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.