Staining
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225804709
2008-07-15T14:09:32Z
141.214.17.5
{{otheruses4|dyeing organic matter in the laboratory|the wood treatment|wood staining|the village in Lancashire|Staining, Lancashire}}
[[Image:Microscope with stained slide.jpg|right|thumb|300px|A stained histologic specimen positioned on the stage of a light microscope.]]
'''Staining''' is a [[biochemistry|biochemical]] technique of adding a class-specific ([[DNA]], [[protein]]s, [[lipid]]s, [[carbohydrate]]s) dye to a substrate to qualify or quantify the presence of a specific compound. It is similar to [[fluorescent tag]]ging.
'''Stains''' and [[dye]]s are frequently used in [[biology]] and [[medicine]] to highlight structures in [[biological tissue]]s for viewing, often with the aid of different [[microscope]]s. Stains may be used to define and examine bulk tissues (highlighting, for example, [[muscle fiber]]s or [[connective tissue]]), [[cell (biology)|cell]] populations (classifying different [[blood cell]]s, for instance), or [[organelle]]s within individual cells.
Biological staining is also used to mark cells in [[flow cytometry]], and to flag [[protein]]s or [[nucleic acid]]s in [[gel electrophoresis]].
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== ''In vitro'' staining ==
[[In vitro]] 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 (histology)|fixation]] and sample preparation, scientists and physicians can use these standard techniques as consistent, repeatable diagnostic tools. A [[counterstain]] is stain that makes cells or structures more visible, when not completely visible with 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.
=== Methods of in vitro staining ===
==== •Preparation ====
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 [[surfactant]]. This treatment will dissolve the [[cell membrane]]s, and allow larger dye molecules access to the cell's interior.
'''[[Fixation (histology)|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 is used to kill, adhere, and alter the specimen so it will accept stains. Most chemical fixatives (chemicals causing fixation) generate [[chemical bond]]s between [[protein]]s and other substances within the sample, increasing their rigidity. Common fixatives 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 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]]: a chemical compound which reacts with the stain to form an insoluble, coloured [[Precipitation (chemistry)|precipitate]]. When excess dye solution is washed away, the mordanted stain remains.
==== •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]]. This can be achieved simply by smearing the sample on to the slide, followed by an application of [[nigrosin]] (Indian ink). 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.
=== Specific techniques ===
==== •Gram staining ====
[[Gram staining]] is used to determine gram status to classify bacteria broadly. It is based on the composition of their [[cell wall]]. Gram staining uses [[methyl violet|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 [[antibiotic]]s.
[[Gram-positive]] bacteria stain dark blue or violet. Their [[cell wall]] is typically <!-- but not always !! --> 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;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 staining]] is used to stain species of ''[[Mycobacterium tuberculosis]]'' that do not stain with the standard laboratory staining procedures like Gram staining.
The stains used are the red colored [[Carbol fuchsin]] that stains the bacteria and a counter stain like [[Methylene blue]] or [[Malachite green]].
==== •Haematoxylin and eosin (H&E) staining ====
[[Image:Emphysema H and E.jpg|right|300px|thumb|Histologic specimen of human [[lung]] tissue stained with [[hematoxylin]] and [[eosin]].]]
[[Haematoxylin and eosin stain]]ing protocol is used frequently in [[histology]] to examine thin sections of tissue. [[Haematoxylin]] stains cell nuclei blue, while [[eosin]] stains cytoplasm, connective tissue and other extracellular substances pink or red. Eosin is strongly absorbed by [[red blood cell]]s, colouring them bright red.
==== •Papanicolaou staining ====
[[Papanicolaou stain]]ing, or Pap staining, is a frequently used method for examining cell samples from various bodily secretions. It is frequently used to stain the [[Pap smear]] specimens. It uses a combination of [[haematoxylin]], [[Orange G]], [[eosin Y]], [[Light Green SF yellowish]], and sometimes [[Bismarck Brown Y]].
==== •PAS staining ====
[[Periodic acid-Schiff]] staining is used to mark [[carbohydrate]]s ([[glycogen]], [[glycoprotein]], [[proteoglycan]]s). It is used to distinguish different types of glycogen storage diseases.
==== •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 nucleus|cell nuclei]].
==== •Romanowsky stains ====
The [[Romanowsky stain]]s are all based on a combination of eosinate (chemically [[reduction (chemistry)|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 [[white blood cells|leukocytes]] (white blood cells) can be readily distinguished. All are also suited to examination of blood to detect blood-borne parasites like [[malaria]].
==== •Silver staining ====
[[Silver stain]]ing is the use of [[silver]] to stain [[histologic section]]s. This kind of staining is important especially to show [[protein]]s (for example type III [[collagen]]) and [[DNA]]. It is used to show both substances inside and outside [[Cell (biology)|cell]]s. Silver staining is also used in [[temperature gradient gel electrophoresis]].
Some cells are ''argentaffin''. These [[Redox|reduce]] silver solution to metallic silver after [[formalin]] [[fixation (histology)|fixation]]. This method was discovered by Italian [[Camillo Golgi]], by using a reaction between [[silver nitrate]] and [[potassium dichromate]], thus precipitating silver chromate in some cells (see [[Golgi's method]]). Other cells are ''argyrophilic''. These reduce silver solution to metallic silver after being exposed to the stain that contains a [[Reducing agent|reductant]], for example [[hydroquinone]] or formalin.
==== •Sudan staining ====
[[Sudan stain]]ing is the use of Sudan dyes to stain sudanophilic substances, usually [[lipid]]s. [[Sudan III]], [[Sudan IV]], [[Oil Red O]], and [[Sudan Black B]] are often used. Sudan staining is often used to determine the level of [[fecal fat]] to diagnose [[steatorrhea]].
==== •Conklin's staining ====
Special technique designed for staining true endospores with the use of malachite green dye, once stained, they do not decolourize.
== ''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 cells or 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.
== 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]] (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.
==== •Bismarck brown ====
[[Bismarck brown]] (also Bismarck brown Y or Manchester brown) imparts a yellow colour to acid [[mucin]]s. 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|Coomassie blue]] (also brilliant blue) nonspecifically stains proteins a strong blue colour. It is often used in gel electrophoresis.
==== •Crystal violet ====
[[Methyl violet|Crystal violet]], when combined with a suitable mordant, stains [[cell wall]]s purple. Crystal violet is an important component in Gram staining.
==== •DAPI ====
[[DAPI]] is a [[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 haematoxylin, imparting a pink or red colour to [[cytoplasm]]ic material, [[cell membrane]]s, and some extracellular structures. It also imparts a strong red colour to [[red blood cell]]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]] [[intercalation (chemistry)|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]] - such cells have much more permeable [[biological membrane|membrane]]s. Consequently, ethidium bromide is often used as a marker for apoptosis in cells populations and to locate bands of DNA in [[gel electrophoresis]]. The stain may also be used in conjunction with [[acridine orange]] (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.
==== •Fuchsin ====
[[Fuchsin]] may be used to stain collagen, smooth muscle, or [[mitochondrion|mitochondria]].
Acid fuchsin is commonly used in Masson's trichrome and van Gieson's picro-fuchsin, and was used in an older method to stain mitochondria.
==== •Haematoxylin ====
[[Haematoxylin]] (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]].
==== •Hoechst stains ====
[[Hoechst stain|Hoechst]] is a ''bis''-benzimidazole derivative compound which 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 stain|Hoechst]]: ''Hoechst 33258'' and ''Hoechst 33342''. The two compounds are functionally similar, but with a little difference in structure. Hoechst 33258 contains a terminal [[hydroxyl]] group and is thus more soluble in aqueous solution, however this characteristics reduces its ability to penetrate the [[plasma membrane]]. Hoechst 33342 contains a [[ethyl]] substitution on the terminal hydroxyl group (i.e. an ethylether group) making it more hydrophobic for easier plasma membrane passage.
==== •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 iodine|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 [[cell nucleus|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 stain|Gimenez staining technique]] for bacteria. It also can be used to directly stain [[endospore|spore]]s.
==== •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.
==== •Osmium tetroxide ====
[[Osmium tetroxide]] is used in optical microscopy to stain [[lipid]]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.
==== •Rhodamine ====
[[Rhodamine]] is a protein specific fluorescent stain commonly used in fluorescence microscopy.
==== •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.
== Electron microscopy ==
Similar to light microscopy, stains can be used to selectively highlight cellular structures in [[transmission electron microscopy]]. Electron-dense compounds of heavy metals are typically used. For example, [[phosphotungstic acid]] is a common [[negative stain]] for [[virus]]es, [[nerve]]s, [[polysaccharide]]s, and other biological tissue materials.
Other chemicals used in electron microscopy staining include:
[[ammonium molybdate]], [[cadmium iodide]], [[carbohydrazide]], [[ferric chloride]], [[hexamine]], [[indium trichloride]], [[lanthanum nitrate]], [[lead acetate]], [[lead citrate]], [[lead(II) nitrate]], [[osmium tetroxide]], [[periodic acid]], [[phosphomolybdic acid]], [[potassium ferricyanide]], [[potassium ferrocyanide]], [[Ruthenium Red]], [[silver nitrate]], [[sodium chloroaurate]], [[thallium nitrate]], [[thiosemicarbazide]], [[uranyl acetate]], [[uranyl nitrate]], and [[vanadyl sulfate]]. [http://www.2spi.com/catalog/chem/stain.shtml]
== See also ==
*[[Derivatization]]
*[[Cytology]]: the study of cells
*[[Histology]]: the study of tissues
*[[Immunohistochemistry]]: the use of antisera to label specific antigens
*[[Microscopy]]
*Article categories for [[:Category:Dyes|dyes]] and [[:Category:Pigments|pigments]]
*[[Ruthenium(II) tris(bathophenanthroline disulfonate)]], a protein dye.
*[[:Category:Staining dyes]]: category of articles on various dyes used for staining in microbiology and histology.
== External links ==
*[http://stainsfile.info/StainsFile/ StainsFile] reference for dyes and staining techniques
*[http://www.microscopy-uk.org.uk/mag/artfeb00/rhvital.html Vital Staining for Protozoa and Related Temporary Mounting Techniques] ~ Howey, 2000
*[http://www.microscopy-uk.net/mag/artoct00/fixation.html Speaking of Fixation: Part 1] and [http://www.microscopy-uk.net/mag/artdec00/fixation2.html Part 2] - by M. Halit Umar
*[http://www.histology-world.com/stains/stains.htm Photomicrographs of Histology Stains]
[[Category:Staining| ]]
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