Staining (biology)
Overview
 
Staining is an auxiliary technique used in microscopy
Microscopy
Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...

 to enhance contrast in the microscopic
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

 image. Stains and dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....

s are frequently used in biology
Biology
Biology is a natural science concerned with the study of life and living organisms, including their structure, function, growth, origin, evolution, distribution, and taxonomy. Biology is a vast subject containing many subdivisions, topics, and disciplines...

 and medicine
Medicine
Medicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....

 to highlight structures in biological tissue
Biological tissue
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...

s for viewing, often with the aid of different microscope
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

s. Stains may be used to define and examine bulk tissues (highlighting, for example, muscle fibers or connective tissue
Connective tissue
"Connective tissue" is a fibrous tissue. It is one of the four traditional classes of tissues . Connective Tissue is found throughout the body.In fact the whole framework of the skeleton and the different specialized connective tissues from the crown of the head to the toes determine the form of...

), 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....

 populations (classifying different blood cell
Blood cell
A blood cell, also called a hematocyte, is a cell normally found in blood. In mammals, these fall into three general categories:* red blood cells — Erythrocytes* white blood cells — Leukocytes* platelets — Thrombocytes...

s, for instance), or organelle
Organelle
In cell biology, an organelle is a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own lipid bilayer....

s within individual cells.

In biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

 it involves adding a class-specific (DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

, protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s, lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...

s, carbohydrate
Carbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...

s) dye to a substrate to qualify or quantify the presence of a specific compound.
Discussions
Encyclopedia
Staining is an auxiliary technique used in microscopy
Microscopy
Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...

 to enhance contrast in the microscopic
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

 image. Stains and dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....

s are frequently used in biology
Biology
Biology is a natural science concerned with the study of life and living organisms, including their structure, function, growth, origin, evolution, distribution, and taxonomy. Biology is a vast subject containing many subdivisions, topics, and disciplines...

 and medicine
Medicine
Medicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....

 to highlight structures in biological tissue
Biological tissue
Tissue is a cellular organizational level intermediate between cells and a complete organism. A tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function. These are called tissues because of their identical functioning...

s for viewing, often with the aid of different microscope
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

s. Stains may be used to define and examine bulk tissues (highlighting, for example, muscle fibers or connective tissue
Connective tissue
"Connective tissue" is a fibrous tissue. It is one of the four traditional classes of tissues . Connective Tissue is found throughout the body.In fact the whole framework of the skeleton and the different specialized connective tissues from the crown of the head to the toes determine the form of...

), 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....

 populations (classifying different blood cell
Blood cell
A blood cell, also called a hematocyte, is a cell normally found in blood. In mammals, these fall into three general categories:* red blood cells — Erythrocytes* white blood cells — Leukocytes* platelets — Thrombocytes...

s, for instance), or organelle
Organelle
In cell biology, an organelle is a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own lipid bilayer....

s within individual cells.

In biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...

 it involves adding a class-specific (DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

, protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s, lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...

s, carbohydrate
Carbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...

s) dye to a substrate to qualify or quantify the presence of a specific compound. Staining and fluorescent tag
Fluorescent tag
In molecular biology and biotechnology, a fluorescent tag is a part of a molecule that researchers have attached chemically to aid in detection of the molecule to which it has been attached. The tag is some kind of fluorescent molecule...

ging can serve similar purposes. Biological staining is also used to mark cells in flow cytometry
Flow cytometry
Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...

, and to flag protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s or nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...

s in gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...

.

Staining is not limited to biological materials, it can also be used to study the morphology
Morphology (biology)
In biology, morphology is a branch of bioscience dealing with the study of the form and structure of organisms and their specific structural features....

 of other materials for example the lamellar structures of semi-crystalline polymer
Polymer
A polymer is a large molecule composed of repeating structural units. These subunits are typically connected by covalent chemical bonds...

s or the domain structures of block copolymers.

In vivo vs In vitro

In vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...

staining ( Vital Staining ) is the process of dyeing living tissues—in vivo means "in life" (compare with in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...

staining). By causing certain cells or structures to take on contrasting colour(s), their form (morphology) 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
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...

staining involves colouring cells or structures that are no longer living. Certain stains are often combined to reveal more details and features than a single stain alone. Combined with specific protocols for fixation
Fixation (histology)
In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...

 and sample preparation, scientists and physicians can use these standard techniques as consistent, repeatable diagnostic tools. A counterstain
Counterstain
A counterstain is a stain with color contrasting to the principal stain, making the stained structure more easily visible.An example is 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.
  • For example, crystal violet
    Crystal violet
    Crystal violet or Gentian violet is a triarylmethane dye. The dye is used as a histological stain and in Gram’s method of classifying bacteria. Crystal violet has antibacterial, antifungal, and anthelmintic properties and was formerly important as a topical antiseptic...

     stains only Gram-positive
    Gram-positive
    Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain and appearing red or pink...

     bacteria in Gram staining
    Gram staining
    Gram staining is a method of differentiating bacterial species into two large groups ....

    . A safranin
    Safranin
    Safranin is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. This is the classic counterstain in a Gram stain...

     counterstain is applied which stains all cells, allowing the identification of Gram-negative bacteria as well.


Often these stains are called vital stains. They are introduced to the organism while the cells are still living. However, these stains are eventually toxic to the organism, some more so than others. 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.

Fixation
Fixation (histology)
In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...

–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 is used to kill, adhere, and alter the specimen so it will accept stains. Most chemical fixatives (chemicals causing fixation) generate chemical bond
Chemical bond
A chemical bond is an attraction between atoms that allows the formation of chemical substances that contain two or more atoms. The bond is caused by the electromagnetic force attraction between opposite charges, either between electrons and nuclei, or as the result of a dipole attraction...

s between protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s and other substances within the sample, increasing their rigidity. Common fixatives include formaldehyde
Formaldehyde
Formaldehyde is an organic compound with the formula CH2O. It is the simplest aldehyde, hence its systematic name methanal.Formaldehyde is a colorless gas with a characteristic pungent odor. It is an important precursor to many other chemical compounds, especially for polymers...

, ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...

, 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...

, and/or picric acid
Picric acid
Picric acid is the chemical compound formally called 2,4,6-trinitrophenol . This yellow crystalline solid is one of the most acidic phenols. Like other highly nitrated compounds such as TNT, picric acid is an explosive...

. Pieces of tissue may be embedded in paraffin
Paraffin
In chemistry, paraffin is a term that can be used synonymously with "alkane", indicating hydrocarbons with the general formula CnH2n+2. Paraffin wax refers to a mixture of alkanes that falls within the 20 ≤ n ≤ 40 range; they are found in the solid state at room temperature and begin to enter the...

 wax to increase their mechanical strength and stability and to make them easier to cut into thin slices.

Permeabilization involves treatment of cells with (usually) a mild surfactant
Surfactant
Surfactants are compounds that lower the surface tension of a liquid, the interfacial tension between two liquids, or that between a liquid and a solid...

. This treatment will dissolve the cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...

s, and allow larger dye molecules access to 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
Pap smear
The Papanicolaou test is a screening test used in to detect pre-cancerous and cancerous processes in the endocervical canal of the female reproductive system. Changes can be treated, thus preventing cervical cancer...

) the sample can be directly applied to a slide. For larger pieces of tissue, thin sections (slices) are made using a microtome
Microtome
A microtome is a sectioning instrument that allows for the cutting of extremely thin slices of material, known as sections. Microtomes are an important device in microscopy preparation, allowing for the preparation of samples for observation under transmitted light or electron radiation...

; these slices can then be mounted and inspected.

Staining proper

At its simplest, the actual staining process may involve immersing the sample (before or after fixation and mounting) in dye solution, followed by rinsing and observation.
Many dyes, however, require the use of a mordant
Mordant
A mordant is a substance used to set dyes on fabrics or tissue sections 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 cell or tissue preparations. The term mordant comes from the Latin...

: a chemical compound which reacts with the stain to form an insoluble, coloured precipitate
Precipitation (chemistry)
Precipitation is the formation of a solid in a solution or inside anothersolid during a chemical reaction or by diffusion in a solid. When the reaction occurs in a liquid, the solid formed is called the precipitate, or when compacted by a centrifuge, a pellet. The liquid remaining above the solid...

. When excess dye solution is washed away, the mordanted stain remains.

Most of the dyes commonly used in microscopy are available as certified stains. This means that samples of the manufacturer's batch have been tested by an independent body, the Biological Stain Commission
Biological Stain Commission
The BSC is an 85 year-old organization well known to many thousands of scientists, worldwide but especially in N America, who buy certified dyes for staining of microscopic preparations or for making up selective culture media for bacteria. Manufacturers and other vendors submit samples from their...

 and found to be meet or exceed certain standards of purity, dye content and performance in staining techniques. These standards are published in detail in the journal Biotechnic & Histochemistry
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 bimonthly in print and online by Informa on behalf of the Biological Stain Commission...

 Many dyes are inconsistent in composition from one supplier to another. The use of certified stains eliminates a source of unexpected results.

Negative staining

A simple staining method for bacteria which is usually successful even when the "positive staining" methods detailed below fail, is to employ a negative stain
Negative stain
Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid....

. This can be achieved simply by smearing the sample on to the slide, followed by an application of nigrosin
Nigrosin
Nigrosin is a mixture of synthetic black dyes made by heating a mixture of nitrobenzene, aniline and aniline hydrochloride in the presence of a copper or iron catalyst. Its main industrial uses are as a colorant for lacquers and varnishes and in marker-pen inks...

 (a black synthetic dye) or Indian 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. Note: negative staining is a mild technique which may not destroy the microorganisms therefore it is unsuitable for studying pathogens.

Gram staining

Gram staining
Gram staining
Gram staining is a method of differentiating bacterial species into two large groups ....

 is used to determine gram status to classify bacteria broadly. It is based on the composition of their cell wall
Cell wall
The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...

. Gram staining uses crystal violet to stain cell walls, iodine
Iodine
Iodine is a chemical element with the symbol I and atomic number 53. The name is pronounced , , or . The name is from the , meaning violet or purple, due to the color of elemental iodine vapor....

 as a mordant, and a fuchsin or safranin
Safranin
Safranin is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. This is the classic counterstain in a Gram stain...

 counterstain to mark all bacteria. Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium's susceptibility to some antibiotic
Antibiotic
An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic; today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic has come to denote a broader range of...

s.

Gram-positive
Gram-positive
Gram-positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counterstain and appearing red or pink...

 bacteria stain dark blue or violet. Their cell wall
Cell wall
The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...

 is typically rich with peptidoglycan
Peptidoglycan
Peptidoglycan, also known as murein, is a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria , forming the cell wall. The sugar component consists of alternating residues of β- linked N-acetylglucosamine and N-acetylmuramic acid...

 and lacks the secondary membrane and lipopolysaccharide
Lipopolysaccharide
Lipopolysaccharides , also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals.-Functions:LPS is the major...

 layer found in Gram-negative bacteria.

On most Gram-stained preparations, Gram-negative
Gram-negative
Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color...

 organisms will appear red or pink because they are counterstained. Due to 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. Also, 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.

Ziehl-Neelsen stain

Ziehl-Neelsen stain
Ziehl-Neelsen stain
The Ziehl–Neelsen stain, also known as the acid-fast stain, was first described by two German doctors; Franz Ziehl , a bacteriologist and Friedrich Neelsen , a pathologist. It is a special bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria...

ing is used to stain species of Mycobacterium tuberculosis
Mycobacterium tuberculosis
Mycobacterium tuberculosis is a pathogenic bacterial species in the genus Mycobacterium and the causative agent of most cases of tuberculosis . First discovered in 1882 by Robert Koch, M...

that do not stain with the standard laboratory staining procedures like Gram staining.

The stains used are the red coloured Carbol fuchsin
Carbol fuchsin
Carbol fuchsin, carbol-fuchsin, or carbolfuchsin, is a mixture of phenol and basic fuchsin, used in bacterial staining procedures. It is commonly used in the staining of mycobacteria as it has an affinity for the mycolic acids found in their cell walls.It is a component of Ziehl-Neelsen...

 that stains the bacteria and a counter stain like 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...

 or Malachite green
Malachite green
Malachite green is an organic compound that is used as a dyestuff and has emerged as a controversial agent in aquaculture. Malachite green is traditionally used as a dye for materials such as silk, leather, and paper...

.

Haematoxylin and eosin (H&E) staining

Haematoxylin and eosin staining protocol is used frequently in histology
Histology
Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...

 to examine thin sections of tissue. Haematoxylin
Haematoxylin
Haematoxylin, hematoxylin, Natural Black 1, or C.I. 75290 is extracted from the heartwood of the logwood tree. When oxidized it forms haematein, a compound that forms strongly coloured complexes with certain metal ions, the most notable ones being Fe and Al salts. Metal-haematein complexes are used...

 stains cell nuclei blue, while 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....

 stains cytoplasm, connective tissue and other extracellular substances pink or red. Eosin is strongly absorbed by red blood cell
Red blood cell
Red blood cells are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen to the body tissues via the blood flow through the circulatory system...

s, colouring them bright red. In a skilfully made H & E preparation the red blood cells are almost orange, and collagen and cytoplasm (especially muscle) acquire different shades of pink. When the staining is done by a machine, the subtle differences in eosinophilia are often lost. Hematoxylin stains the cell nucleus and other acidic structures (such as RNA-rich portions of the cytoplasm and the matrix of hyaline cartilage) blue. In contrast, eosin stains the cytoplasm and collagen pink.

Papanicolaou staining

Papanicolaou stain
Papanicolaou stain
Papanicolaou stain is a multichromatic staining histological technique developed by George Papanikolaou, the father of cytopathology....

ing, or Pap staining, is a frequently used method for examining cell samples from various bodily secretions. It is frequently used to stain Pap smear
Pap smear
The Papanicolaou test is a screening test used in to detect pre-cancerous and cancerous processes in the endocervical canal of the female reproductive system. Changes can be treated, thus preventing cervical cancer...

 specimens. It uses a combination of haematoxylin
Haematoxylin
Haematoxylin, hematoxylin, Natural Black 1, or C.I. 75290 is extracted from the heartwood of the logwood tree. When oxidized it forms haematein, a compound that forms strongly coloured complexes with certain metal ions, the most notable ones being Fe and Al salts. Metal-haematein complexes are used...

, Orange G
Orange G
Orange G 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:...

, eosin Y
Eosin Y
Eosin Y is a form of eosin....

, Light Green SF yellowish
Light Green SF yellowish
Light Green SF yellowish, or Light Green, Acid Green, Lissamine green SF, Acid Green 5, Food Green 2, FD&C Green no. 2, Green No. 205, Acid Brilliant Green 5, Pencil Green SF, or C.I. 42095, is a green triarylmethane dye. It is used in histology for staining collagen; for that purpose it is a...

, and sometimes Bismarck Brown Y
Bismarck brown Y
Bismarck brown Y is a diazo dye. It is used in histology for staining tissues. It stains acid mucins to yellow color. It can be used with live cells...

.

PAS staining

Periodic acid-Schiff
Periodic acid-Schiff
Periodic acid-Schiff is a staining method used to detect glycogen and other polysaccharides in tissues. The reaction of periodic acid oxidizes the diol functional groups in glucose and other sugars, creating aldehydes that react with the Schiff reagent to give a purple-magenta color...

 staining is used to mark carbohydrate
Carbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...

s (glycogen
Glycogen
Glycogen is a molecule that serves as the secondary long-term energy storage in animal and fungal cells, with the primary energy stores being held in adipose tissue...

, glycoprotein
Glycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...

, proteoglycan
Proteoglycan
Proteoglycans are proteins that are heavily glycosylated. The basic proteoglycan unit consists of a "core protein" with one or more covalently attached glycosaminoglycan chain. The point of attachment is a Ser residue to which the glycosaminoglycan is joined through a tetrasaccharide bridge...

s). It is used to distinguish different types of glycogen storage diseases.

Masson's trichrome

Masson's trichrome
Masson's trichrome
Masson's trichrome is a three-colour staining protocol used in histology. The recipes evolved from Claude L. Pierre Masson's original formulation to different specific applications, but all are suited for distinguishing cells from surrounding connective tissue....

 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
"Connective tissue" is a fibrous tissue. It is one of the four traditional classes of tissues . Connective Tissue is found throughout the body.In fact the whole framework of the skeleton and the different specialized connective tissues from the crown of the head to the toes determine the form of...

. Most recipes will produce red keratin
Keratin
Keratin refers to a family of fibrous structural proteins. Keratin is the key of structural material making up the outer layer of human skin. It is also the key structural component of hair and nails...

 and muscle fibers, blue or green staining of collagen
Collagen
Collagen is a group of naturally occurring proteins found in animals, especially in the flesh and connective tissues of mammals. It is the main component of connective tissue, and is the most abundant protein in mammals, making up about 25% to 35% of the whole-body protein content...

 and bone
Bone
Bones are rigid organs that constitute part of the endoskeleton of vertebrates. They support, and protect the various organs of the body, produce red and white blood cells and store minerals. Bone tissue is a type of dense connective tissue...

, light red or pink staining of cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...

, and black cell nuclei
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...

.

Romanowsky stains

The Romanowsky stain
Romanowsky stain
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 cells in pathologic specimens....

s are all based on a combination of eosinate (chemically reduced 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....

) and 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...

 (sometimes with its oxidation products 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....

 and azure B). Common variants include Wright's stain
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...

, Jenner's stain
Jenner's stain
Jenner's Stain is used in microscopy for staining blood smears....

, Leishman stain
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...

 and Giemsa stain
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:...

.

All are used to examine blood
Blood
Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....

 or bone marrow
Bone marrow
Bone marrow is the flexible tissue found in the interior of bones. In humans, bone marrow in large bones produces new blood cells. On average, bone marrow constitutes 4% of the total body mass of humans; in adults weighing 65 kg , bone marrow accounts for approximately 2.6 kg...

 samples. They are preferred over H&E for inspection of blood cells because different types of leukocytes (white blood cells) can be readily distinguished. All are also suited to examination of blood to detect blood-borne parasites like malaria
Malaria
Malaria is a mosquito-borne infectious disease of humans and other animals caused by eukaryotic protists of the genus Plasmodium. The disease results from the multiplication of Plasmodium parasites within red blood cells, causing symptoms that typically include fever and headache, in severe cases...

.

Silver staining

Silver stain
Silver stain
Silver staining is the use of silver to selectively alter the appearance of the target.-Use in medicine:It is used to stain histologic sections. This kind of staining is important especially to show proteins and DNA. It is used to show both substances inside and outside cells...

ing is the use of silver
Silver
Silver is a metallic chemical element with the chemical symbol Ag and atomic number 47. A soft, white, lustrous transition metal, it has the highest electrical conductivity of any element and the highest thermal conductivity of any metal...

 to stain histologic sections. This kind of staining is important especially to show protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s (for example type III collagen
Collagen
Collagen is a group of naturally occurring proteins found in animals, especially in the flesh and connective tissues of mammals. It is the main component of connective tissue, and is the most abundant protein in mammals, making up about 25% to 35% of the whole-body protein content...

) and DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

. It is used to show both substances inside and outside 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. Silver staining is also used in temperature gradient gel electrophoresis
Temperature gradient gel electrophoresis
Temperature Gradient Gel Electrophoresis and Denaturing Gradient Gel Electrophoresis are forms of electrophoresis which use either a temperature or chemical gradient to denature the sample as it moves across an acrylamide gel. TGGE and DGGE can be applied to nucleic acids such as DNA and RNA,...

.

Some cells are argentaffin. These reduce
Redox
Redox reactions describe all chemical reactions in which atoms have their oxidation state changed....

 silver solution to metallic silver after formalin fixation
Fixation (histology)
In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological tissues are preserved from decay, thereby preventing autolysis or putrefaction...

. This method was discovered by Italian Camillo Golgi
Camillo Golgi
Camillo Golgi was an Italian physician, pathologist, scientist, and Nobel laureate.-Biography:Camillo Golgi was born in the village of Corteno, Lombardy, then part of the Austrian Empire. The village is now named Corteno Golgi in his honour. His father was a physician and district medical officer...

, by using a reaction between silver nitrate
Silver nitrate
Silver nitrate is an inorganic compound with chemical formula . This compound 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...

 and potassium dichromate, thus precipitating silver chromate in some cells (see Golgi's method
Golgi's method
Golgi's method is a nervous tissue staining technique discovered by Italian physician and scientist Camillo Golgi in 1873. It was initially named the black reaction by Golgi, but it became better known as the Golgi stain or later, Golgi method.Golgi' staining was famously used by Spanish...

). Other cells are argyrophilic. These reduce silver solution to metallic silver after being exposed to the stain that contains a reductant
Reducing agent
A reducing agent is the element or compound in a reduction-oxidation reaction that donates an electron to another species; however, since the reducer loses an electron we say it is "oxidized"...

, for example hydroquinone
Hydroquinone
Hydroquinone, also benzene-1,4-diol or quinol, is an aromatic organic compound that is a type of phenol, having the chemical formula C6H42. Its chemical structure, shown in the table at right, has two hydroxyl groups bonded to a benzene ring in a para position. It is a white granular solid...

 or formalin.

Sudan staining

Sudan stain
Sudan stain
Sudan staining is the use of Sudan dyes to stain sudanophilic substances, usually lipids. Sudan lysochromes are used....

ing is the use of Sudan dyes to stain sudanophilic substances, usually lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...

s. Sudan III
Sudan III
Sudan III is a lysochrome diazo dye used for staining of triglycerides in frozen sections, and some protein bound lipids and lipoproteins on paraffin sections. It has the appearance of reddish brown crystals and a maximum absorption at 507 nm.Sudan III is a dye used for Sudan staining...

, Sudan IV
Sudan IV
Sudan IV is a lysochrome 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 maximum absorption at 520 nm.Sudan IV is one of the dyes used for Sudan staining. Similar...

, Oil Red O
Oil Red O
Oil Red O is a lysochrome diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections...

, and Sudan Black B
Sudan Black B
Sudan Black B is a lysochrome diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. It has the appearance of a dark brown to black powder with maximum absorption at 596-605 nm and melting point 120-124 °C...

 are often used. Sudan staining is often used to determine the level of fecal fat
Fecal fat
In medicine, the fecal fat test is a diagnostic test for fat malabsorption conditions, which lead to excess fat in the feces .-Background:...

 to diagnose steatorrhea
Steatorrhea
Steatorrhea is the presence of excess fat in feces. Stools may also float due to excess lipid, have an oily appearance and be especially foul-smelling. An oily anal leakage or some level of fecal incontinence may occur. There is increased fat excretion, which can be measured by determining the...

.

Conklin's staining

Special technique designed for staining true endospores with the use of malachite green dye, once stained, they do not decolourize.

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
Acridine orange 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 respectively. When bound to DNA, it is very similar spectrally to fluorescein, with an...

 (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
Bismarck brown
Bismarck brown may refer to:* Bismarck brown R, basic brown 4* Bismarck brown Y, basic brown 1...

 (also Bismarck brown Y or Manchester brown) imparts a yellow colour to acid mucin
Mucin
Mucins are a family of high molecular weight, heavily glycosylated proteins produced by epithelial tissues in most metazoans. Mucins' key characteristic is their ability to form gels; therefore they are a key component in most gel-like secretions, serving functions from lubrication to cell...

s. Bismarck brown may be used with live cells.

Carmine

Carmine
Carmine
Carmine , also called Crimson Lake, Cochineal, Natural Red #4, C.I. 75470, or E120, is a pigment of a bright-red color obtained from the aluminum salt of carminic acid, which is produced by some scale insects, such as the cochineal beetle and the Polish cochineal, and is used as a general term for...

 is an intensely red dye which may be used to stain glycogen
Glycogen
Glycogen is a molecule that serves as the secondary long-term energy storage in animal and fungal cells, with the primary energy stores being held in adipose tissue...

, while Carmine alum is a nuclear stain. Carmine stains require the use of a mordant, usually aluminum.

Coomassie blue

Coomassie blue (also brilliant blue) nonspecifically stains proteins a strong blue colour. It is often used in gel electrophoresis.

Crystal violet

Crystal violet
Methyl violet
Methyl violet is a family of organic compounds that are mainly used as dyes. Depending on the amount of attached methyl groups, the color of the dye can be altered. Its main use is as a purple dye for textiles and to give deep violet colors in paint and ink...

, when combined with a suitable mordant, stains cell wall
Cell wall
The cell wall is the tough, usually flexible but sometimes fairly rigid layer that surrounds some types of cells. It is located outside the cell membrane and provides these cells with structural support and protection, and also acts as a filtering mechanism. A major function of the cell wall is to...

s purple. Crystal violet is an important component in Gram staining.

DAPI

DAPI
DAPI
DAPI or 4',6-diamidino-2-phenylindole is a fluorescent stain that binds strongly to A-T rich regions in DNA. It is used extensively in fluorescence microscopy...

 is a fluorescent nuclear stain, excited by ultraviolet
Ultraviolet
Ultraviolet light is electromagnetic radiation with a wavelength shorter than that of visible light, but longer than X-rays, in the range 10 nm to 400 nm, and energies from 3 eV to 124 eV...

 light and showing strong blue fluorescence when bound to DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

. 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
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....

 is most often used as a counterstain to haematoxylin, imparting a pink or red colour to cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...

ic material, cell membrane
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...

s, and some extracellular structures. It also imparts a strong red colour to red blood cell
Red blood cell
Red blood cells are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen to the body tissues via the blood flow through the circulatory system...

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 (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
Ethidium bromide is an intercalating agent commonly used as a fluorescent tag in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane...

 intercalates
Intercalation (chemistry)
In chemistry, intercalation is the reversible inclusion of a molecule between two other molecules . Examples include DNA intercalation and graphite intercalation compounds.- DNA intercalation :...

 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
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...

 - such cells have much more permeable membrane
Biological membrane
A biological membrane or biomembrane is an enclosing or separatingmembrane that acts as a selective barrier, within or around a cell. It consists of a lipid bilayer with embedded proteins that may constitute close to 50% of membrane content...

s. 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
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...

. The stain may also be used in conjunction with acridine orange
Acridine orange
Acridine orange 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 respectively. When bound to DNA, it is very similar spectrally to fluorescein, with an...

 (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 fuchsine

Acid 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....

 may be used to stain collagen, smooth muscle, or mitochondria
Mitochondrion
In cell biology, a mitochondrion is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 1.0 micrometers in diameter...

.
Acid fuchsine is used as the nuclear and cytoplasmic stain in Mallory's trichrome method. Acid fuchsine stains cytoplasm in some variants of Masson's trichrome. In Van Gieson's picro-fuchsine, acid fuchsine imparts its red colour to collagen fibres. Acid fuchsine is also a traditional stain for mitochondria (Altmann's method).

Haematoxylin

Haematoxylin
Haematoxylin
Haematoxylin, hematoxylin, Natural Black 1, or C.I. 75290 is extracted from the heartwood of the logwood tree. When oxidized it forms haematein, a compound that forms strongly coloured complexes with certain metal ions, the most notable ones being Fe and Al salts. Metal-haematein complexes are used...

 (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 H&E (haematoxylin and eosin) staining—one of the most common procedures in histology
Histology
Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...

.

Hoechst stains

Hoechst
Hoechst stain
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These Bis-benzimides were originally developed by the Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33342nd compound made by the company. There are three related Hoechst stains:...

 is a bis-benzimidazole derivative compound which binds to the minor groove of DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

. 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 stain
Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These Bis-benzimides were originally developed by the Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33342nd compound made by the company. There are three related Hoechst stains:...

: Hoechst 33258 and Hoechst 33342. The two compounds are functionally similar, but with a little difference in structure. Hoechst 33258 contains a terminal hydroxyl
Hydroxyl
A hydroxyl is a chemical group containing an oxygen atom covalently bonded with a hydrogen atom. In inorganic chemistry, the hydroxyl group is known as the hydroxide ion, and scientists and reference works generally use these different terms though they refer to the same chemical structure in...

 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 group
In chemistry, an ethyl group is an alkyl substituent derived from ethane . It has the formula -C2H5 and is very often abbreviated -Et.Ethylation is the formation of a compound by introduction of the ethyl functional group, C2H5....

 substitution on the terminal hydroxyl group (i.e. an ethylether group) making it more hydrophobic for easier plasma membrane passage

Iodine

Iodine
Iodine
Iodine is a chemical element with the symbol I and atomic number 53. The name is pronounced , , or . The name is from the , meaning violet or purple, due to the color of elemental iodine vapor....

 is used in chemistry
Chemistry
Chemistry is the science of matter, especially its chemical reactions, but also its composition, structure and properties. Chemistry is concerned with atoms and their interactions with other atoms, and particularly with the properties of chemical bonds....

 as an indicator for starch
Starch
Starch or amylum is a carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. This polysaccharide is produced by all green plants as an energy store...

. 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
Gram staining
Gram staining is a method of differentiating bacterial species into two large groups ....

, used in microbiology
Microbiology
Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...

.
Lugol's solution
Lugol's iodine
Lugol's iodine, also known as Lugol's solution, first made in 1829, is a solution of elemental iodine and potassium iodide in water, named after the French physician J.G.A. Lugol. Lugol's iodine solution is often used as an antiseptic and disinfectant, for emergency disinfection of drinking water,...

 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
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...

 more visible. Iodine is also used as a mordant in Gram's staining, it enhances dye to enter through the pore present in the cell wall/membrane.

Malachite green

Malachite green
Malachite green
Malachite green is an organic compound that is used as a dyestuff and has emerged as a controversial agent in aquaculture. Malachite green is traditionally used as a dye for materials such as silk, leather, and paper...

 (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
Gimenez stain
The Gimenez staining technique uses biological stains to detect and identify bacterial infections in tissue samples. Although largely superseded by techniques like Giemsa staining, the Gimenez technique may be valuable for detecting certain slow-growing or fastidious bacteria.Basic fuchsin stain ...

 for bacteria. It also can be used to directly stain spore
Endospore
An endospore is a dormant, tough, and temporarily non-reproductive structure produced by certain bacteria from the Firmicute phylum. The name "endospore" is suggestive of a spore or seed-like form , but it is not a true spore . It is a stripped-down, dormant form to which the bacterium can reduce...

s.

Methyl green

Methyl green is used commonly with bright-field microscopes to dye the chromatin of cells so that they are more easily viewed.

Methylene blue

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...

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

Neutral red

Neutral red
Neutral red
Neutral Red 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 staining methods. Together with Janus Green B, it is used to stain embryonal tissues and supravital...

 (or toluylene red) stains Nissl substance
Nissl body
A Nissl body is a large granular body found in neurons. These granules are rough endoplasmic reticulum and are the site of protein synthesis...

 red. It is usually used as a counterstain in combination with other dyes.

Nile blue

Nile blue
Nile blue
Nile blue is a stain used in biology and histology. It may be used with live or fixed cells, and imparts a blue colour to cell nuclei....

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

Nile red

Nile red
Nile red
Nile red is a lipophilic stain. It is produced by boiling a solution of Nile blue with sulfuric acid. As can be seen from the structural formulae, this process replaces an amino group with a carbonyl group. Nile red stains intracellular lipid droplets red...

 (also known as Nile blue oxazone) is formed by boiling Nile blue with sulfuric acid
Sulfuric acid
Sulfuric acid is a strong mineral acid with the molecular formula . Its historical name is oil of vitriol. Pure sulfuric acid is a highly corrosive, colorless, viscous liquid. The salts of sulfuric acid are called sulfates...

. This produces a mix of Nile red and Nile blue. Nile red is a lipophilic
Lipophilic
Lipophilicity, , refers to the ability of a chemical compound to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene. These non-polar solvents are themselves lipophilic — the axiom that like dissolves like generally holds true...

 stain; it will accumulate in lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...

 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 is used in optical microscopy to stain lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...

s. It dissolves in fats, and is reduced by organic materials to elemental osmium, an easily visible black substance.

Rhodamine

Rhodamine
Rhodamine
Rhodamine is a family of related chemical compounds, fluorone dyes. Examples are Rhodamine 6G and Rhodamine B. They are used as a dye and as a dye laser gain medium. They are often used as a tracer dye within water to determine the rate and direction of flow and transport...

 is a protein specific fluorescent stain commonly used in fluorescence microscopy.

Safranin

Safranin
Safranin
Safranin is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. This is the classic counterstain in a Gram stain...

 (or Safranin O) is a nuclear stain. It produces red nuclei, and is used primarily as a counterstain. Safranin may also be used to give a yellow colour to collagen.

Stainability of tissues

Positive affinity for a specific stain may be designated by the suffix -philic. For example, tissues that stain with an azure
Azure
In heraldry, azure is the tincture with the colour blue, and belongs to the class of tinctures called "colours". In engraving, it is sometimes depicted as a region of horizontal lines or else marked with either az. or b. as an abbreviation....

 dye may be referred to as azurophil
Azurophil
An azurophil is an object readily stained with an azure dye. Azurophils include certain cytoplasmic granules in white blood cells and hyperchromatin, imparting a burgundy or merlot coloration. Neutrophils in particular are known for containing azurophils loaded with a wide variety of...

ic. This may also be used for more generalized staining properties, such as acidophilic
Acidophile (histology)
An acidophile describes is a term used by histologists to describe a particular staining pattern of cells and tissues when using haematoxylin and eosin stains...

 for tissues that stain by acidic stains (most notably 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....

), basophilic
Basophilic
Basophilic is a technical term used by histologists. It describes the microscopic appearance of cells and tissues, as seen down the microscope, after a histological section has been stained with a basic dye. The most common such dye is haematoxylin....

 when staining in basic
Base (chemistry)
For the term in genetics, see base A base in chemistry is a substance that can accept hydrogen ions or more generally, donate electron pairs. A soluble base is referred to as an alkali if it contains and releases hydroxide ions quantitatively...

 dyes and amphophilic when staining with either acid or basic dyes. In contrast, Chromophobic
Chromophobe
The term chromophobe refers to histological structures which do not stain readily, and thus appear more relatively pale under the microscope—hence their "fear" of "color" .-Cancer:...

 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
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...

. Electron-dense compounds of heavy metals are typically used.

Phosphotungstic acid

Phosphotungstic acid
Phosphotungstic acid
Phosphotungstic acid , tungstophosphoric acid , is a heteropoly acid with the chemical formula 31240. It normally present as a hydrate. EPTA is the name of ethanolic phosphotungstic acid, its alcohol solution used in biology. It has the appearance of small, colorless-grayish or slightly...

 is a common negative stain
Negative stain
Negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid....

 for virus
Virus
A virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...

es, nerve
Nerve
A peripheral nerve, or simply nerve, is an enclosed, cable-like bundle of peripheral axons . A nerve provides a common pathway for the electrochemical nerve impulses that are transmitted along each of the axons. Nerves are found only in the peripheral nervous system...

s, polysaccharide
Polysaccharide
Polysaccharides are long carbohydrate molecules, of repeated monomer units joined together by glycosidic bonds. They range in structure from linear to highly branched. Polysaccharides are often quite heterogeneous, containing slight modifications of the repeating unit. Depending on the structure,...

s, and other biological tissue materials.

Osmium tetroxide

Osmium tetroxide is used in optical microscopy to stain lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...

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
Ruthenium tetroxide is a diamagnetic tetrahedral ruthenium compound. As expected for a charge-neutral symmetrical oxide, it is quite volatile. The analogous OsO4 is more widely used and better known...

 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
Ammonium heptamolybdate is an odourless crystalline compound ranging in colour from white to yellow-green. It is usually encountered as the tetrahydrate, whose chemical formula is 6Mo7O24·4H2O...

, cadmium iodide
Cadmium iodide
Cadmium iodide, CdI2, is a chemical compound of cadmium and iodine. It is notable for its crystal structure, which is typical for compounds of the form MX2 with strong polarization effects.-Uses:...

, carbohydrazide
Carbohydrazide
Carbohydrazide is used as an oxygen scavenger in water treatment for boilers. It is an alternative to the hazardous and potentially carcinogenic hydrazine. Carbohydrazide reacts with oxygen to make water and nitrogen and urea...

, ferric chloride, hexamine
Hexamine
Hexamethylenetetramine is a heterocyclic organic compound with the formula 6N4. This white crystalline compound is highly soluble in water and polar organic solvents. It has a cage-like structure similar to adamantane. It is useful in the synthesis of other chemical compounds, e.g. plastics,...

, indium trichloride, lanthanum nitrate, lead acetate
Lead acetate
Lead acetate can refer to:* Lead acetate , Pb4* Lead acetate , Pb2...

, lead citrate, lead(II) nitrate
Lead(II) nitrate
Lead nitrate is an inorganic compound with the chemical formula Pb2. It commonly occurs as a colourless crystal or white powder and, unlike most other lead salts, is soluble in water....

, periodic acid
Periodic acid
Periodic acid, or iodic acid is an oxoacid of iodine having chemical formula HIO4 or H5IO6.In dilute aqueous solution, periodic acid exists as discrete hydronium and metaperiodate ions. When more concentrated, orthoperiodic acid, H5IO6, is formed; this dissociates into hydronium and...

, phosphomolybdic acid
Phosphomolybdic acid
Phosphomolybdic acid, also known as dodeca molybdophosphoric acid or PMA is a component of Masson's trichrome stain. It is a yellow-green compound, freely soluble in water and polar organic solvents such as ethanol...

, potassium ferricyanide
Potassium ferricyanide
Potassium ferricyanide is the chemical compound with the formula K3[Fe6]. This bright red salt contains the octahedrally coordinated [Fe6]3− ion. It is soluble in water and its solution shows some green-yellow fluorescence.-Preparation:...

, potassium ferrocyanide
Potassium ferrocyanide
Potassium ferrocyanide is the inorganic compound with formula K4[Fe6]•3H2O. It is the potassium salt of the coordination complex [Fe6]4-. This salt forms lemon-yellow monoclinic crystals.-Synthesis:...

, ruthenium red
Ruthenium red
The inorganic dye ammoniated ruthenium oxychloride, also known as Ruthenium Red, is used in histology to stain aldehyde fixed mucopolysaccharides....

, silver nitrate
Silver nitrate
Silver nitrate is an inorganic compound with chemical formula . This compound 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...

, silver proteinate, sodium chloroaurate, thallium nitrate, thiosemicarbazide, uranyl acetate
Uranyl acetate
Uranyl acetate is the acetate salt of uranium and is a yellow crystalline solid made up of yellow rhombic crystals and has a slight acetic odor. Uranyl acetate is slightly radioactive, the precise radioactivity depends on the isotopes of uranium present...

, uranyl nitrate
Uranyl nitrate
Uranyl nitrate is a water soluble yellow uranium salt. The yellow-green crystals of uranium nitrate hexahydrate are triboluminescent.Uranyl nitrate can be prepared by reaction of uranium salts with nitric acid...

, and vanadyl sulfate. http://www.2spi.com/catalog/chem/stain.shtml

See also

  • Cytology
    Cell biology
    Cell biology is a scientific discipline that studies cells – their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level...

    : the study of cells
  • Histology
    Histology
    Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...

    : the study of tissues
  • Immunohistochemistry
    Immunohistochemistry
    Immunohistochemistry or IHC refers to the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and...

    : the use of antisera to label specific antigens
  • Ruthenium(II) tris(bathophenanthroline disulfonate)
    Ruthenium(II) tris(bathophenanthroline disulfonate)
    Tetrasodium trisruthenium is a coordination compound containing a ruthenium center. In this form, it is the salt of a sulfonic acid. This compound is an extension of the well known phenanthroline series of coordination compounds...

    , a protein dye.
  • Vital stain
    Vital stain
    A vital stain is 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...

    : stains that do not kill cells

External links

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