Principles of Staining

7/30/2019 Principles of Staining

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The process of applying dyes on the sections tosee and study the architectural pattern of thetissue and physical characteristics of the cells.

This is made possible because different tissuesand different cells display varying affinities formost dyes and stains used during process, sothat they become more visible, morphological

changes are more easily identified, and thepresence or absence of disease process can beestablished.


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Histological Staining The process whereby the tissue constituents are

demonstrated in sections by direct interaction with a

dye or staining solution, producing coloration of theactive tissue component.

Includes:

Micro-anatomic stains

Bacterial stains Specific tissue stains (muscles, connective tissue and

neurologic stains.)


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Histochemical Staining(Histochemistry) The process whereby various constituents of tissues

are studied thru chemical reactions that will permitmicroscopic localization of a specific tissuesubstance.

Examples of such type of stains are Perls Prussian

Blue reaction for hemoglobin, and Periodic AcidSchiff staining for carbohydrates.


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Immunohistochemical Staining A combination of immunologic and histochemical

techniques that allows phenotypic markers to be

detected to be detected and demonstrated under themicroscope, using a wide range of polyclonal ormonoclonal, fluorescent labeled or enzyme-labeledantibodies.


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Direct Staining

Process of giving color to the sections using aqueousor alcoholic dye solutions. (ex. Eosin, Methylene

Blue).


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Indirect Staining The process whereby the action of the dye is

intensified by adding another agent or MORDANTwhich serves as a link or bridge between the tissue

and the dye, to make staining reaction possible. The mordant combines with the tissue to form a

TISSUE-MORDANT-DYE-COMPLEX, which in turncombines with the tissue to form aqueous andalcoholic solvents.

Examples of Mordant are: Potassium alum withhematoxylin in Erlichs hematoxylin, and Iron inWeigerts hematoxylin.


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Accentuator, on the other hand, is not essential tothe chemical union of the tissue and the dye. Itdoes not participate in the staining reaction,

but merely accelerates or hastens the speed ofthe staining reaction by increasing the stainingpower and selectivity of the dye.

Examples are: Potassium hydroxide in

Loefflers methylene blue and Phenol in Carbolthionine and carbol fuchsin.


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The process whereby tissue elements arestained in a definite sequence, and stainingsolution is applied for specific periods of time,

or until desired intensity of coloring of thedifferent tissue elements is attained.

Once the dye is taken up by tissue, it is notwash or decolorized. The differentiation or

distinction of tissue details relies solely on theselective affinity of the dye for different cellularelements.


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With this technique, the tissue is firstoverstained to obliterate the cellular details,and the excess stain is removed or decolorized

from unwanted parts of the tissue, until thedesired intensity of color is obtained.


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The selective removal of excess stain from thetissue during regressive staining in order that aspecific substance may be stained distinctly

from the surrounding tissues. This is done by washing the section in simple

solution (water, alcohol) or by the use ofoxidizing agents and acids.


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It entails the use of specific dyes whichdifferentiate particular substances by stainingthem with a color that is different from that of

the stain itself. Tissue components combine with these dyes to

form a different color from the surroundingtissues, epithelial mucins, mast cell

granules,and amyloid.


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Metachromatic dyes are basic dyes belonging tothe thizine and triphenylmethane groups, such as: Methyl violet or crystal violet

Cresyl blue Safranin

Bismarck brown

Basic fuchsin

Methylene blue Thionine

Toluidine blue

Azure A, B, C


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Application of different color or stain toprovide contrast and background to thestaining of the structural components to be

demonstrated.


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The following are most common counterstainsused in the laboratory:

1. Cytoplamic stainsRed Yellow Green

Eosin Y picric acid light green SFEosin B orange G Lissamine greenPhloxine B rose bengal2. Nuclear Stains

Red Blue

Nuetral red methylene blueSafranin O toluidine blueCarmine celestine blueHematoxylin


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A process where specific tissue elements aredemonstrated, not by stains, but by colorlesssolutions of metallic salts which are thereby

reduced by the tissues, producing a opaque,usually black deposit on the surface of thetissue or bacteria

It is different from stains because it is not

absorbed by tissues , but I held physically onthe surface as precipitate or as a reductionproduct in certain tissue components.


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A selective staining of living cell constituents,demonstrating cytoplasmic structures byphagocytosis of the dye particle, as in vital

staining of reticulo-endothelial system withtrypan blue or by staining of pre- existingcellular components.


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Done by injecting the dye into any part of theanimal body, producing specific coloration ofcertain cells, particularly those of the reticulo-

endothelial systems. Common dyes are: lithium, carmine, india ink


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Used to stain living cells immediately afterremoval from the living body. Thin slices of tissueare place in small staining dishes and enoughstaining solutions is added to cover the tissue.

Common dyes are: Neutral red- the best supravital stain

Janus green- recommended for mitochondria

Trypan blue Nile blue

Thionine

Toluidine blue


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After the section is cut and mounted on theslide, it must be drained and dried thoroughlyto ensure that all moisture between the section

and slide has evaporated so that the section isfirmly attached to the slide. If drying is notcomplete, the section may become detachedfrom the slide during the process of staining,

most likely after using acid differentiator.


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Paraffin wax is poorly permeable to moststaining solutions and should therefore beremoved from the section before staining. This

is usually done by immersing the paraffinsection in a solvent, two times, at 1-2 minutesduration each, for sections up to 10 micra thick.


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Coplin Jar- holds 5-9 slides

Slotted Staining Dishes- holds 5 to 19 slides,over which different solutions are poured.

Metals or Glass Staining Racks or Carrier-holds 10-30 slides upright.

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Principles of Staining