Everything about Staining totally explained
Staining is a
biochemical technique of adding a class-specific (
DNA,
proteins,
lipids,
carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. It is similar to
fluorescent tagging.
Stains and
dyes are frequently used in
biology and
medicine to highlight structures in
biological tissues for viewing, often with the aid of different
microscopes. Stains may be used to define and examine bulk tissues (highlighting, for example,
muscle fibers or
connective tissue),
cell populations (classifying different
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 acids in
gel electrophoresis.
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 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.
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 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. Sometimes heat is used to kill, adhere, and alter the specimen so it'll accept stains. Most chemical fixatives (chemicals causing fixation) generate
chemical bonds between
proteins 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
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.
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's unsuitable for studying pathogens.
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
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're 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 isn't 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.
Haematoxylin and eosin (H&E) staining
Haematoxylin and eosin staining 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 cells, colouring them bright red.
Papanicolaou staining
Papanicolaou staining, or Pap staining, is a frequently used method for examining cell samples from various bodily secretions. It is frequently used to stain thkhpige
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
carbohydrates (
glycogen,
glycoprotein,
proteoglycans). 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 nuclei.
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 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
formalin 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
reductant, for example
hydroquinone or formalin.
Sudan staining
Sudan staining is the use of Sudan dyes to stain sudanophilic substances, usually
lipids.
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 don't 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.
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 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's very similar spectrally to fluorescein.
Bismarck brown
Bismarck brown (also Bismarck brown Y or Manchester brown) imparts a yellow colour to acid
mucins. 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 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
fluorescent nuclear stain, excited by
ultraviolet light and showing strong blue fluorescence when bound to
DNA. DAPI isn't 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
cytoplasmic material,
cell membranes, and some extracellular structures. It also imparts a strong red colour to
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, providing a fluorescent red-orange stain. Although it won't stain healthy cells, it can be used to identify cells that are in the final stages of
apoptosis - such cells have much more permeable
membranes. 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
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 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:
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 (for example 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 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'll 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
lipids. It dissolves in fats, and is reduced by organic materials to elemental osmium, an easily visible black substance. Because it's a heavy metal that absorbs electrons, it's 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
viruses,
nerves,
polysaccharides, 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.
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