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Pigment & mineral

Staining

QuestionAnswer
Artifact pigment Formed as a result of chemical action - fixation, processing, usually on tissue, not w/i cell, e.g. formalin, mercury, chrome pigments
Exogenous pigment From outside the body, carbon pigment, asbestos fibers, tattoo pigments, metals - normal (iron) or pathologic (copper)
Carbon pigment Seen in lung & lymph node, resists bleaching & extration, insol. in conc'd sulfuric acid - use to differentiate
Asbestos fibers Birefringent, magnesium silicate, in body fibers coated w/ iron-containing protein, known as asbestos bodies, lose birefringence demo w/ Prussian blue
Endogenous hematogenous pigments From blood, e.g. hemoglobin, hemosiderin, biliverdin
Hemoglobin Conjugated protein, anionic, stains w/ acid dyes (eosin), hemoglobin breaks into globin protein & heme, heme splits into iron & biliverdin
Hemosiderin Yellow to brown pigment, stored iron (Fe2+), usu. in marrow, pathologic elsewhere (liver), differentiate from other yellow-brown pigments w/ Prussian blue rxn
Hemochromatosis Disease caused by excessive absorption of dietary iron, excessive hemosiderin deposits in liver, pancreas, damaging to tissue
Biliverdin Greenish bile pigment, prod'd from destruction of rbc, liver red. to bilirubin - remove from circulation, abnormal accumulation - jaundice, pigment in bile canaliculi, cytoplasm of Kupffer cells & hepatocytes
Hematoidin Similar to bilirubin, ox. to bilirubin, formed as result of hemorrhage & red'd O2 tension
Endogenous nonhematogenous pigments Lipid - e.g. lipofucsin, ceroid, nonlipidic - e.g. melanin
Melanin pigment Formed by ox. tyrosine, histochem. demo melanocytes, bleach w/ H2O2, potassium permanganate & oxalic acid, dissolved by strong alkali, insol. weak acid, base, organic solvents, argentaffin, positive Schmorl
Lipofuscin pigment "Wear & tear" pigment, yellow-brown, in more permanent cells (heart, liver, neurons)
Ceroid pigment Brownish-yellow, hepatocytes & macrophages of rats w/ cirrhosis, rarely seen in humans
What stains for lipofuscin & ceroid pigments? Oil red O, Sudan black B, & PAS, differentiate - ceroid is acid-fast, lipofsucin is not
Engodenous deposits Urate crytals (gouty tophi), chronic gout may deposit in soft tissue too, sol. in water, alcoholic fix., birefringent w/ polarizing, demo w/ argentaffin rxn
Minerals Special stains can demo: Ca2+, Fe2+/Fe3+, Cu2+, PO43+, CO32+, silver, lead, copper, gold, microincineration can be used to ID inorganic components
Cytoplasmic granules Adrenal chromaffin granules, pancreatic endocrine cells, GI enterochromaffin cells, "C" cells of thyroid, some pituitary cells, are APUD cells demo w/ argyrophil or argentaffin rxns, fixative v. important
APUD cells Have high uptake of amine-precursors & ability to decarboxylate
Best fixative for chromaffin granules? Primary chromate fixatives, e.g. Orth
Argentaffin granules of GI are destroyed by? Alcoholic fixatives
Paneth cell granules are destroyed by? Acetic acid
Prussian blue stain purpose Detect ferric (Fe3+) iron, usu. small amount in marrow & spleen, large deposits - hemochromatosis, hemosiderosis
Prussian blue stain facts Fixative: alcohol or 10% NBF, QC: section w/ Fe3+, don't want excess iron, chemically clean glassware, iron-free reagents
Prussian blue stain solutions potassium ferrocyanide 2% (bind Fe3+) HCl 2% (acidify) nuclear-fast red
Prussian blue stain results nuclei & hemofuchsin - bright red hemosiderin (iron) - blue background - pink
Nuclear-fast red solution aluminum sulfate nuclear-fast red thymol (preservative) dH2O
Fixation for iron demo: Some acidic fixatives & decal. solns. dissolve iron, can demo iron after Zenker w/ acetic acid, but mercury - bad, best to use another fixative & decal briefly in acetic or formic acid
Turnball blue stain purpose Detect ferrous (Fe2+) iron, ferrous iron v. toxic, not normally stored, easily absorbed by intestine but converted to ferric, used w/ Schmorl technique
Turnball blue stain facts Fixative: alcohol or 10% NBF QC: section w/ Fe2+
Turnball blue stain solutions potassium ferricyanide staining soln. (bind Fe2+) acetic acid 1% (post-stain rinse) nuclear-fast red
Turnball blue stain results ferrous iron - blue background - pink-red
Potassium ferricyanide staining solution potassium ferricyanide HCl 0.06N
Schmorl technique purpose Indicate reducing substances - will red. ferric ions, stains melanin, argentaffin granules, formalin pigment, mostly replaced by IHC
Schmorl technique facts Fixative: 10% NBF QC: section w/ melanin or argentaffin granules
Schmorl technique solutions ferric chloride-potassium ferricyanide working soln. pH 2.4 Mayer mucicarmine soln. or metanil yellow
Schmorl technique results reducing/argentaffin substances - blue-green goblet cells, mucin - rose background - yellow-green (less better)
Mayer mucicarmine solution carmine alum lake aluminum hydroxide anhy. aluminum chloride 50% ethanol 25% ethanol
Fontana-Masson & microwave Fontana-Masson stains purpose Demo argentaffin substances, e.g. melanin, argentaffin granules of carcinoid tumors, some neurosecretory granules, demos other red'ing substances (formalin pigment)
Fontana-Masson & microwave Fontana-Masson stains facts Fixative: 10% NBF, avoid alcohol QC: skin for melanin, small intestine or appendix for argentaffin granules, Melanoma, tumor w/ argentaffin granules, chemically clean glassware, nonmetallic instruments
Fontana-Masson stain solutions Fontana silver nitrate w/ NH4OH gold chloride sodium thiosulfate nuclear-fast red
Fontana-Masson stain results melanin, argentaffin granules - black nuclei - pink overstaining gives gray background & loss of contrast
Microwave Fontana-Masson stain solutions Fontana silver nitrate w/ NH4OH gold chloride sodium thiosulfate nuclear-fast red
Microwave Fontana-Masson stain results argentaffin cell granules, chromaffin granules, melanin, other argentaffin substances - black nuclei - pink
Grimelius stain purpose Demo argyrophil granules in neurosecretory tumors, also stains argentaffin substances
Grimelius stain facts QC: argyrophil-positive carcinoid tumor preferred, can use small intestine, chemically clean glassware, nonmetallic forceps
Grimelius stain solutions working silver solution reducing solution nuclear-fast red
Grimelius stain results argentaffin & argyrophil granules - dark brown to black nuclei - red background - pale yellow-brown
Grimelius working silver solution acetic acid-acetate buffer pH 5.6 silver nitrate 1% dH2O
Grimelius, Churukian-Schenk, microwave Churukian-Schenk reducing solution hydroquinone sodium sulfite dH2O
How to differentiate between argentaffin & argyophil substances? Stain w/ both Grimelius (argyophil) & Fontana-Masson (argentaffin) & compare
Churukian-Schenk & microwave Churukian-Schenk methods purpose Demo argyrophil granules in neurosecretory tumors
Churukian-Schenk & microwave Churukian-Schenk methods facts QC: argyrophil-positive carcinoid tumor preferred, can use small intestine, chemically clean glassware, nonmetallic forceps
Churukian-Schenk method solutions acidified water pH 4.0 to 4.2 silver nitrate 0.5% reducing solution nuclear-fast red
Churukian-Schenk method results argyophil granules, argentaffin substances - black nuclei - red background - yellow-brown
Microwave Churukian-Schenk method solutions citric acid-glycine soln. silver nitrate 0.5% reducing solution nuclear-fast red
Microwave Churukian-Schenk method results argyrophil & argentaffin cells - black nuclei - orange to red background - light yellow-orange
Gomori methenamine-silver method purpose Demo urates, may demo large calcium deposits
Gomori methenamine-silver method facts Fixative: abs. alcohol QC: section w/ urates
Gomori methenamine-silver method solutions working methenamine-silver nitrate sodium thiosulfate light green w/ acetic acid
Gomori methenamine-silver method results urates - black background - green
Bile stain purpose Detect bilirubin in tissue, aka Hall stain, aka Fouchet rxn
Bile stain solutions Fouchet reagent van Gieson
Bile stain results bile, bilirubin - emerald green to olive drab background - yellow
Fouchet reagent solution trichloracetic acid ferric chloride dH2O
van Gieson solution acid fuchsin picric acid
von Kossa stain purpose ID calcium in tissue, detects anions bound to calcium, not the calcium itself
von Kossa stain facts Fixative: alcohol preferred, 10% NBF ok
von Kossa stain solutions silver nitrate sunlight sodium thiosulfate nuclear-fast red
von Kossa stain results calcium salts - black background - red
Alizarin Red S stain purpose ID calcium in tissue, reaction product is birefringent
Alizarin Red S stain facts Fixative: alcoholic formalin or 10% NBF
Alizarin Red S staining solution Alizarin red S (chelate w/ calcium) dH2O NH4OH to pH 4.1 to 4.3 - critical
Alizarin Red S stain results calcium deposits - orange-red
Rhodanine & microwave Rhodanine methods purpose Detect copper in tissue, esp. liver - Wilson disease, aka Lindquist rhodanine
Rhodanine & microwave Rhondanine methods facts Fixative: 10% NBF Sections: paraffin 6-8 ┬Ám QC: section w/ copper (fetal liver)
Rhodanine method solutions working rhodanine soln. Mayer hematoxylin w/ dH2O borax (blue Mayer)
Rhodanine method results copper - bright red to red yellow nuclei - light blue If copper conc. low, stain may fade after coverslipping
Microwave Rhodanine method solutions working Rhodanine w/ sodium acetate-formalin Mayer hematoxylin w/ dH2O borax
Microwave Rhodanine method results copper - bright red or rust-red intracytoplasmic granules nuclei - blue
Working Rhodanine solution 5-(p-dimethylamino-benyzlidine) rhodanine abs. ethanol dH2O filter
Pigment A substance that has enough color to be visible w/o any further staining
What are some diseases associated w/ melanin? Melanomas, Addison's disease
Prussian blue vs. Turnball blue Prussian - ferrocyanide for ferric ions Turnball - ferricyanide for ferrous ions
Fontana Masson w/ melanin bleach To compare, deparaffinize & hydrate 2 slides, leave 1 in dH2O, 1 in potassium permanganate, wash w/ tap & dH2O, in oxalic acid, wash w/ tap & dH2O, stain both w/ Fontana Masson protocol
Argentaffin stains Gomori methenamine-silver, Grocott methenamine-silver, Fontana Masson, von Kossa
Argyrophil stains Reticulin, Holmes, Bielschowsky, Warthin-Starry, Dieterle, Steiner & Steiner, Grimelius, Churukian-Schenk
Formalin pigment Is a reducing substance, may give positive rxn to Schmorl & Fontana-Masson
Chloroacetate Esterase (Leder) stain purpose Detection of neutrophils
Stained starch granules show: A characteristic Maltese cross configuration when polarized, may be introduced from talcum powder of gloves
Ferrous ions Fe2+, toxic, not normally stored, Turnbull blue
Ferric ions Fe3+, normally present in tissue, usu. bound to protein (hemosiderin), in marrow, spleen, decaying, hemorrhagic material, excess - hemachromatosis, Prussian blue
Created by: CCF
 

 



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