Stains For Medical Students

1. Stains For Medical Students 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. 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.tissue after processing.

2. Tissue stains stain whatever is left in the embedded tissue after processing. Standard processing of formalin-fixed tissue removes small non-protein molecules, especially lipids. Clinicians need to know only a little about stains used by pathologists in establishing diagnosis. The following will come in handy in understanding pathology slides and reports

3. Haematolxylin Basic dye Blue-purple colour Stains nuclei, ribosomes & rough ER (DNA & RNA - acidic) Haematoxylin is a metal chelator & is used with aluminum ions, which link it to fixed phosphate groups. Haematoxylin also stains calcium salts, and bacteria blue-purple. Structures that stain with haematolxylin are termed basophilic Eosin Acid dye Pink-red colour Stains most cytoplasm proteins which are mostly basic Eosin (red) binds to free amino groups and thus stains arginine and lysine pink to red. Air, water, fat, and carbohydrate end up unstained. Structures that stain with eosin are termed eosinophilic Haematoxylin & Eosin (H&E)‘Standard Tissue Stain’Most common technique in animal histology & routine pathologyIn general, nuclei stain blue & cytolplasm stains pink to red

4. Periodic acid-Shift reaction (PAS)used to mark carbohydrates (glycogen, glycoprotein, proteoglycans) • PAS is a ‘Histochemical Staining Technique’: • staining techniques which specifically stain components of cells & tissues • invaluable for understanding of cell & tissue structure & function, and for diagnosis of diseased tissue • The reaction of periodic acid selectively oxidizes the glucose residues & creates aldehydes that react with the Schiff reagent and creates a purple-magenta color. • PAS staining is mainly used for staining structures containing a high proportion of carbohydrate macromolecules (glycogen, glycoprotein, proteoglycans), typically found in connective tissues, mucus, and basal laminae. • PAS staining can be used to distinguish between different types of glycogen storage disease, as well as being indicative of certain forms of cancer, such as Paget's disease.

5. Anything with a cis-dihydroxy group, i.e., insoluble sugar compounds, gets stained deep red-magnenta. • Examples include: • carbohydrate/glycogen • mucin (produced by goblet cells of GIT & respiratory tract) • basement membranes & brush borders of kidney tubules & intestines • cartilage • reticulin • type I collagen (weak) • all amyloids (weak)

6. Prussian Blue uses a special solution of ferrocyanide. • By the familiar reaction (college chemistry, laundry bluing, old-fashioned blueprints), it stains all exposed ferric ions very blue. • PB is a common stain used by pathologists to detect the presence of iron in biopsy specimens, such as on bone marrow. • PB's ability to incorporate +1 cations makes it useful as a sequestering agent for certain heavy metals ions Congo Red is a special dye that fits tightly into beta-pleated proteins of all sorts. • Beta-pleated proteins in humans are abnormal and are called amyloids. • Congo Red stains all amyloids brick red, and everything else pink. • Because of the way beta-pleated sheets line up Congo Red, amyloids also acquire an apple green birefringence when stained with Congo Red and then examined under polarized light

7. Sudan / Oil Red O are oil-based stains that can only be used on thin slices of tissue from which fat has not been removed (frozen sections). • These stain only fat (usually black or bright red). • The principle is of course hydrophobic bonding. Romanowsky stain are all based on a combination of eosinate (chemically reduced eosin) and methylene blue • Common variants include Wright's stain, Jenner's stain, Leishman stain and Giemsa stain. • All are used to examine blood or bone marrow samples. • Nuclei stained dark blue/violet, erythrocytes pale pink & cytoplasm pale blue • They are preferred over H&E for inspection of blood cells because different types of leukocytes can be readily distinguished. • All are also suited to examination of blood to detect blood-borne parasites like malaria.

8. Mucicarmine is a special dye that stains only epithelial mucin (usually red). The actual chemistry remains mysterious. • Alcian blue stains certain mucoid substances (hyaluronic acid, sulfomucin, maybe carboxymucin depending on the recipe). • Trichrome uses familiar aniline dyes to stain collagen (type I, also basement membranes) blue or green and everything else some other color. The stain depends on the special way collagen is woven. • Acid-fast stains (ZN, auramine O, others) stain certain waxes a permanent red (or some other color). This shows up mycobacteria (TB bugs) and certain other rare substances. • Argentaffin stains test the ability of cell structures to bind and reduce silver, while argyrophil stains demonstrate all sites of silver binding, whether or not reduction occurs. (Everything that is argentaffin is argyrophil, but not vice-versa.) • Methenamine silver is the most sensitive and specific common stain for fungi and pneumocystis. It stains them black.

9. Elastic stains (Verhoeff, Van Gieson) selectively stain elastic fibers (typically black). • Metachromatic stains take advantage of molecule-stacking. A single dye will impart a variety of hues to different structures. The most important metachromatic dyes are those use to stain blood and bone marrow smears (the various azures); * Bismark brown imparts a metachromatic yellow on pap smears. • Light green stains RNA green. • Orange G stains disulfide bonds orange. Pathologists often add a bit of this to their eosin stain, as it gives keratin a more orange hue than most other proteins. • Papanicolaou's stain is used for cytology (i.e., smears of cells on glass slides, "Pap" smears). It contains hematoxylin, eosin, light green, orange G, and sometimes Bismark brown. • Methyl green pyronine stains RNA red and everything else green. It is of historic interest. • Immunostaining (immunofluorescence, immunoperoxidase) uses monoclonal antibodies to demonstrate specific antigens (i.e., specific proteins) in tissues.

10. Gram-Staining • Gram staining is used to determine gram status to classify bacteria broadly. • It is based on the composition of bacterial 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. • Gram-negative organisms will usually appear red or pink (counterstained). • Due to the 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.