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Q. Why do we need to stain?
A. In bright field microscopy, the background is brighter than the specimen. The image observed is the product of light that has passed through the specimen. Staining improves the refractive index, making the sub-cellular structure more visible. This is essential for bright field microscopy.
Q. What are special stains?
A. Special stains are the stains that are used to visualize specific tissues and cellular structures. These are dyes that bind to the cellular components either physically or by chemical bonds.
Q. What is routine staining?
A. Sections are routinely stained with Hematoxylin and Eosin. Hematoxylin stains the nuclei in cells and other cationic elements purple. Eosin is an anionic dye and binds to cytoplasmic proteins below pH6 giving a pink color.
Q. What is a counterstain?
A. Counterstain is defined as a stain with a color that contrasts to the primary stain. For example, Hematoxylin is commonly used as a counterstain in Immunohistochemistry.
Detection of microorganisms in tissues
Q. What is the Acid Fast staining?
A. Acid fast staining is a method of differential staining that detects the presence of the lipoid capsule that takes up carbol fuchsin, and is particularly useful for identifying Mycobacterium species. Once the cell is stained, it resists the decolorization by diluting mineral acid; hence the name acid fast staining.
Q. What is the Gram staining?
A. Gram staining differentiates bacteria into two classes: Gram positive and Gram negative depending on their cell wall structure and composition. Crystal violet is first applied to the sections as a primary stain, followed by iodine mordant forming a dye lake.
The sections are then decolorized using an alcohol/acetone mixture. Gram positive cells do not decolorize at this step; however Gram negative bacteria will not retain crystal violet. On counterstaining with safranin, the gram negative bacteria stain red.
Q. What is the Geimsa staining?
A. Geimsa is a polychromatic stain that permits the differentiation of cells present in hematopoietic tissues and for the demonstration of certain microorganisms. It is used for the detection of Helicobacter pylori. The H. Pylori and nuclei stain dark blue and cytoplasm stain pink with this stain.
Q. What is the PAS staining for Fungi?
A. The fungal cell wall contains polysaccharides that are oxidized by the periodic acid to aldehydes. The aldehydes react with Schiff's reagent to stain the fungi rose pink.
Q. What is the GMS staining?
A. The GMS staining is a silver staining technique for demonstrating fungi in tissue sections. It is primarily based on staining the polysaccharides in fungal cell walls.
Detection of different tissue and cellular components
Polysaccharides, Mucins, Mucopolysaccahrides and Amyloid
Q. What is a PAS Kit and what does it stain?
A. The PAS staining is used to demonstrate presence of polysaccharides, neutral mucosubstances and basement membrane in tissue sections. The reaction is based on the oxidation of glycol group to aldehydes by periodate. The schiff's reagent selectively stains the aldehydes to a rose pink color.
Q. What is PAS with Diastase?
A. PAS with Diastase staining is used for the demonstration of glycogen in tissue sections. Diastase and alpha-Amylase are enzymes that digest glycogen polymer to maltose and glucose that are easily washed from the sections thus giving a negative reaction with PAS.
Q. What is the Alcian blue staining?
A. Alcian blue is a copper phthalocyanin basic dye that is water-soluble and colored blue due to its copper content. It is believed to form salt linkages with the acid groups of acidic mucopolysaccharides staining them blue.
Q. What is the Alcian Blue/PAS staining?
A. In Alcian Blue/PAS staining, acidic mucopolysaccharides are stained blue with Alcian blue, whereas neutral polysaccharides stain rose pink with the PAS reaction.
Q. What is the Mucicarmine staining?
A. Mucicarmine is used for demonstrating epithelial mucin in tissue sections. Aluminum is believed to form a chelation complex with the carmine, and the complex has a net positive charge allowing it to bind with the acid substrates of low density such as mucins.
Q. What is the Amyloid staining?
A. Congo red method is used for staining Amyloid. Congo red, a metachromatic dye, is a benzidine derivative that can react with cellulose. It is a linear molecule allowing the azo and amine groups of the dye to form hydrogen bonds with similarly spaced hydroxyl radicals of the amyloid. The amyloid stains red to deep pink. When seen in polarized light, the apple green birefringence is visualized in stained amyloid.
Lipids
Q. What is the Oil red O stain?
A. The Oil Red O stain is used for the demonstration of neutral fat in frozen sections. It is a physical method of staining as the dye is more soluble in the lipoid substances than hydroalcoholic dye solvents. The fat in tissue sections stains intense red.
Q. What is the Sudan Black stain?
A. Sudan Black, the most sensitive of lipid dyes, is used for demonstrating lipids and phospholipids in frozen tissue sections. In addition to its solubility in neutral fat, Sudan black is slightly basic and will combine with acidic groups in compound lipids.
Fibers
Q. What is the Elastic stain?
A. Verhoeff's Elastic stain is used to demonstrate pathologic conditions such as atrophy, breaks, thinning, loss etc. in elastic fibers. It stains the elastic fibers blue to black, nuclei stain blue, collagen stains pink-red, and other tissue elements stain yellow.
Q. What is the Reticulum stain?
A. Reticulum stain is used to demonstrate reticular fibers in tissue sections by ammoniacal silver stain. It can aid in the differential diagnosis of carcinomas, sarcomas, and lymphosarcomas. In certain liver diseases where a change from normal reticular fiber pattern is seen, this stain can be of help.
Q. What is the Masson Trichrome stain?
A. Masson Trichrome, a mixture of three dyes, is used to differentiate between collagen and smooth muscle in tumors, and increases in collagen in cirrhosis. The three dyes used are aniline blue (stains collagen and mucus to blue or blue green) Beibrich Scarlet (stains cytoplasm, muscle and keratin red) and Weigert's Iron Hematoxylin (stains nuclei blue to black).
Q. What is the Iron Stain?
A. Prussian blue stain is used for the demonstration of ferric iron in the tissue sections. This stain can aid in identifying hemochromatosis and hemosederosis where abnormal large deposits of iron may be seen. In Prussian blue staining, the sections are treated with an acidic solution of potassium ferrocyanide and any ferric iron present reacts to form an insoluble bright blue pigment called Prussian blue.
Q. What is Papanicolaou staining?
A. PAP (Papanicolaou) stain is a polychromatic stain used to demonstrate the variations of cellular maturity and metabolic state. It is used to detect cancer cells routinely in cytological preparations from cells that are scraped or brushed off the cervix in women. The combination of dyes gives the subtle range of green blue and pink hues to the cell cytoplasm according to the maturity level of the cells. Non-keratinized, normal superficial and intermediate squamous cells are stained green and keratinized cells are stained orange to pink. Cancer cells are usually green in cytoplasm and stained blue in the nucleus.
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