STAINING Flashcards by FRANCHESCA MAUREEN CUISON

The process whereby
tissue components are
made visible in
microscopic sections by
direct interaction with a
dye or staining
solution.

STAINING

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A colored compound is
used to produce
contrast.

STAINING

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Physical characteristics can be evaluated.

Structural relationships of tissues and their
cells.

Morphologic changes are more easily
identified.

Presence or absence of disease can be
established.

STAINING

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Acidic (nucleus)

BASIC STAIN

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Purified form of a coloring agent or crude
dye that is generally applied in an aqueous
solution

HISTOLOGIC STAIN

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A chemical compound that reacts with the
stain to form an insoluble, colored
precipitate in the tissue.

Makes the staining reaction possible.

MORDANT

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Color of stains are not the real color of a
particular tissue.

TRUE

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Majority/ Routine staining > Hematoxylin &
Eosin.

TRUE

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nuclear detail

hematoxylin

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cytoplasmic detail

eosin

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Paraffin wax is poorly permeable to stains.

TRUE

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The process whereby the tissue constituents are
demonstrated in sections by direct interaction with a
dye or staining solution

Active tissue component is colored.

HISTOLOGICAL STAIN

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The process whereby various constituents of tissues
are studied through chemical reactions that permit
microscopic localization of specific tissue substances.

HISTOCHEMICAL STAINING

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Act as a substrate for the enzyme.

REAGENT

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From substrate, not the tissue.

FINAL COLORATION

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A combination of immunologic and histochemical
techniques that allow phenotypic markers to be
detected by antibodies (e.g. polyclonal, monoclonal,
enzyme-labeled or fluorescent-labeled).

IMMUNOHISTOCHEMICAL

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Visualization of Antigen-antibody complex
○ Antibody conjugated w/ an enzyme >
enzyme catalyze color-producing reaction
○ Antibody w/ fluorophore

IMMUNOHISTOCHEMICAL

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a.k.a.
“simple stainingˮ

● Uses aqueous or alcoholic dye solutions (e.g.
methylene blue, eosin) to produce a color.

● Only 1 dye, washed away after 3060 sec.

DIRECT STAINING

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Uses a mordant or another agent to intensify the
action of the dye used.

INDIRECT

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Serves as a link or bridge between the tissue and the
dye.

INDIRECT + MORDANT

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The dye may stain weakly by itself, therefore the
mordant combines with the dye forming a colored
“lakeˮ which would combine with the tissue forming an
insoluble “tissue-mordant-dye-complexˮ which would
allow subsequent counterstaining and dehydration to
be easy.

INDIRECT + MORDANT

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Not essential and does not participate in the
chemical reaction of the tissue and dye

INDIRECT + ACCENTUATOR

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Accelerates the speed of the staining reaction by
increasing the staining power and selectivity of the
dye.

INDIRECT + ACCENTUATOR

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Tissue elements are stained in a definite sequence.

● The staining with specific periods of time or until
desired color is attained.

● Not washed or decolorized.

● The distinction of tissue detail relies solely on the
selective affinity of the dye for various cellular
elements.

PROGRESSIVE

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Tissue overstained initially to obliterate cellular details. ● Excess stain is removed or decolorized from unwanted tissue parts until the desired color is obtained. ● Routine H&E for microanatomical studies.

REGRESSIVE

The selective removal of excess stain from the tissue during regressive staining so that specific substance may stain distinctly from the surrounding tissue.

DIFFERENTIATION OR DECOLORIZATION

Uses more than one stain for differentiation. Usually done by washing the section in simple solution (e.g. water or alcohol) or use of acids and oxidizing agents.

DIFFERENTIATION DECOLORIZATION

Primary stain= basic dye

differentiation = acidic solution

Primary stain = acidic dye Differentiation = alkaline solution

TRUE

Differentiator for both acidic and basic dyes by dissolving excess dye,

ALCOHOL

A differentiating agent. ● Can oxidize hematoxylin to a soluble, colorless compound.

MORDANT

Disadvantage: if a mordant stained section is allowed to remain in a differentiating agent such as 1% or 2% alcohol, all of the dye will be removed.

MORDANT

Makes use of specific dyes which differentiate particular substances by staining it with a color that is different from that of the stain itself (metachromasia)

METACHROMATIC STAINING

Usually employed in staining cartilage, connective tissue, epithelial mucins, amyloid and mast cell granules.

METACHROMATIC

necessary for most metachromatic staining techniques

WATER

Usually lost if the section is dehydrated in alcohol after staining. Satisfactorily seen in formalin-fixed tissues.

METACHROMASIA

Belongs to thizine and triphenylmethane groups:

METACHROMATIC DYES BASIC

process where specific tissue elements are demonstrated not by stains but by colorless solutions of metallic salts which are thereby reduced by the tissue > opaque, black deposits on the surface

METALLIC IMPREGANTION

It is not absorbed by the tissues, could be a precipitate or a reduction product on certain tissues.

METALLIC IMPREGNATION

The selective staining of living cell constituents.

VITAL

Demonstrates cytoplasmic structures.

VITAL

By engulfment/phagocytosis of the dye particle

VITAL

By staining of pre-existing cellular components.

VITAL

excluded by living tissue, but taken ● up by dead cells. (staining of RES w/ trypan blue)

VITAL

Nucleus is resistant to vital stains.

TRUE

injecting the dye into any part of the body ● Examples: lithium, carmine and India ink

INTRAVITAL

Used immediately after removal of cells from the living body ● Supravital stains: taken up by living cells, may be toxic > dilute amounts only ● Examples: Neutral red (best), Janus green (mitochondria), Trypan blue, Nile blue, Thionine and Toluidine Blue

SUPRAVITAL

Application of a different color or stain to provide contrast and background to the staining of the structural components to be demonstrated.

COUNTERSTAINING

EOSIN Y EOSIN B PHLOXINE B

Red

PICRIC ACID ORANGE G ROSE BENGAL

yellow

Lt. green sf lissamine green

green

NEUTRAL RED SAFRANIN CARMINE

RED NUCLEAR STAINS

METHYLENE TOLUIDINE CELESTINE HEMATOXYLIN

BLUE NUCLEAR

Clear paraffin embedded sections in first xylene bath for 3 minutes.

FIRST STEP

2. Transfer to a second xylene bath for 2 to 3 minutes. 3. Immerse in the first bath of absolute ethyl alcohol for 2 minutes.

Transfer to a bath of 95% ethyl alcohol for 1 to 2 minutes

REHYDRATION

5. Rinse in running water for a minute. 6. Stain with Harris Alum Hematoxylin for 5 minutes (Ehrlichʼs hematoxylin requires 1530 minutes).

blue to blue black

NUCLEI

KARYOSOME

DRK BLUE

CYTOPLASM

pale pink

RBCs, eosinophilic granules, keratin

bright orange red

Calcium and decalcified bone

purplish blue

Decalcified bone matrix, collage, and osteoid

PINK

Muscle fibers

deep pink

Similar to paraffin sections although duration may be shorter. Sections may be mounted in aqueous medium directly from water. Stained by picking up sections on albuminized slide ff by quick drying or direct staining on a wet slide using a dropper.

FROZEN SECTION

Possible staining methods ○ Hematoxylin-Eosin method ○ Thionine method ○ Polychrome Methylene Blue method ○ Alcoholic Pinacyanol method

FROZEN SECTION

Stains may be used again as long as they have their staining properties

TRUE

Derived from plants and animals Mostly plants) ○ Hematoxylin; ○ Cochineal dyes ; ○ Orcein; ○ Saffron.

NATURAL DYE

Hematoxylin campechianum (tree) deep rd or violet in color

HEMATOXYLIN

Hematoxylin campechianum (tree) deep rd or violet in color

HEMATOXYLIN

Require oxidation of hematoxylin through the process of“Ripeningˮ active coloring agent

HEMATIN

Powerful nuclear and chromatin staining capacity ● Polychrome property (produced with differentiation) ● Not a true basic dye

HEMTOXYLIN

Frequently used with mordants ○ Alum, Iron, Chromium, Copper

HEMATOXYLIN

Expose the substance to air and sunlight A slow process, 34 months

NATURAL RIPENING

Hydrogen peroxide, mercuric oxide, potassium permanganate, Sodium perborate, sodium iodate Used immediately > shorter shelf-life

ARTIFICIAL RIPENING OR CHEMICAL OXIDATION

Excessive oxidation > production of useless compound

OVER RIPENING

cochineal bug (Coccus cacti)

COCHINEAL

COCHINEAL + ALUM

CARMINE DYE

COCHINEAL + PICRIC ACID NEUROPATHOLOGICAL STAIN

PICROCARMINE

COCHINEAL + ALUMINUM CHLORIDE DEMONSTRATION OF GLYCOGEN

BEST CARMINE

Colorless, treated with ammonia, exposed to air > blue or violet color ● Weak acid, soluble in alkali ● Used for staining elastic fibers

ORCEIN

Vegetable dye (lichens)

ORCEIN

“coal tar dyesˮ ● Derived from hydrocarbon benzene. Collectively known as “Aniline dyesˮ

ARTIFICIAL DYE

coloring property

CHROMOPHORE

dyeing property

AUXICHROME

Attached to a benzene ring

chromophore and auxochrome

Substances that are capable of producing visible color but is not permanent and can be easily removed. ● From chromogens

CHROMOPHORE

Substances that are added to a chromogen, which alter the property of the chromogen by altering its shade, enabling it to form salts with another compound and enables it to retain its color in the tissue.

AUXOCHROME

affinity to cytoplasm

ACID

affinity to the nucleus

BASIC

Formed by combining aqueous solutions of acid and basic dyes. ● Stains the cytoplasm and nucleus simultaneously and Differentially. ○ Since they are composed of acid and basic, they stain almost everything. ● Insoluble to barely soluble in water, soluble in alcohol.

NEUTRAL

Progressive and regressive staining.

aluminum hematoxylin

sodium iodate

ERLICHS

Stains mucopolysaccharide substances (e.g. cartilages, cement lines of bones), tissues subjected in acid decalcification, tissues stored in formalin ● Problem: not an ideal stain for frozen sections

ERLICHS

Good regressive stain ● Routine stain for nuclear staining Dark purple color when ripened with mercuric chloride

HARRIS

Needs filtering prior to use Ripened with alcoholic iodine

COLE

Regressive and progressive stain Nuclear counterstain sodium iodate din parang erlichs

MAYER

Used only for differential or regressive staining. ○ Not used for progressive staining since it will cause intense colorization. ● Produce blue-black lakes. ● Iron is an active oxidizing agent. ● May be used for all fixatives.

IRON HEMATOXYLIN

Standard iron hematoxylin. ● Used in demonstrating muscle fibers and connective tissue.

WEIGERTS

Regressive staining > for nuclear and cytoplasmic inclusions

HEIDENHAINS

Color: reddish brown to purple ● Nuclei, Fibrin, Muscle striations (blue) ● Bone and cartilage (orange-red, brownish-red to deep brick-red stain) Natural ripening achieved with light and air. PROGRESSIVE

PTAH

A red acid dye that combines w/ Hgb > orange ● Routinely used as a counterstain after hematoxylin and before methylene blue ● Stains connective tissues and cytoplasm differentially ● Mostly used for demonstrating cytoplasmic detail

EOSIN

Deeper red color

EOSIN B, ERYTHROSIN B

EOSIN S, EOSIN ALCOHOLSOLUBLE

ETHYL EOSIN

RARELY USED TODAY

EOSIN B AND S

Combination of eosin and methylene blue. ● Variants: ○ Wrightʼs; ○ Giemsa; ○ Jennerʼs stain; ○ Leishman stain. Used for blood or bone marrow > different types of WBC can be readily distinguished.

ROMANOWSKY STAIN