STAINING

                          STAINING.

.Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. 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(, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells, for instance), or organelles within individual cells. A stained histologic specimen, sandwiched between a glass microscope slide and coverslip, mounted on the stage of a light microscope.

In biochemistry it involves adding a class-specific (DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. Staining and fluorescent tagging can serve similar purposes. Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis.

Staining is not limited to biological materials, it can also be used to study the morphology of other materials for example the lamellar structures of semi-crystalline polymers or the domain structures of block copolymers.

A stained histologic specimen, sandwiched between a glass microscope slide and coverslip, mounted on the stage of a light microscope

 In vivo vs In vitro:

In vivo staining is the process of dyeing living tissues-in vivo means "in life" . By causing certain cells or structures to take on contrasting colour, 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 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.

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 .

IN VITRO METHODS:

 Preparation:

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

Fixation–which may itself consist of several steps–aims to preserve the shape of the cells or tissue involved as much as possible. Sometimes heat fixation is used to kill, adhere, and alter the specimen so it will 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.

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.

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 precipitate. 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 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 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. This can be achieved simply by smearing the sample on to the slide, followed by an application of nigrosin (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.

 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 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. 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 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:

Microscopic view of a histologic specimen of human lung tissue stained with hematoxylin and eosin.

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. 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.

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 Pap smear specimens. It uses a combination of haematoxylin, Orange G, eosin Y, Light Green SF yellowish, and sometimes Bismarck Brown Y.

PAS staining:

PAS diastase showing the fungus Histoplasma.

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 stain demonstrating histoplasma (black round balls).

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 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 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).

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 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 binds with A=T rich repeats of chromosomes.DAPI also 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 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 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 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.

Acid fuchsine:

Acid fuchsine may be used to stain collagen, smooth muscle, or mitochondria. 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. Acid fuchsine is also a traditional stain for mitochondria (Altmann's method).

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

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

Methylene blue:

Methylene blue 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 (or toluylene red) stains Nissl substance red. It is usually used as a counterstain in combination with other dyes.

Nile blue:

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

 Nile red:

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

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.

Stainability of tissues:

Positive affinity for a specific stain may be designated by the suffix -philic. For example, tissues that stain with an azure dye may be referred to as azurophilic. This may also be used for more generalized staining properties, such as acidophilic for tissues that stain by acidic stain (most notably eosin), basophilic when staining in basic dyes and amphophilic when staining with either acid or basic dyes. In contrast, Chromophobic tissues do not take up colored dye readily.

Electron microscopy:

As in light microscopy, stains can be used to enhance contrast in transmission electron microscopy. Electron-dense compounds of heavy metals are typically used.

Phosphotungstic acid:

Phosphotungstic acid is a common negative stain for viruses, nerves, polysaccharides, and other biological tissue materials.

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 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. OsO₄ 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, leav ing behind a deposit of non-volatile osmium in a lower oxidation state.

 Ruthenium tetroxide:

Ruthenium tetroxide 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, cadmium iodide, carbohydrazide, ferric chloride, hexamine, indium trichloride, lanthanum nitrate, lead acetate, lead citrate, lead(II) nitrate, periodic acid, phosphomolybdic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate, sodium chloroaurate, thallium nitrate, thiosemicarbazide, uranyl acetate, uranyl nitrate, and vanadyl sulfate