====== Biological Staining ======

Staining is a biochemical technique of adding a class-specific ([[DNA]], [[protein|proteins]], [[lipid|lipids]], [[carbohydrate|carbohydrates]]) dye to a substrate to qualify or quantify the presence of a specific compound. It is similar to [[fluorescence|fluorescent]] tagging.

Stains and [[dye|dyes]] are frequently used in [[biology]] and [[medicine]] to highlight structures in [[tissue|biological tissues]] for viewing, often with the aid of different [[microscope|microscopes]]. Stains may be used to define and examine bulk tissues (highlighting, for example, [[muscle]] fibers or [[connective tissue]]), cell populations (classifying different [[blood cell|blood cells]], for instance), or organelles within individual cells.

Biological staining is also used to mark cells in [[flow cytometry]], and to flag proteins or [[nucleic acid|nucleic acids]] in [[gel electrophoresis]].

{{ emphysema_h_and_e.jpg?200|Lung tissue from emphysema patient, stained with haematoxylin and eosin}}

===== In vitro Staining =====

In vitro staining involves colouring cells or structures that are no longer living. In vitro means literally "in glass"; compare with in vivo. 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]] is stain added which makes visible cells or structures not coloured by the principal stain. For example, [[crystal violet]] stains only Gram-positive [[bacteria]] in Gram staining. A safranin counterstain is applied which stains all cells, allowing the identification of Gram-negative bacteria as well.

The preparatory steps involved depend on the type of analysis planned; some or all of the following procedures may be required.

**Permeabilization** involves treatment of cells with (usually) a mild [[detergent]]. This detergent treatment will dissolve the cell [[membrane|membranes]], and allow larger dye molecules access to the cell's interior.

**[[Fixation]]**–which may itself consist of several steps–aims to preserve the shape of the cells or tissue involved as much as possible. Most fixatives (chemicals causing fixation) generate chemical bonds between proteins and other substances within the sample, increasing their rigidity. Common fixative solutions often include [[formaldehyde]], [[ethanol]], [[methanol]], and/or [[picric acid]]. Pieces of tissue may be embedded in [[paraffin]] wax to increase their mechanical strength and stability and to make them easier to cut into thin slices.

**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 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.

**Staining**

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]]: a chemical compound which reacts with the stain to form an insoluble, coloured [[precipitate]]. When excess dye solution is washed away, the mordanted stain remains.

**Haematoxylin and Eosin (H&E) Staining**

This staining protocol is used frequently in [[histology]] to examine thin sections of [[tissue]]. [[Hematoxylin]] stains [[cell]] [[nucleus|nuclei]] blue, while [[eosin]] stains [[cytoplasm]] and [[connective tissue]] pink or red. Eosin is strongly absorbed by [[red blood cell|red blood cells]], colouring them bright red.

**Masson's Trichrome**

Masson's trichrome 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]]. Most recipes will produce red keratin and [[muscle]] fibers, blue or green staining of [[collagen]] and [[bone]], light red or pink staining of cytoplasm, and black cell nuclei.

**Gram Staining**

Gram staining is used to determine gram status classify [[bacteria]] broadly. It is based on the composition of their cell wall. Gram staining uses [[crystal violet]] to stain [[cell]] walls, [[iodine]] as a [[mordant]], and a [[fuchsin]] or [[safranin]] [[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 antibiotics.

Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich with peptidoglycan and lacks the secondary [[membrane]] and lipopolysaccharide layer found in Gram-negative bacteria.

On most Gram-stained preparations, Gram-negative organisms will appear red or pink because they are counterstained. 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.

**Romanowsky Stains**

The Romanowsky stains are all based on a combination of eosinate (chemically reduced [[eosin]]) and [[methylene blue]] (sometimes with its oxidation products azure A and azure B). Common variants include Wright's stain, Jenner's stain, Leishman stain and Giemsa stain.

All are used to examine [[blood]] or [[bone marrow]] samples. They are preferred over H&E for inspection of blood cells because different types of leukocytes (white [[blood cell|blood cells]]) can be readily distinguished. All are also suited to examination of blood to detect blood-borne parasites like malaria.

**Silver Staining**

Silver staining is the use of [[silver]] to stain histologic sections. This kind of staining is important especially to show proteins (for example type III [[collagen]]) and [[DNA]]. It is used to show both substances inside and outside cells. Silver staining is also used in temperature gradient [[gel electrophoresis]].

Some cells are argentaffin. These reduce silver solution to metallic silver after {formaldehyde|formalin]] [[fixation]]. Other cells are argyrophilic. These ones reduce silver solution to metallic silver after being exposed to the stain that contains a reductant, for example hydroquinone or [[formaldehyde|formalin]].

===== In vivo Staining =====

In vivo staining is the process of dyeing living tissues—in vivo means "in life" (compare with in vitro staining). By causing certain cells or structures to take on contrasting color(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 [[cell|cells]] or [[tissue|tissues]].

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 1:5,000 to 1:500,000 (Howey, 2000). Note that many stains may be used in both living and fixed cells.

===== Basic 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 [[dye|dyes]] may be used with fixed cells and tissues; vital dyes (suitable for use with living organisms) are noted.

**Bismarck brown**

Bismarck brown (also Bismarck brown Y or Manchester brown) imparts a yellow colour to acid mucins. Mast Cells may be stained metachromatically with Bismark Brown. Bismarck brown may be used with live cells.

**Carmine**

Carmine is an intensely red dye which may be used to stain [[glycogen]], 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 [[protein|proteins]] a strong blue colour. It is often used in [[gel electrophoresis]].

**Crystal violet**

Crystal violet, when combined with a suitable [[mordant]], stains cell walls purple. Crystal violet is an important component in Gram staining.

**DAPI**

DAPI is a [[fluorescence|fluorescent]] nuclear stain, excited by [[ultraviolet]] light and showing strong blue [[fluorescence]] when bound to [[DNA]]. DAPI is not visible with regular transmission microscopy. It may be used in living or fixed cells.

**Eosin**

Eosin is most often used as a [[counterstain]] to [[hematoxylin]], imparting a pink or red colour to [[cytoplasm|cytoplasmic]] material, cell membranes, and some extracellular structures. It also imparts a strong red colour to [[red blood cell|red blood cells]]. 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 intercalates and stains [[DNA]]. It is a [[fluorescence|fluorescent]] red-orange stain. Although it will not stain healthy cells, it can be used to identify cells in the late stages of [[apoptosis]]; such cells have much more permeable [[membrane|membranes]]. Consequently, ethidium bromide is often used as a marker for apoptosis in populations of cells. Ethidium bromide is also used to locate bands of DNA in gel electrophoresis.

**Fuchsin**

The name "fuchsin" is applied to two dyes.  Acid fuchsin is anionic and is frequently used to stain cytoplasmic structures and fibres made predominantly from basic proteins, such as [[collagen]], [[smooth muscle]], or [[mitochondria]]. It is frequently used as part of Masson's trichrome. 

Basic Fuchsin is an older name applied to a mixture of cationic dyes, one of which [[pararosaniline]] is an important constituent of Schiff's reagent (Used to detect aldehydes in the Periodic Schiff method.)  Other basic fuchsin mixtures are used to stain bacteria such as the acid alcohol fast TB and acid fast leprosy bacilli.

**Haematoxylin**

Haematoxylin is natural dye extracted from a tree which grows in Central and South America, //Hematoxylum campechianum//.  Hematoxylin itself is not usable to stain and must first be oxidised to the active dye hematein.  This dye is usually used with metal salts as mordants for the staining of tissue components such as nuclei, mitochondria, myelin, and muscle striations. 

The two commonest mordants are salts of aluminum and ferric iron.  With aluminum as the mordant, nuclei are stained deep blue.  With ferric iron, nuclei are black and resist extraction with mild acids better than the aluminum mordanted stains do.  The commonest use for hematoxylin is in the form of a [[hemalum]], //i.e.// a solution of aluminum mordanted hematein, used to stain nuclei blue in contrast to eosin stained cytoplasm.  This is the hematoxylin and eosin stain (H&E), arguably the most common procedure in [[histology]].

**Hoechst stains**

Hoechst 33258 and Hoechst 33342 are two closely related [[fluorescence|fluorescent]] stains. They fluoresce strongly when bound to [[DNA]], but are not visible under transmitted light. The two compounds are functionally very similar, and both may be used in living cells.

**Iodine**

Iodine is used in chemistry as an indicator for starch. When starch is mixed with iodine in solution, an intensely dark blue color 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, used in microbiology.

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.

**Malachite green**

Malachite green (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 also can be used to directly stain spores.

**Methyl green**

Methyl green is chemically related to crystal violet, sporting an extra methyl or ethyl group.

**Methylene blue**

Methylene blue is used to stain animal cells, such as human cheek cells, to make their nuclei more observable.

**Neutral red**

Neutral red (or toluylene red) stains nuclei 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 (also known as Nile blue oxazone) is formed by boiling Nile blue with sulfuric acid. This produces a mix of Nile red and Nile blue. Nile red is a lipophilic stain; it will accumulate in lipid globules inside cells, staining them red. Nile red can be used with living cells.

**Rhodamine**

Rhodamine is a fluorescent stain.

**Safranin**

Safranin (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]].

===== External Links =====

[[http://stainsfile.info/|StainFile]] - Reference for dyes and staining techniques