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153 Cards in this Set
- Front
- Back
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H&E steps
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1. hematoxylin staining step
2. differentiation 3. bluing step 4. washing 5. eosin counterstain |
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H&E hematoxylin staining step
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Aluminium hematoxylin
basic dye regressive stain mordant indirect |
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H&E differentiation
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0.5%-1.0% acid alcohol
red nuclei everything else colourless |
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H&E bluing step
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Ammonia H2O
nuclei become blue again |
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H&E wash
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H2O and then alcohol
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H&E counterstain step
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Eosin
acidic dye direct quinoid |
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H&E results
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nuclei- blue
cytoplasm-pink Basic tissue- pink to red calcium, cartilage, RNA- blue |
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H&E demonstrates and principle
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routine stain general morphology
Ionic bonding -salt linkage |
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alcian blue steps
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1. pre-rinse step
2. alcian blue staing step 3. nuclear counterstaining step |
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alcian blue pre- rinse step
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acetic acid
changes the pH of the tissue to the same as the dye allowing it to pick up the dye |
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Alcian blue demonstrates and principle
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acid mucins
ionic bonding and salt linkage |
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alcian blue staining step
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alcian blue
basic dye member of the copper phthalocyanine dyes H2O soluble electrostatic bonds occur between the pos charged basic dyes and negative tissue group |
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alcian blue counterstain
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Neutral red
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alcian blue results
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pH 2.5 -sulphated&carboxylated mucin- blue turquoise
pH1.0- only sulphated mucin Nuclei- Blue- hematoxylin Red- neutral red |
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Luxol fast blue steps
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1. myelin staining step
2. differentiation 3. counterstain |
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Luxol fast blue myelin staining step
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luxol fast blue
basic dye copper phthalocyanine chromogen soluble in alcohol slow reaction use heat (37*-60*) forms a myelin-LFB complex |
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Luxol fast blue differentiation
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0.05% lithium carbonate and 70% ethanol
lithium carbonate is weak base ethanol slows down or stops process check micro |
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LFB counterstain
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1% eosin
acid dye |
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LFB results
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myelin (white matter)- blue
background (grey matter)- pink |
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LFB demo and principle
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myelin
base replacement with salt formation |
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Cresyl echt violet steps
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1. nervous tissue staining
2. differentiation |
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cresyl echt violet demo and principle
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nissl substance
RNA is basophillic and stains with basic aniline dyes by ionic bonding |
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cresyl echt violet nervous tissue staining
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0.5%-1.0% cresyl echt violet
basic pH 2.5 will only dye DNA&RNA containing structures |
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cresyl echt violet differentiation
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100% alcohol
check micro |
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cresyl echt violet results
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nissl substance- dark purple
neurons/nuclei- purple background-lilac/clear |
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Gordon and sweets steps
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1. oxidation
2. sensitization 3. silver impregnation 4. reduction 5. toning (optional) 6. silver fixing 7. counterstain |
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Gordon and sweets demo and principle
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reticulin
argyrophilic silver impregnation |
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gordon and sweets oxidation
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potassium permanganate (KMnO4)
leaves brown pigment bleach with oxalic acid oxidation exposes aldehyde groups |
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gordon and sweets sensitization
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iron alum
acts like a mordant aldehydes are coated with ferric ions wash well with dH2O |
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gordon and sweets silver impregnation
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ammoniacal silver
sensitive and reactive AgNO3+NH4OH+NaOH |
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gordon and sweets reduction
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unbuffered formaldehyde
extraneous reducer changes silver to blacken metallic state stop reaction with H2O |
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gordon and sweets toning
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0.2% gold cholride
improves the quality of the technique gold replaces the silver giving a finer precipitate producing a permanent, neutral black colour of high density in the reticulin |
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gordon and sweets silver (fixing)
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5% sodium thiosulfate
stops all chemical activity removes all excess silver and gold ions |
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gordon and sweets counterstain
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1% neutral red
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gordon and sweets results
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reticulin - black
nuclei - red cytoplasm - pink |
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periodic acid methenamine silver method (PAMS) steps
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1. oxidation
2. impregnation 3. toning 4. fixing step 5. counterstain |
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pams demo and principle
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basement membrane
argentaffin silver reaction |
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pams oxidation
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periodic acid
oxidizes basement membrane carbohydrate to aldehyde groups |
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pams impregnation
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methenamine silver
silver ions are bound to aldehydes and reduced to visible metallic silver by aldehyde groups used to speed up the reaction |
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pams toning
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gold chloride
replace silver with a finer denser, more permanent black coating |
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pams fixing step
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sodium thiosulfate
stops all reaction cleans away excess silver and gold |
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pams counterstain
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light green
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pams results
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basement membrane- black
background- green |
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pams thickness
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2um
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Fontanna masson steps
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1. silver impregnation step
2. toning step 3. fixing 4. counterstain |
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fontanna masson demo and principle
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melanin and argentaffin granules
argentaffin silver method |
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fontanna masson silver impregnation step
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ammoniacal or methenamine silver
no oxidization needed because melanin and argentaffin granules are strong reducing agents heat to 60* to increase speed of reaction check microscopically |
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fontanna masson toning step
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gold chloride
replaces silver with a finer denser precipitate causing black coating |
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fontanna masson fixing step
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sodium thiosulfate
stops all reactions cleans excess silver and gold |
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fontanna masson counterstain
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nuclear fast red
basic |
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fontana masson results
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melanin and argentaffin granules - brown/black
nuclei and cytoplasm- pink |
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Von Kossa steps
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1. silver impregnation
2. fixing 3. counterstain |
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von kossa demo and principle
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calcium
metal substitution and argyrophilic reduction |
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von kossa silver impregnation
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5% silver nitrate and light
after slides are put in a solution of silver nitrate then exposed to bright light stop reaction when calcium salts are brown to black |
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von kossa fixing
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sodium thiosulfate
stops reaction |
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von kossa counterstain
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nuclear fast red
basic dye |
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von kossa results
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calcium- brown/black
background- pink nuclei/cytoplasm- pink |
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Gomori's Methenamine silver steps
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1. oxidization
2. bleaching 3. silver impregnation 4. toning 5. silver 'fixing' 6. counterstain |
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Gomori's methenamine silver demonstrates and principle
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fungi
argentaffin silver reaction |
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Gomori's Methenamine Silver oxidization
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5% chromic acid
very strong overoxidizes all other carbs and non reactive acids makes more specific for fungal cell walls |
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Gomori's Methenamine Silver bleaching
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1% sodium bisulfite
removes chromic acid |
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Gomori's Methenamine Silver- silver impregnation
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methenamine silver nitrate
methenamine-provides an alkaline condition and chelates silver ions to produce silver amine groups silver nitrate- source of silver ions borax- buffer maintains an alkaline pH done at 60* |
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Gomori's Methenamine Silver toning
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0.1% gold chloride
improves contrast stabilizes |
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GMS removal of unreduced silver fixing
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2% sodium thiosulfate
stops the chemical reaction |
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Gomori's Methenamine Silver counterstain
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light green
acid dye bound by electrostatic attraction |
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Gomori's Methenamine Silver results
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fungal cell walls- black
inner parts of mycelia- grey mucin- grey other tissue components- green |
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Gomori's aldehyde fuchsin method steps
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1. elastic staining
2. washing 3. counterstain |
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Gomori's Aldehyde Fuchsin demo and principle
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elastic fibers
hydrogen bonding and van der waals forces progressive |
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Gomori's Aldehyde Fuchsin elastic staining
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aldehyde fuchsin
basic fuchsin, 70% alcohol, HCl, paraldehyde not specific for elastic stains progressively no differentiation necessary |
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Gomori's Aldehyde Fuchsin washing
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70% alcohol
removes excess colour from the background |
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Gomori's Aldehyde Fuchsin counterstain
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light green
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Gomori's Aldehyde Fuchsin results
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elastic fibers- purple
mast cell granules, beta-cell granules, beta-cell islets of langerhans, cheif cells & mucins- purple collagen and other tissue components- green |
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Verhoeffs steps
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1. elastic staining
2. differentiation 3. background clearing step 4. counterstain |
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verhoeffs demo and principle
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elastic fibers
hydrogen bonding and van der waals forces regressive |
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verhoeffs elastic staining
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verhoeffs hematoxylin
hematoxylin/ferric chloride/iodine lake iodine increases affinity of elastic fibers for iron hematoxylin and acts as a trapping agent no stabilizer present |
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verhoeffs differentiation
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2% ferric chloride
mordant differentiation breaks tissue- mordant dye complex check micro reaction stopped by wash step |
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verhoeffs background clearing step
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2.5% sodium thiosulfate
removes excess iodine (colour) wash well in H2O to remove alkaline pH which would otherwise inhibit the counterstain acid dyes |
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verhoeffs counterstain
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eosin
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verhoeffs results
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elastic fibers- black
nuclei- grey to black collagen- pink RBC- red other tissue - pink |
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Congo red steps
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1. nuclear staining
2. suppression of ionizing step 3. amyloid staining step |
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congo red demo and principle
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amyloid
hydrogen bonding |
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congo red thickness
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8-10um
polarizing microscope shows a apple green birefringence |
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congo red nuclear staining
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harris' hematoxylin
ionic bonding aluminium mordant |
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congo red suppression of ionizing step
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alkaline salt solution
depresses the ionization of amyloid releases hydrogen bonds |
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congo red amyloid staining step
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congo red
acid dye binds to amyloid through hydrogen bonds |
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congo red results
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amyloid- deep pink to red
elastic- pale pink nuceli -blue |
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Toluidine blue steps
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1. metachromatic staining
2. counterstaining |
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toluidine blue demo and principle
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mast cells
metachromatic |
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toluidine blue metachromatic staining
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toludine blue
pH 5.0 |
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toluidine blue counterstain
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light green
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toluidine blue results
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mast cells- deep violet
acid mucins- purple/ pink nuclei- blue cytoplasm- light green RBC- light green to pale yellow |
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periodic acid schiffs steps
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1. oxidizing
2. schiffs reagent 3.washing step 4. nuclear counterstain |
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PAS demo and principle
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basement membrane
histochemical |
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PAS oxidation
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periodic acid
mild/slow will not overoxidize the aldehyde groups |
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PAS schiffs reagent
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basic fuchsin (red)
sulfurous acid (to destroy the chromophore) demonstrates aldehydes cannot be reversed |
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PAS washing
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tap H2O
causes further oxidation develops colour and removes excess schiffs |
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PAS nuclear counterstain
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aluminium hematoxylin
(richard allen regressive) |
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PAS results
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PAS Pos- magenta
basement membrane- magenta nuclei- blue |
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perls prussian blue steps
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1. hemosiderin demonstration
2. counterstain |
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PPB demo and principle
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iron
histochemical |
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PPB hemosiderin demo
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potassium ferrocyanide and HCl
HCl breaks down hemosiderin into ferric iron and protien Ferric reacts with K ferrocyanide to form an insoluble blue pigment(prussian blue or ferric ferocyanide) |
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PPB counterstain
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nuclear fast red or eosin
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PPB results
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hemosiderin- blue
background - pink |
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Giemsa solution steps
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1. bacterial staining
2. differentiation |
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Giemsa demo and principle
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helicobacter pylori
polychromasia |
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giemsa bacterial staining
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giemsa solution
(eosin links with basic methylene blue to break down azure A, azure B & methyl violet) acid tissue stains metachromatic and background stains orthochromatically |
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giemsa differentiation
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dilute acetic acid (vinegar)
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giemsa results
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microorganisms- blue
nuclei- blue mast cells- purple background- pink to pale blue |
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Massons trichrome steps
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1. post fixation
2. nuclear stain 3. cytoplasm stain 4. differentiation 5. collagen stain 6. remove excess collagen stain |
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massons trichrome demo and principle
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collagen
porosity of tissues and molecular weight of dye |
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massons trichrome post fixation
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bouins solution
enhances stain |
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massons trichrome nuclear stain
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weigerts hematoxylin
must be an iron hematoxylin ferric chloride acts as an oxidizer and mordant black dye lake progressive |
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massons trichrome cytoplasm stain
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biebrich scarlet and acid fuchsion
acid or anionic dyes create competition for binding sites fuchsin (acid dye) stains collagen biebrichs (acid dye) stains muscle and cytoplasm |
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masson trichrome differentiation
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PMA/PTA
strong acid large molecular weight remove acid fuchsin from collagen only penetrate collagen |
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masson trichrome collagen stain
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light green or aniline blue
selectively stains collagen |
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masson trichrome removal of excess collagen stain
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1%acetic acid
sharpens cytoplasmic stain |
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masson trichrome results
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nuclei- dark grey
cytoplasm- shades of red muscle- bright red RBC- bright red collagen- green or blue |
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Van gieson steps
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1. nuclear stain
2. cytoplasmic stain |
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van gieson demo and principle
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collagen
porosity of tissue and molecular weight of the dye |
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van giesons nuclear stain
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weigerts iron hematoxylin
progressive |
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van gieson cytoplasmic stain
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van giesons solution
(picric acid and acid fuchsin) picric acid- small molecular weight- muscle acid fuchsin-large molecular weight-collagen |
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van gieson results
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nuclei- grey black
cytoplasm, muscle, RBC- yellow collagen- red |
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Gram stain steps
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1. primary stain
2. production of high molecular weight complex 3. decolourizing step 4. secondary stain 5. counterstain/dehydration 6. dehydration and clearing |
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Gram demonstration and principle
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gram pos/neg
nature of cell wall |
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gram primary stain
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crystal violet
basic dye reacts with RNA of positive organisms |
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gram production of HMW complex
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iodine
all purple trapping agent crystal violet/RNA is precipitated to form a HMWC in bacteria |
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gram decolourizing step
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acetone/alcohol
+ stay purple, -washes out= clear background |
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gram secondary stain
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basic fuchsin
- turns red background=red |
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gram counterstain/dehydration
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picric acid/acetone
picric acid binds to cytoplasm by electrostatic attraction acetone-differentiator/dehydrator |
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gram dehydration and clearing
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acetone/xylene
completes dehydration and begin clearing |
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gram results
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gram pos- deep blue/purple
gram neg- red nuclei-red cytoplasm, muscle, collagen, RBC-yellow fibrin, keratin, calcium, some fungi- deep blue |
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Ziel Neelsen steps
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1. bacteria staining
2. decolourizing step 3. counterstain |
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Ziel neelsen demo and principle
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acid fast bacilli
nature of cell wall |
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ZN bacterial staining
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carbol fuchsin
(carbolic acid, basic fuchsin, alcohol) carbolic acid- accentuator-increases selectivity, and rate of reaction carbolic fuchsin is done at 60* |
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ZN decolourizing
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1% acid alcohol
breaks the bond btw basic dye and tissue cant break the basic fuchsin-mycolic acid complex |
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ZN counterstain
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methylene blue
basic dye |
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ZN results
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acid fast bacilli- red/hot pink
nuclei-dark blue background-pale blue |
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Oil red O steps
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1. neutral lipid staining
2. differentiation 3. counterstain 4. counterstain enhancement |
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Oil red O demo and principle
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lipids
selective solubility |
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oil red O thickness, control and special requirement
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10-18 um frozen
control not required aqueous media needed dont press out bubbles |
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oil red O neutral lipid stain
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oil red O
weak red, acid dye dye solvent= propylene gylcol or isopropanol dye attracted by adsorption dye dissolves in the lipid (lysochrome-azodye) |
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oil red O differentiation
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60% isopropanol
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oil red O counterstain
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harris' hematoxylin (aluminium)
nuclei stained blue by electrostatic attraction cytoplasm=pale blue by physical attraction |
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oil red O counterstain enhancement
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ammoinia water
blueing agent increases contrast between lipid and nuclei |
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oil red O results
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neutral lipids- orange/red
phospholipid- pink background- blue nuclei- blue |
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Rapid H&E
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done in under 10 min
progressive stain fresh frozen tissue alcoholic eosin used to start dehydrating while counterstaining |
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affinity for silver
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reticulin, nerve fibers, melanin, fungi, calcium
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potassium permanganate (KMnO4)
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very strong oxidizing agent
often used in demonstration of reticulin |
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argentaffin
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can reduce silvers by itself
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argyrophilic
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needs an extranuous reducer
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silver substitution
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silver ions replace calcium ions which are linked with phospate or carbonate in the bone
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silver source in ammoniacal silver solution
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silver nitrate
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advantages/disadvantages to Gomori's Aldehyde Fuchsin
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ad- no differentiation step
solution can be stored identifies btw young and old elastic- young-delicate old-thick dis- not specific for elastic |
