LESSON 8: STAINING Flashcards
process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cells
Staining
tissues and cells display varying affinities for most dyes and stains used during the process
Staining
Acidic structures—greater affinity for
Basic structures—greater affinity for
basic dyes
acidic dyes
related procedure that makes use of heavy metal salts which are selectively precipitated on certain cellular and tissue components
Impregnation
used for silver staining of nervous tissue and demonstration of reticulin
Impregnation
Impregnation most commonly used agent:—may also be used as a staining agent
silver nitrate
THREE MAJOR TYPES OF STAINING
- HISTOLOGICAL STAINING
- HISTOCHEMICAL STAINING/HISTOCHEMISTRY
- IMMUNOHISTOCHEMICAL STAINING
METHODS OF STAINING
- DIRECT STAINING
- INDIRECT STAINING
- PROGRESSIVE STAINING
- REGRESSIVE STAINING
- METACHROMATIC STAINING
- COUNTERSTAINING
- METALLIC IMPREGNATION
- VITAL STAINING
- VITAL STAINING:
INTRAVITAL STAINING
SUPRAVITAL STAINING
tissue constituents are demonstrated in sections by direct interaction with a dye or staining solution
HISTOLOGICAL STAINING
producing coloration of the active tissue component
HISTOLOGICAL STAINING
employed to demonstrate the general relationship of tissues and cells with differentiation of nucleus and cytoplasm
HISTOLOGICAL STAINING
Examples: microanatomic stains, bacterial stains and specific tissue stains (muscles, CT, and neurologic stains)
HISTOLOGICAL STAINING
tissue constituents are studied through chemical reactions that will permit microscopic localization of a specific tissue substance
HISTOCHEMICAL STAINING/HISTOCHEMISTRY
Examples: Perl’s Prussian blue reaction for hemoglobin, Periodic Acid Schiff staining for carbohydrates
HISTOCHEMICAL STAINING/HISTOCHEMISTRY
Enzyme histochemistry: active reagent serves as the substrate upon which the enzymes act
HISTOCHEMICAL STAINING/HISTOCHEMISTRY
final opacity of coloration produced from the substrate rather than the tissue
HISTOCHEMICAL STAINING/HISTOCHEMISTRY
combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated
IMMUNOHISTOCHEMICAL STAINING
makes use of different labels: monoclonal/polyclonal, fluorescent-labeled, enzyme-labeled antibodies
IMMUNOHISTOCHEMICAL STAINING
process of giving color to the sections by using aqueous or alcoholic dyes
- DIRECT STAINING
process whereby action of dye is intensified by adding another reagent (MORDANT) which serves as a link/bridge between tissue and dye making staining reaction possible
- INDIRECT STAINING
may be applied to tissue before staining or may be included in the staining process, or may be incorporated as part of the dye solution itself
MORDANT
MORDANT Eg.
potassium alum with hematoxylin in Ehrlich’s hematoxylin and iron in Weigert’s hematoxylin
not essential to the chemical union of tissue and dye
ACCENTUATOR
but merely accelerates or hastens the speed of staining reaction by increasing the staining power and selectivity of the dye
ACCENTUATOR
ACCENTUATOR Eg:
potassium hydroxide in Loeffler’s methylene blue and phenol in carbol thionine and carbol fuchsin
process whereby tissue elements are stained in a definite sequence, and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained
PROGRESSIVE STAINING
no decolorization or washing after the application of the dye
PROGRESSIVE STAINING
tissue is first over stained to obliterate cellular details, and excess stain is removed or decolorized from unwanted parts of the tissue, until the desired intensity of color is obtained
- REGRESSIVE STAINING
: selective removal of excess stain from the tissue so that a specific substance may be stained distinctly from surrounding tissues
DIFFERENTIATION/DECOLORIZATION
use of dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself
METACHROMATIC STAINING
basic dyes belonging to the thiazine and triphenylmethane groups
METACHROMATIC STAINING
METACHROMATIC STAINING Examples:
methyl violet or crystal violet Cresyl blue for reticulocytes Safranin Bismarck brown
stain tissues in color shades that are similar to the color of the dye itself
ORTHOCHROMATIC STAINING
application of a different color or stain to provide contrast and background to the staining of structural components to be demonstrated
COUNTERSTAINING
Cytoplasmic stains:
Red eosin Y, Eosin B, Phloxine B
Yellow piciric acid, orange G, Rose Bengal
Green light green SF, Lissamine green
Nuclear stains:
Red neutral red, safranin 0, carmine, hematoxylin
Blue methylene blue, toluidine blue, Celestine blue
process where specific tissue elements are demonstrated, not by stains, but by colorless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, usually black deposit on the surface of the tissue
METALLIC IMPREGNATION
agent is not absorbed by the tissue, but is held physically on the surface as a precipitate or as a reduction product
METALLIC IMPREGNATION
Examples: ammoniacal silver and gold chloride
METALLIC IMPREGNATION
reduced by argentaffin cells (melanin and intestinal glands) Gold chloride
Ammoniacal silver
selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle
VITAL STAINING
**nucleus is resistant to staining
VITAL STAINING
***if nuclear structures are demonstrated > death of cell already
VITAL STAINING
Examples: trypan blue>reticuloendothelial cells; Janus green>mitochondria
VITAL STAINING
done by injecting dye into any part of the body (intravenous, intraperitoneal or subcutaneous), producing specific color
INTRAVITAL STAINING
Examples: lithium, carmine and India ink
INTRAVITAL STAINING
stain living cells immediately after removal from the living body
SUPRAVITAL STAINING
Examples: neutral red, Janus green, trypan blue, etc.
SUPRAVITAL STAINING
Regressive staining due to the decolorization step using acid alcohol
H and E Staining Technique
H and E Staining Technique Major steps:
Deparaffinization (Step 1)
Hydration (Step 2)
Nuclear staining (Step 4)
Differentiation (Step 6)
Bluing (Step 8)
Counterstaining (Step 10)
Dehydration (Step 11)
Clearing (Step 12)
Nuclei:
blue to blue black
Karyosome:
dark blue
Cytoplasm, proteins in edema fluid:
pale pink
RBCs, eosinophilic granules, keratin:
bright orange-red
Basophilic cytoplasm, plasma cells, osteoblast:
purplish pink
Cartilage:
pink or light blue to dark blue
Calcium and calcified bone:
purplish blue
Decalcified bone matrix, collagen, osteoid:
pink
Muscle fibers:
deep pink
extracted from the core/heartwood of a Mexican tree (Hematoxylin campechianum)
HEMATOXYLIN
Ripening/Oxidizing: exposure to air & sunlight or to strong oxidizing agents
HEMATOXYLIN
Aluminum Hematoxylin:
Iron Hematoxylin:
Phosphotungstic Acid Hematoxylin:
Sodium iodate
Ehrlich’s
Tissues subjected to acid decalcification and acidic tissues
Ehrlich’s
Mercuric chloride
Harris’
Routine nuclear staining, exfoliative cytology and staining sex chromosomes
Harris’
Alcoholic iodine
Cole’s
Routine purposes, esp in sequence to Celestine blue
Cole’s
Sodium iodate
Mayer’s
