STAINING TECHNIQUES — Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains or dyes are used in biology and medicine to highlight structures in biological tissues for viewing with microscope. — Cell staining is a technique that can be used to better visualize cells and cell components under a microscope. Using different stains, it is possible to stain preferentially certain cell components, such as a nucleus or a cell wall, or the entire cell. — Most stains can be used on fixed, or non-living cells, while only some can be used on living cells; some stains can be used on either living or non-living cells. — In biochemistry, staining involves adding a class specific (DNA, lipids, proteins or carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound. — Staining and fluorescence tagging can serve similar purposes Purposes of Staining — The most basic reason that cells are stained is to enhance visualization of the cell or certain cellular components under a microscope.

— Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample.

— Cells may also be enumerated by staining cells to determine biomass in an environment of interest.

— Stains may be used to define and examine bulk tissues (e.g. muscle fibers or connective tissues), cell populations (different blood cells) or organelles within individual cells.

— Biological staining is also used to mark cells in flow cytometry, flag proteins or nucleic acids on gel electrophoresis

— Staining is not limited to biological materials, it can also be used to study the morphology (form) of other materials e.g. the lamellar structure of semi crystalline polymers or domain structures of block co-polymers — Staining can be: — In vivo: also known as vital staining which involves dyeing living tissues. Certain cells or structures take up contrasting colors, the morphology or position within a cell or tissue can be readily seen and studied. In in vivo staining, the cytological details that are not apparent are revealed. It also reveals where certain chemicals or specific chemical reactions are taking place within cells or tissues.

— In vitro staining: involves coloring of cells or structures that have been removed from their biological context. Here certain stains are often combined to reveal more details and features than a single stain alone. This staining can be used by scientists and physicians for consistent and repeatable diagnostic tools. — General Staining Techniques — Simple stain techniques.

This can be performed with basic dyes such as crystal violet or methylene blue, positively charged dyes that are attracted to the negatively charged materials of the microbial cytoplasm.

Such a procedure is the simple stain procedure. An alternative is to use a dye (nigrosin, Congo red) repelled by the negatively charged cytoplasm and gather around the cells.

Therefore leaving the cells clear and unstained. This technique is called the negative stain technique. — General Staining Techniques — Differential stain techniques.

This distinguishes two kinds of organisms. Example is the Gram stain technique which separates bacteria into two groups: Gram- positive bacteria and Gram-negative bacteria.

Crystal violet is first applied, followed by the mordant iodine, which fixes the stain. Then the slide is washed with alcohol, and the Gram-positive bacteria retain the crystal-violet iodine stain while the Gram-negative bacteria lose the stain.

The Gram-negative bacteria subsequently stain with the safranin dye, the counterstain, used next. These bacteria appear red under the oil-immersion lens, while Gram-positive bacteria appear blue or purple, reflecting the crystal violet retained during the washing step. — Another differential stain technique is the acid-fast technique.

This technique differentiates species of Mycobacterium from other bacteria.

Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. Then the cells are washed with a dilute acid-alcohol solution.

Mycobacterium species resist the effect of the acid-alcohol and retain the carbolfuchsin stain (bright red). Other bacteria lose the stain and take on the subsequent methylene blue stain (blue).

Thus, the acid-fast bacteria appear bright red, while the nonacid-fast bacteria appear blue when observed under oil-immersion microscopy. — Common Biological Stains — Stains react or concentrate in different parts of a cell or tissue in order to reveal specific parts or areas.

— Acridine Orange (AO): A nucleic acid selective fluorescent cationic dye for cell cycle determination. It is cell permeable and interacts with DNA and RNA by intercalation or electrostatic attractions.

— Ethidium bromide: Intercalates and stains DNA providing a fluorescent red orange stain. Usually used to stain dead cells but can also be used to identify cells in final stages of apoptosis therefore serves as marker for apoptosis in cell population. In gel eletrophoresis, Etbr is used to locate bands of DNA. The stain may also be used in combination with AO in viable cell counting. The combined stain causes live cells to fluoresce and apoptotic cells retain red orange fluorescence. — DAPI (4’-6-Diamidino-2-phenylindole): A fluorescent nuclear stain which is excited by ultraviolet light and showing strong blue fluorescence when bound to DNA. DAPI bind within the A=T rich repeats of chromosome. It may be used in living or fixed cells. DAPI stained cells are used for cell counting and useful in various cytochemical investigations. DAPI is a DNA binding compound, it is therefore likely to have some level of mutagenic properties. — Hoechst stains: This is a benzimidazole derivative compound which bind to the minor groove of DNA of sequences rich in A=T. It is often used in fluorescence microscopy for DNA staining. Hoechst stains appear yellow when dissolved in aqueous solutions and emit blue under UV excitation. There are two major types of Hoechst stain: Hoechst 33258 and Hoechst 33342. These two stains differ in structure but similar in function. Hoechst 33258 contains a terminal hydroxyl group therefore more soluble in aqueous solution but reduced ability to penetrate the plasma membrane. Hoechst 33342 contains an ethyl substitution on the terminal hydroxyl group making it more hydrophobic for easier plasma membrane passage.

— Hoechst dyes are commonly used to stain genomic DNA in applications such as fluorescence microscopy and immunohistochemistry, flow cytometry for cell counting or sorting, detection of DNA in the presence RNA in agarose gels and chromosome sorting.

— Hoechst stains are mutagenic and carcinogenic in nature as they bind to DNA and can interfere with DNA replication during cell division — Bismarck Brown Y (Manchester Brown): This is a diazo dye used in histology for staining tissues. It imparts a yellow colour to acid mucins. This dye may be used in living cells. It can also be used to stain cartilage in bone specimens. Bismarck Brown Y is one of the Kasten’s Schiff-type reagents in the periodic acid-schiff stain to stain cellulose and in Feulgen stain to stain DNA. It can also be used as a counter stain for Victoria blue R for staining of acid-fast microorganisms

— Carmine: is a pigment of a bright red color obtained from aluminium salt of carminic acid produced by some scale insects such as cochineal scale and Polish cochineal. It is an intensely red dye used to stain glycogen using a mordant usually aluminium.

— Coomassie Brilliant Blue: This is stain from two similar triphenylmethane dyes initially developed for use in textile industry but now commonly used for staining proteins in analytical biochemistry. The two forms which differ based on the addition two methyl groups are the Coomassie Brilliant Blue G-250 and Coomassie Brilliant Blue R-250. The stain non specifically stains proteins to a strong blue color giving the protein an overall negative charge and often used in gel electrophoresis. — Crystal Violet: In combination with suitable mordant, stains cell walls purple. It is an important component of Gram staining.

— Haematoxylin: nuclear stain used with a mordant. It stains nuclei blue-violet or brown and often used with eosin in haematoxylin staining in histology.

— Eosin: This is a fluorescent red dye resulting from the action of bromine on fluorescein. Used as counter stain to haematoxylin in H&E staining commonly used in histology, imparting a pink or red color to cytoplasmic material, cell membrane and some extracellular structures. Structures that stain readily with eosin are termed eosinophilic. Tissue stained with haematoxylin and eosin shows cytoplasm stained pink-orange and nuclei stained darkly either blue or purple. Eosin also stains red blood cells intensely red — Acid Fuchsine: Used to stain collagen, smooth muscle or mitochondria. It is used as the nuclear and cytoplasmic stain in Masson’s trichrome stain. In Van Gieson’s picro fuchsine, acid fuchsine imparts its red color to collagen fibers and in Altmann’s method acid fuchsine is a stain for mitochondria. — 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. — Iodine: Used in chemistry as starch indicator. An intensely dark blue color is developed when mixed with starch. 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 of the component of 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 to stain cell, 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/membrane. — Malachite green: Malachite green (also known as diamond green B or victoria green B) is an organic compound that is used as a dye stuff. Traditionally, malachite green is used as a dye for materials like silk, leather and paper. It 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 a heterocyclic aromatic chemical compound used to stain animal cells, such as human cheek cells, to make their nuclei more observable. Also used to stain 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 red: Nile red (also known as Nile blue oxazone) is formed by boiling Nile blue with sulfuric acid producing a mix of Nile red and Nile blue. Nile red is a lipophilic stain which accumulates 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: 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. — Stainability of tissues — Positive affinity for a specific stain may be designated by the suffix -philic. — Tissues that stain with an azure dye may be referred to as azurophilic.

— Tissues that stain with acidic stains (most notably eosin) are referred to as acidophilic.

— Tissues that stain with basic dyes are basophilic

— Tissues that stain with either acid or basic dyes are amphophilic. In contrast, Chromophobic tissues do not take up coloured dye readily. — Stains used in 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: Common negative stain for viruses, nerves, polysaccharides, and other biological tissue materials. — Osmium tetroxide: Used in optical microscopy to stain lipids. It dissolves in fats, and is reduced by organic materials to elemental osmium which is an easily visible black substance. The ability of this heavy metal to absorb electrons makes it 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. — Ruthenium tetroxide: Ruthenium tetroxide is equally volatile more aggressive than osmium tetraoxide as it is 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(II) nitrate/citrate/acetate — Periodic acid — Phosphomolybdic acid — Potassium ferricyanide/ferrocyanide — Ruthenium red — Silver nitrate — Thallium nitrate — Thiosemicarbazide — Uranyl nitrate/acetate — Vanadyl sulfate. Stain preparation

The type of analysis to be carried out determine the preparatory steps. However some or all of the procedures may be required

Fixation: This may consist of several steps but all the steps sums to preserve the shape of the cells or tissues involved as much as possible. Chemical fixatives generate chemical bonds between proteins and other substances within the sample in order to increase their rigidity. Heat fixation is used to kill, adhere and alter the specimen so it will accept stains

Common fixatives include formaldehyde, ethanol, methanol, picrric acid.

Tissue pieces can be embedded in parafilm wax to increase their mechanical strength and stability and make them easier to cut into small sizes. — Permeabilization: this is treatment of cells with mild surfactant in order to dissolve the cell membrane and allow larger dye molecules gain access to the internal part of the cell.

— Mounting: Involves attaching the sample to a glass microscope slide for imaging. Sometimes cells may be grown directly on a slide. Loose samples, such as blood smear or pap smear can be applied directly to a slide while thin sections of larger tissue are made using the microtome. — Specific Staining Techniques

— Gram Staining: This is used to determine gram status to classify bacteria broadly. This is based on the composition of their cell wall. Gram staining utilizes crystal violet to stain the cell walls, iodine as mordant which reacts with the stain to form an insoluble colored precipitate. Fuchsin or safranin is then used as counterstain to mark all bacteria.

— The Gram status of bacteria is very important in medicine as the presence or absence of cell wall can alter the susceptibility of the bacterium to antibiotics.

— The Gram positive bacteria stain dark blue/violet because the cell wall is rich in peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer which is found in Gram negative bacteria.

— Gram negative bacteria will appear red/pink because they are counterstained. Gram negative bacteria have only a few layers of pepetidoglycan and secondary cell wall made of lipopolysaccharides. The porosity of the cell wall is increased due to high lipid content of the cell wall hence, after alcohol treatment, the crystal violet-iodine (CVI) complex can pass through. Thus the primary stain is not retained. Gram Stain

Procedure Reagent Gram Positive Gram Negative cell cell Fixed cells on Colorless Colorless slide Primary stain Crystal violet Purple Purple Mordant Iodine Purple Purple Decolorizer Alcohol Purple Colorless Counterstain Safranin Purple Red — Giemsa Staining: Named after German microbiologis, Gustav Giemsa is used in cytogenetics and histopathological diagnosis of malaria and other parasites. The Giemsa stain solution is a mixture of methylene blue, eosin and azure B. A thin film of the specimen on a microscope is fixed in pure methanol for 30 seconds by immersing it or by putting a few drops of methanol on the slide. The slide is immersed n a freshly prepared 5% Giemsa stain solution for 20-30 minutes then flushed with tap water and left to dry. — Giemsa stain is a classic stain for blood film for peripheral blood smear and bone marrow specimens. Erythrocytes stain pink, platelets show a light pale pink. The lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue while leukocyte nuclear membrane stains magenta. — Giemsa stain is also a differential stain and can be used to study the adherence of pathogenic bacteria to human cells. It differentially stains human and bacteria cells purple and pink respectively. It can be used for histopathological diagnosis of malaria and some other protozoan blood parasites. — Giemsa stain is also used to visualize chromosomes, and to identify mast cells — Ziehl-Neelsen Stain: Used to stain species of mycobacterium tuberculosis that do not stain with the standard laboratory staining procedures. The stains used are the red red colored fuchsin and a countestain like methylene blue or malachite green. — Haematoxylin and Eosin (H&E) Staining: Used frequently in histology to examine thin sections of tissue. Haematoxylin stains cell nuclei and other acidic structures like RNA rich portions of cytoplasm blue while eosin stains cytoplasm, connective tissue and other extracellular substances pink or red. Red blood cell strongly absorbs eosin and give a bright red color. — Romanowsky Stains: based on combinations of eosinate (reduced eosin) and methylene blue. Used to examine blood or bone marrow. It is better than H&E for inspection of blood cells because it distinguishes different types of leukocytes. — Papanicolaou Staining: Also known as pap staining and is frequently used to examine cell samples from bodily separations. It is used to stain pap smear specimens by using combination of haematoxylin, orange G and eosin Y, light green SF yellowish and sometimes Bismarck brown Y. — Periodic Acid Schiff (PAS) Staining: Used to mark carbohydrates such as glycogen, glycoproteins and proteoglycans. It distinguishes different type of glycogen storage diseases — Sudan Staining: This is the use of Sudan dyes to stain sudanophilic substances usually lipids. Dyes such as Sudan lysochromes (Sudan II, Sudan III, Sudan IV, Oil Red O and Sudan Black are used. The dyes have high affinity to fats and are therefore used to demonstrate triglycerides, lipids and lipoproteins. Sudan stain test is often used to determine the level of fecal fat to diagnose steatorrhea. Normally a stool sample should show only a few drops of red-orange stained fat under the microscope. The simplicity of the method makes it suitable for screening even though it is only semiquantitative — Silver Staining: Involves the use of silver to stain histologic sections. This stain show proteins especially the type III collagen and DNA outside and within the cells. Silver staining is also used in temperature gradient gel electrophoresis. Some cells are argentaffin because they reduce silver solution to metallic silver after formalin fixation. This method was discovered by Camilla Golgi when he used reaction between silver nitrate and potassium dichromate causing precipitation in some cells. On the other hand some cells are argyrophillic because they reduce silver solution to metalic silver on exposure to reductant such a shydroquinone or formalin in stain.