals with melting point 199 °C and m ...
,
Oil Red O Oil Red O (Solvent Red 27, Sudan Red 5B, C.I. 26125, C26H24N4O) is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. It has the appearance ...
,
Osmium tetroxide Osmium tetroxide (also osmium(VIII) oxide) is the chemical compound with the formula OsO4. The compound is noteworthy for its many uses, despite its toxicity and the rarity of osmium. It also has a number of unusual properties, one being that the ...
, and Sudan Black B are often used. Sudan staining is often used to determine the level of
fecal fat In medicine, the fecal fat test is a diagnostic test for fat malabsorption conditions, which lead to excess fat in the feces (steatorrhea). Background In the duodenum, dietary fat (primarily triglycerides) is digested by enzymes such as pancreat ...
in diagnosing steatorrhea.


Wirtz-Conklin

The Wirtz-Conklin stain is a special technique designed for staining true endospores with the use of malachite green dye as the primary stain and safranin as the counterstain. Once stained, they do not decolourize. The addition of heat during the staining process is a huge contributing factor. Heat helps open the spore's membrane so the dye can enter. The main purpose of this stain is to show germination of bacterial spores. If the process of germination is taking place, then the spore will turn green in color due to malachite green and the surrounding cell will be red from the safranin. This stain can also help determine the orientation of the spore within the bacterial cell; whether it being terminal (at the tip), subterminal (within the cell), or central (completely in the middle of the cell).


Collagen hybridizing peptide

Collagen Hybridizing Peptide (CHP) staining allows for an easy, direct way to stain denatured collagens of any type (Type I, II, IV, etc.) regardless if they were damaged or degraded via enzymatic, mechanical, chemical, or thermal means. They work by refolding into the collagen triple helix with the available single strands in the tissue. CHPs can be visualized by a simple
fluorescence microscope A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microsc ...
.


Common biological stains

Different stains react or concentrate in different parts of a cell or tissue, and these properties are used to advantage to reveal specific parts or areas. Some of the most common biological stains are listed below. Unless otherwise marked, all of these dyes may be used with fixed cells and tissues; vital dyes (suitable for use with living organisms) are noted.


Acridine orange

Acridine orange Acridine orange is an organic compound that serves as a nucleic acid-selective fluorescent dye with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows the dye to interact with DNA by interca ...
(AO) is a nucleic acid selective fluorescent cationic dye useful for cell cycle determination. It is cell-permeable, and interacts with DNA and RNA by intercalation or electrostatic attractions. When bound to DNA, it is very similar spectrally to fluorescein. Like fluorescein, it is also useful as a non-specific stain for backlighting conventionally stained cells on the surface of a solid sample of tissue (fluorescence backlighted staining).


Bismarck brown

Bismarck brown (also Bismarck brown Y or Manchester brown) imparts a yellow colour to acid
mucin Mucins () are a family of high molecular weight, heavily glycosylated proteins (glycoconjugates) produced by epithelial tissues in most animals. Mucins' key characteristic is their ability to form gels; therefore they are a key component in most ...
s. and an intense brown color to mast cells. One default of this stain is that it blots out any other structure surrounding it and makes the quality of the contrast low. It has to be paired with other stains  in order to be useful. Some complementing stains used alongside Bismark brown are Hematoxylin and Toluidine blue which provide better contrast within the histology sample.


Carmine

Carmine is an intensely red dye used to stain
glycogen Glycogen is a multibranched polysaccharide of glucose that serves as a form of energy storage in animals, fungi, and bacteria. The polysaccharide structure represents the main storage form of glucose in the body. Glycogen functions as one o ...
, while Carmine alum is a nuclear stain. Carmine stains require the use of a mordant, usually
aluminum Aluminium (aluminum in American and Canadian English) is a chemical element with the symbol Al and atomic number 13. Aluminium has a density lower than those of other common metals, at approximately one third that of steel. It has ...
.


Coomassie blue

Coomassie blue Coomassie brilliant blue is the name of two similar triphenylmethane dyes that were developed for use in the textile industry but are now commonly used for staining proteins in analytical biochemistry. Coomassie brilliant blue G-250 differs from ...
(also brilliant blue) nonspecifically stains proteins a strong blue colour. It is often used in gel electrophoresis.


Cresyl violet

Cresyl violet stains the acidic components of the neuronal cytoplasm a violet colour, specifically nissl bodies. Often used in brain research.


Crystal violet

Crystal violet Crystal violet or gentian violet, also known as methyl violet 10B or hexamethyl pararosaniline chloride, is a triarylmethane dye used as a histological stain and in Gram's method of classifying bacteria. Crystal violet has antibacterial, antif ...
, when combined with a suitable mordant, stains
cell wall 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 ...
s purple. Crystal violet is the stain used in Gram staining.


DAPI

DAPI DAPI (pronounced 'DAPPY', /ˈdæpiː/), or 4′,6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA. It is used extensively in fluorescence microscopy. As DAPI can pass through an inta ...
is a
fluorescent Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
nuclear stain, excited by
ultraviolet Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nanometer, nm (with a corresponding frequency around 30 Hertz, PHz) to 400 nm (750 Hertz, THz), shorter than that of visible light, but longer than ...
light and showing strong blue fluorescence when bound to DNA. DAPI binds with A=T rich repeats of chromosomes. DAPI is also not visible with regular transmission microscopy. It may be used in living or fixed cells. DAPI-stained cells are especially appropriate for cell counting.


Eosin

Eosin Eosin is the name of several fluorescent acidic compounds which bind to and form salts with basic, or eosinophilic, compounds like proteins containing amino acid residues such as arginine and lysine, and stains them dark red or pink as a resul ...
is most often used as a counterstain to haematoxylin, imparting a pink or red colour to
cytoplasm In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. The ...
ic material,
cell membrane The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...
s, and some extracellular structures. It also imparts a strong red colour to
red blood cell Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...
s. Eosin may also be used as a counterstain in some variants of Gram staining, and in many other protocols. There are actually two very closely related compounds commonly referred to as eosin. Most often used is
eosin Y Eosin Y, also called C.I. 45380 or C.I. Acid Red 87, is a member of the triarylmethane dyes. It is produced from fluorescein by bromination. Use Eosin Y is commonly used as the red dye in red inks. It is commonly used in histology, most nota ...
(also known as eosin Y ws or eosin yellowish); it has a very slightly yellowish cast. The other eosin compound is eosin B (eosin bluish or imperial red); it has a very faint bluish cast. The two dyes are interchangeable, and the use of one or the other is more a matter of preference and tradition.


Ethidium bromide

Ethidium bromide Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag ( nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It ...
intercalates and stains DNA, providing a fluorescent red-orange stain. Although it will not stain healthy cells, it can be used to identify cells that are in the final stages of
apoptosis Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
– such cells have much more permeable
membranes A membrane is a selective barrier; it allows some things to pass through but stops others. Such things may be molecules, ions, or other small particles. Membranes can be generally classified into synthetic membranes and biological membranes. Bi ...
. Consequently, ethidium bromide is often used as a marker for apoptosis in cells populations and to locate bands of DNA in
gel electrophoresis Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
. The stain may also be used in conjunction with
acridine orange Acridine orange is an organic compound that serves as a nucleic acid-selective fluorescent dye with cationic properties useful for cell cycle determination. Acridine orange is cell-permeable, which allows the dye to interact with DNA by interca ...
(AO) in viable cell counting. This EB/AO combined stain causes live cells to fluoresce green whilst apoptotic cells retain the distinctive red-orange fluorescence.


Acid fuchsin

Acid fuchsine Acid fuchsin or fuchsine acid, (also called Acid Violet 19 and C.I. 42685) is an acidic magenta dye with the chemical formula C20H17N3Na2O9S3. It is a sodium sulfonate derivative of fuchsine. Acid fuchsin has wide use in histology, and is one o ...
may be used to stain collagen, smooth muscle, or
mitochondria A mitochondrion (; ) is an organelle found in the Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...
. Acid fuchsin is used as the nuclear and cytoplasmic stain in Mallory's trichrome method. Acid fuchsin stains cytoplasm in some variants of Masson's trichrome. In Van Gieson's picro-fuchsine, acid fuchsin imparts its red colour to collagen fibres. Acid fuchsin is also a traditional stain for mitochondria (Altmann's method).


Haematoxylin

Haematoxylin Haematoxylin or hematoxylin (), also called natural black 1 or C.I. 75290, is a compound extracted from heartwood of the logwood tree (''Haematoxylum campechianum'') with a chemical formula of . This naturally derived dye has been used as a ...
(hematoxylin in North America) is a nuclear stain. Used with a mordant, haematoxylin stains nuclei blue-violet or brown. It is most often used with eosin in the
H&E stain Hematoxylin and eosin stain ( or haematoxylin and eosin stain or hematoxylin-eosin stain; often abbreviated as H&E stain or HE stain) is one of the principal tissue stains used in histology. It is the most widely used stain in medical diagnos ...
(haematoxylin and eosin) staining, one of the most common procedures in
histology Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
.


Hoechst stains

Hoechst Hoechst, Hochst, or Höchst may refer to: * Hoechst AG, a former German life-sciences company * Hoechst stain, one of a family of fluorescent DNA-binding compounds * Höchst (Frankfurt am Main), a city district of Frankfurt am Main, Germany ** Fra ...
is a ''bis''-benzimidazole derivative compound that binds to the ''minor groove'' of DNA. Often used in fluorescence microscopy for DNA staining, Hoechst stains appear yellow when dissolved in aqueous solutions and emit blue light under UV excitation. There are two major types of
Hoechst Hoechst, Hochst, or Höchst may refer to: * Hoechst AG, a former German life-sciences company * Hoechst stain, one of a family of fluorescent DNA-binding compounds * Höchst (Frankfurt am Main), a city district of Frankfurt am Main, Germany ** Fra ...
: ''Hoechst 33258'' and ''Hoechst 33342''. The two compounds are functionally similar, but with a little difference in structure. Hoechst 33258 contains a terminal
hydroxyl In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy ...
group and is thus more soluble in aqueous solution, however this characteristics reduces its ability to penetrate the plasma membrane. Hoechst 33342 contains an
ethyl Ethyl may refer to: Arts and entertainment * Cold Ethyl, a Swedish rock band *Ethyl Sinclair, a character in the ''Dinosaurs'' television show Science and technology * Ethyl group, an organic chemistry moiety * Ethyl alcohol (or ethanol) * E ...
substitution on the terminal hydroxyl group (i.e. an ethylether group) making it more hydrophobic for easier plasma membrane passage


Iodine

Iodine Iodine is a chemical element with the symbol I and atomic number 53. The heaviest of the stable halogens, it exists as a semi-lustrous, non-metallic solid at standard conditions that melts to form a deep violet liquid at , and boils to a vi ...
is used in
chemistry Chemistry is the science, scientific study of the properties and behavior of matter. It is a natural science that covers the Chemical element, elements that make up matter to the chemical compound, compounds made of atoms, molecules and ions ...
as an indicator for
starch Starch or amylum is a polymeric carbohydrate consisting of numerous glucose units joined by glycosidic bonds. This polysaccharide is produced by most green plants for energy storage. Worldwide, it is the most common carbohydrate in human diets ...
. When starch is mixed with iodine in solution, an intensely dark blue colour develops, representing a starch/iodine complex. Starch is a substance common to most plant cells and so a weak iodine solution will stain starch present in the cells. Iodine is one component in the staining technique known as
Gram staining In microbiology and bacteriology, 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. The name comes from the Danish bac ...
, used in
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 ...
. Used as a
mordant A mordant or dye fixative is a substance used to set (i.e. bind) dyes on fabrics by forming a coordination complex with the dye, which then attaches to the fabric (or tissue). It may be used for dyeing fabrics or for intensifying stains in ...
in Gram's staining, iodine enhances the entrance of the dye through the pores present in the cell wall/membrane. Lugol's solution or Lugol's iodine (IKI) is a brown solution that turns black in the presence of starches and can be used as a cell stain, making the cell nuclei more visible. Used with common vinegar (acetic acid), Lugol's solution is used to identify pre-cancerous and cancerous changes in cervical and vaginal tissues during "Pap smear" follow up examinations in preparation for biopsy. The acetic acid causes the abnormal cells to blanch white, while the normal tissues stain a mahogany brown from the iodine.


Malachite green

Malachite green Malachite green is an organic compound that is used as a dyestuff and controversially as an antimicrobial in aquaculture. Malachite green is traditionally used as a dye for materials such as silk, leather, and paper. Despite its name the dye is ...
(also known as diamond green B or victoria green B) can be used as a blue-green counterstain to safranin in the Gimenez staining technique for bacteria. It can also be used to directly stain
spores In biology, a spore is a unit of sexual or asexual reproduction that may be adapted for dispersal and for survival, often for extended periods of time, in unfavourable conditions. Spores form part of the life cycles of many plants, algae, f ...
.


Methyl green

Methyl green Methyl green (CI 42585) is a cationic or positive charged stain, related to Ethyl Green, that has been used for staining DNA since the 19th century. It has been used for staining cell nuclei either as a part of the classical Unna-Pappenheim s ...
is used commonly with bright-field, as well as fluorescence microscopes to dye the chromatin of cells so that they are more easily viewed.


Methylene blue

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 ...
is used to stain animal cells, such as human cheek cells, to make their nuclei more observable. Also used to stain blood films in cytology.


Neutral red

Neutral red Neutral red (toluylene red, Basic Red 5, or C.I. 50040) is a eurhodin dye used for staining in histology. It stains lysosomes red. It is used as a general stain in histology, as a counterstain in combination with other dyes, and for many stai ...
(or toluylene red) stains Nissl substance red. It is usually used as a counterstain in combination with other dyes.


Nile blue

Nile blue (or Nile blue A) stains nuclei blue. It may be used with living cells.


Nile red

Nile red The Nile, , Bohairic , lg, Kiira , Nobiin: Áman Dawū is a major north-flowing river in northeastern Africa. It flows into the Mediterranean Sea. The Nile is the longest river in Africa and has historically been considered the longest rive ...
(also known as Nile blue oxazone) is formed by boiling Nile blue with
sulfuric acid Sulfuric acid (American spelling and the preferred IUPAC name) or sulphuric acid ( Commonwealth spelling), known in antiquity as oil of vitriol, is a mineral acid composed of the elements sulfur, oxygen and hydrogen, with the molecular formu ...
. This produces a mix of Nile red and Nile blue. Nile red is a
lipophilic Lipophilicity (from Greek λίπος "fat" and φίλος "friendly"), refers to the ability of a chemical compound to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene. Such non-polar solvents are themselves lipop ...
stain; it will accumulate in
lipid Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...
globules inside cells, staining them red. Nile red can be used with living cells. It fluoresces strongly when partitioned into lipids, but practically not at all in aqueous solution.


Osmium tetroxide (formal name: osmium tetraoxide)

Osmium tetraoxide Osmium tetroxide (also osmium(VIII) oxide) is the chemical compound with the Chemical formula, formula OsO4. The compound is noteworthy for its many uses, despite its toxicity and the rarity of osmium. It also has a number of unusual properties, o ...
is used in optical microscopy to stain
lipid Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...
s. It dissolves in fats, and is reduced by organic materials to elemental osmium, an easily visible black substance.


Propidium iodide

Propidium iodide Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. When in an aqueous solution, PI has a fluo ...
is a fluorescent intercalating agent that can be used to stain cells. Propidium iodide is used as a DNA stain in flow cytometry to evaluate cell viability or DNA content in cell cycle analysis, or in microscopy to visualise the nucleus and other DNA-containing organelles. Propidium Iodide cannot cross the membrane of live cells, making it useful to differentiate necrotic, apoptotic and healthy cells. PI also binds to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA staining


Rhodamine

Rhodamine Rhodamine is a family of related dyes, a subset of the triarylmethane dyes. They are derivatives of xanthene. Important members of the rhodamine family are Rhodamine 6G, Rhodamine 123, and Rhodamine B. They are mainly used to dye paper and inks ...
is a protein specific fluorescent stain commonly used in fluorescence microscopy.


Safranine

Safranine Safranin (Safranin O or basic red 2) is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endos ...
(or Safranine O) is a red cationic dye. It binds to nuclei (DNA) and other tissue
polyanions Polyelectrolytes are polymers whose repeating units bear an electrolyte group. Polycations and polyanions are polyelectrolytes. These groups dissociate in aqueous solutions (water), making the polymers charged. Polyelectrolyte properties are th ...
, including glycosaminoglycans in cartilage and mast cells, and components of lignin and plastids in plant tissues. Safranine should not be confused with saffron, an expensive natural dye that is used in some methods to impart a yellow colour to collagen, to contrast with blue and red colours imparted by other dyes to nuclei and cytoplasm in animal (including human) tissues. The incorrect spelling "safranin" is in common use. The -ine ending is appropriate for safranine O because this dye is an amine.


Stainability of tissues

Tissues which take up stains are called chromatic.
Chromosomes A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
were so named because of their ability to absorb a violet stain. Positive affinity for a specific stain may be designated by the suffix ''-philic''. For example, tissues that stain with an
azure stain Romanowsky staining, also known as Romanowsky–Giemsa staining, is a prototypical staining technique that was the forerunner of several distinct but similar stains widely used in hematology (the study of blood) and cytopathology (the study of di ...
may be referred to as
azurophil An azurophilic granule is a cellular object readily stainable with a Romanowsky stain. In white blood cells and hyperchromatin, staining imparts a burgundy or merlot coloration. Neutrophils in particular are known for containing azurophils load ...
ic. This may also be used for more generalized staining properties, such as acidophilic for tissues that stain by
acidic In computer science, ACID ( atomicity, consistency, isolation, durability) is a set of properties of database transactions intended to guarantee data validity despite errors, power failures, and other mishaps. In the context of databases, a sequ ...
stains (most notably
eosin Eosin is the name of several fluorescent acidic compounds which bind to and form salts with basic, or eosinophilic, compounds like proteins containing amino acid residues such as arginine and lysine, and stains them dark red or pink as a resul ...
),
basophilic Basophilic is a technical term used by pathologists. It describes the appearance of cells, tissues and cellular structures as seen through the microscope after a histological section has been stained with a basic dye. The most common such dye i ...
when staining in
basic BASIC (Beginners' All-purpose Symbolic Instruction Code) is a family of general-purpose, high-level programming languages designed for ease of use. The original version was created by John G. Kemeny and Thomas E. Kurtz at Dartmouth College ...
dyes, and ''amphophilic'' when staining with either acid or basic dyes. In contrast, chromophobic tissues do not take up coloured dye readily.


Electron microscopy

As in light microscopy, stains can be used to enhance contrast in
transmission electron microscopy Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a g ...
. Electron-dense compounds of heavy metals are typically used.


Phosphotungstic acid

Phosphotungstic acid Phosphotungstic acid (PTA) or tungstophosphoric acid (TPA), is a heteropoly acid with the chemical formula . It forms hydrates . It is normally isolated as the ''n'' = 24 hydrate but can be desiccated to the hexahydrate (''n'' = 6). EPTA is the na ...
is a common
negative stain In microscopy, negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and ...
for
virus A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsky's 1 ...
es,
nerve A nerve is an enclosed, cable-like bundle of nerve fibers (called axons) in the peripheral nervous system. A nerve transmits electrical impulses. It is the basic unit of the peripheral nervous system. A nerve provides a common pathway for the e ...
s,
polysaccharide Polysaccharides (), or polycarbohydrates, are the most abundant carbohydrates found in food. They are long chain polymeric carbohydrates composed of monosaccharide units bound together by glycosidic linkages. This carbohydrate can react with wa ...
s, and other biological tissue materials. It is mostly used in a .5-2% ph form making it neutral and is paired with water to make an aqueous solution. Phosphotungstic acid is filled with electron dense matter that stains the background surrounding the specimen dark and the specimen itself light. This process is not the normal positive technique for staining where the specimen is dark and the background remains light.


Osmium tetroxide

Osmium tetroxide Osmium tetroxide (also osmium(VIII) oxide) is the chemical compound with the formula OsO4. The compound is noteworthy for its many uses, despite its toxicity and the rarity of osmium. It also has a number of unusual properties, one being that the ...
is used in optical microscopy to stain
lipid Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...
s. It dissolves in fats, and is reduced by organic materials to elemental osmium, an easily visible black substance. Because it is a heavy metal that absorbs electrons, it is perhaps the most common stain used for morphology in biological electron microscopy. It is also used for the staining of various polymers for the study of their morphology by TEM. is very volatile and extremely toxic. It is a strong oxidizing agent as the osmium has an oxidation number of +8. It aggressively oxidizes many materials, leaving behind a deposit of non-volatile osmium in a lower oxidation state.


Ruthenium tetroxide

Ruthenium tetroxide Ruthenium tetroxide is the inorganic compound with the formula RuO4. It is a yellow volatile solid that melts near room temperature. It has the odor of ozone. Samples are typically black due to impurities. The analogous OsO4 is more widely used ...
is equally volatile and even more aggressive than osmium tetraoxide and able to stain even materials that resist the osmium stain, e.g. polyethylene. Other chemicals used in electron microscopy staining include:
ammonium molybdate Ammonium molybdate can refer to: *Ammonium orthomolybdate, (NH4)2MoO4 *Ammonium heptamolybdate, (NH4)6Mo7O24, usually encountered as the tetrahydrate *Ammonium phosphomolybdate, (NH4)3PMo12O40 *Ammonium tetrathiomolybdate, (NH4)2MoS4 this chemical i ...
,
cadmium iodide Cadmium iodide is the inorganic compound with the formula CdI2. It is a white hygroscopic solid. It also can be obtained as a mono- and tetrahydrate. It has few applications. It is notable for its crystal structure, which is typical for compoun ...
,
carbohydrazide Carbohydrazide is the chemical compound with the formula OC(N2H3)2. It is a white, water-soluble solid. It decomposes upon melting. A number of carbazides are known where one or more N-H groups are replaced by other substituents. They occur wi ...
,
ferric chloride Iron(III) chloride is the inorganic compound with the formula . Also called ferric chloride, it is a common compound of iron in the +3 oxidation state. The anhydrous compound is a crystalline solid with a melting point of 307.6 °C. The col ...
,
hexamine Hexamethylenetetramine, also known as methenamine, hexamine, or urotropin, is a heterocyclic organic compound with the formula (CH2)6N4. This white crystalline compound is highly soluble in water and polar organic solvents. It has a cage-like s ...
,
indium trichloride Indium(III) chloride is the chemical compound with the formula Indium, InChlorine, Cl3. This salt (chemistry), salt is a white, flaky solid with applications in organic synthesis as a Lewis acid. It is also the most available soluble derivative ...
, lanthanum(III) nitrate,
lead acetate Lead acetate can refer to: * Lead subacetate (Basic lead acetate), Pb3(OH)4(CH3COO)2 * Lead(IV) acetate Lead(IV) acetate or lead tetraacetate is an organometallic compound with chemical formula . It is a colorless solid that is soluble in nonpo ...
,
lead citrate Lead citrate is a compound of lead and citrate that is primarily used as an enhancer for heavy metal staining in electron microscopy. It binds to osmium and uranyl acetate Uranyl acetate is the acetate salt of uranium oxide, a toxic yellow-gr ...
,
lead(II) nitrate Lead(II) nitrate is an inorganic compound with the chemical formula Pb( NO3)2. It commonly occurs as a colourless crystal or white powder and, unlike most other lead(II) salts, is soluble in water. Known since the Middle Ages by the name plumbu ...
,
periodic acid Periodic acid ( ) is the highest oxoacid of iodine, in which the iodine exists in oxidation state +7. Like all periodates it can exist in two forms: orthoperiodic acid, with the chemical formula , and metaperiodic acid, which has the formula . ...
,
phosphomolybdic acid Phosphomolybdic acid is the heteropolymetalate with the formula . It is a yellow solid, although even slightly impure samples have a greenish coloration. It is also known as dodeca molybdophosphoric acid or PMA, is a yellow-green chemical compound ...
,
potassium ferricyanide Potassium ferricyanide is the chemical compound with the formula K3 e(CN)6 This bright red salt contains the octahedrally coordinated 3−.html" ;"title="e(CN)6sup>3−">e(CN)6sup>3− ion. It is soluble in water and its solution shows some g ...
,
potassium ferrocyanide Potassium ferrocyanide is the inorganic compound with formula K4 e(CN)63H2O. It is the potassium salt of the coordination complex e(CN)6sup>4−. This salt forms lemon-yellow monoclinic crystals. Synthesis In 1752, the French chemist Pie ...
,
ruthenium red The inorganic dye ammoniated ruthenium oxychloride, also known as ruthenium red, is used in histology to stain aldehyde fixed mucopolysaccharides. Ruthenium red (RR) has also been used as a pharmacological tool to study specific cellular mechani ...
,
silver nitrate Silver nitrate is an inorganic compound with chemical formula . It is a versatile precursor to many other silver compounds, such as those used in photography. It is far less sensitive to light than the halides. It was once called ''lunar caustic' ...
,
silver proteinate Silver proteinate (brand name: Protargol) is used in electron microscopy with periodic acid and thiocarbohydrazide or thiosemicarbohydrazide as a positive stain for carbohydrates such as glycogen. It can also be used for light microscopy to stain n ...
, sodium chloroaurate, thallium nitrate,
thiosemicarbazide Thiosemicarbazide is the chemical compound with the formula H2NC(S)NHNH2. A white, odorless solid, it is related to thiourea (H2NC(S)NH2) by the insertion of an NH center. They are commonly used as ligands for transition metals. Many thiosemicar ...
,
uranyl acetate Uranyl acetate is the acetate salt of uranium oxide, a toxic yellow-green powder useful in certain laboratory tests. Structurally, it is a coordination polymer with formula UO2(CH3CO2)2(H2O)·H2O. Structure In the polymer, uranyl (UO22+) ...
,
uranyl nitrate Uranyl nitrate is a water-soluble yellow uranium salt with the formula . The hexa-, tri-, and dihydrates are known. The compound is mainly of interest because it is an intermediate in the preparation of nuclear fuels. Uranyl nitrate can be prepa ...
, and vanadyl sulfate.


See also

*
Biological Stain Commission The Biological Stain Commission (BSC) is an organization that provides third-party testing and certification of dyes and a few other compounds that are used to enhance contrast in specimens examined in biological and medical laboratories. The BS ...
: Third-party quality control and certification of stains *
Cytology Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living an ...
: the study of cells *
Histology Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures vis ...
: the study of tissues *
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 ...
: the use of antisera to label specific antigens * Ruthenium(II) tris(bathophenanthroline disulfonate), a protein dye. *
Vital stain A vital stain in a casual usage may mean a stain that can be applied on living cells without killing them. Vital stains have been useful for diagnostic and surgical techniques in a variety of medical specialties. In supravital staining, living cell ...
: stains that do not kill cells *
PAGE Page most commonly refers to: * Page (paper), one side of a leaf of paper, as in a book Page, PAGE, pages, or paging may also refer to: Roles * Page (assistance occupation), a professional occupation * Page (servant), traditionally a young m ...
: separation of protein molecules *
Barium enema A lower gastrointestinal series is a medical procedure used to examine and diagnose problems with the human colon of the large intestine. Radiographs (X-ray pictures) are taken while barium sulfate, a radiocontrast agent, fills the colon via an ...
- a type of in vivo stain that creates contrast in the x-ray part of the light spectrum * Diaphonization


References


Further reading

* * * *


External links


The Biological Stain commission
is an independent non-profit company that has been testing dyes since the early 1920s and issuing Certificates of approval for batches of dyes that meet internationally recognized standards.
StainsFile
Reference for dyes and staining techniques.

~ Howey, 2000

an

– by M. Halit Umar



at Sridhar Rao P.N's home page {{Clinical microbiology techniques dyes
pigments A pigment is a colored material that is completely or nearly insoluble in water. In contrast, dyes are typically soluble, at least at some stage in their use. Generally dyes are often organic compounds whereas pigments are often inorganic compou ...
Staining dyes Scientific techniques Biological techniques and tools