Special Stains Flashcards
What do the trichrome stains demonstrate
connective tissue
What is the basis for trichrome staining
porosity
what is the pH of trichrome stains
1-3
What type of hematoxylin is normally used with a trichrome stain
Weigerts iron hematoxylin
What should be done for best results of masson trichrome staining
fixed in bouins fluid or premordanted in bouins at 50-60C for an hour
What is the purpose of a masson trichrome stain
differentiate between smooth muscle and collagen in tumors and demonstrate increased collagen in disease states such as cirrhosis
What do biebrich scarlet and acid fuchsin do in masson trichrome stain
stain all acidophilic tissue components red
What does phosphomolybdic acid do in masson trichrome stain
bumps red out of the larger tissue pores and attract aniline blue
What does aniline blue do in masson trichrome stain
stains collagen and other larger pore tissues blue
What colour are nuclei in mason trichrome
grey to black
What colour are cytoplasm, keratin, muscle fibers and erythrocytes in mason trichrome
red
what colour is collagen in masson trichrome
blue
What causes decreased red staining in masson trichrome
old or overused biebrich/scarlet acid solution
What causes decreased blue staining in masson trichrome
prolonged rinse following PMA
old/overused aniline blue
over-differentiation with acetic acid
What cause all staining to be decreased in a masson trichrome
failure to premordant with bouins when using formalin fixed tissue
What are the two common elastin stains
verhoeffs and gomori’s aldehyde fuchsin
What is the theory of elastin stains
elastin contains disulfide bridges which may be oxidized to form sulfonic acid derivatives which are strongly basophilic
What is the theory of verhoeffs elastin stain
tissue is overstained with iodine ferric chloride hematoxylin solution. The ferric chloride works as a mordant, iodine oxidizes the hematoxylin and the elastic fibers. the tissue is then differentiated using excess mordant and iodine is removed with sodium thiosulfate
What colour are elastic fibers in verhoeffs elastic stain
black
What colour is collagen in verhoeffs elastic stain
red
What colour is muscle in verhoeffs elastic stain
yellow
What is the theory of gomoris aldehyde fuchsin
acidified paraldehyde added to an alcoholic basic fuchsin solution forms aldehyde fuchsin which is used progressively
What colour are elastin fibers in gomoris aldehyde fuchsin
purple
What colour are other tissue elements in gomoris aldehyde fuchsin
green
What is the cause of elastin fibers being poorly stained with gomoris aldehyde fuchsin
old or improperly stored aldehyde fuchsin
paraldehyde is old
slides cut too far in advance
What techniques are used to demonstrate reticular fibers
silver stains
What is the preffered method for silver stains
induced argyrophilia
What is the theory of gordon and sweets silver stain
reticular fibers are oxidized to create create reactive sites using potassium permanganate then an oxacilic acid bleaching step removes discolouration caused by the permanganate. Ferric ammonium sulfare is used as a sensitizer and is replaced by silver. Formalin reduces the silver and then gold chloride is used as a toner. Sodium thiosulfate removes any unreacted silver
What colour is reticulin with gordon and sweets
black
What colour are other tissues with gordon and sweets
red
What causes weak reticular staining with gordon and sweets
excess ammonia in silver solution
dirty glassware
use of buffered formalin in reduction step
What causes nuclei stain with silver
oxidization of nuclear material with potassium permanganate
What are common carbohydrate stains
periodic acid schiff or alcian blue
What tissues are positive in alcian blue at pH 2.5
carboxylated mucins and sulfated mucins
What tissues are positive in alcian blue at pH 1.0
sulfated mucin
What tissues are positive in PAS
glycogen and neutral mucin
What tissues are positive in PAS with diastase digestion
neutral mucin
What is the PAS used for
detect glycogen or mucin
detect fungal species
demonstrate basement membranes
demonstrate reticulin, zymogen granules of the pancrease, thyroid colloid and russel bodies of plasma cells
What is the theory of PAS
periodic acid is used to oxidize hydroxyl groups within carbohydrates to form aldehydes. Schiff reagent reacts with free aldehydes created at the oxidization step binding it to the tissue. Rinses remove the loosely bound sulfurous acid group from the central carbon atom
What colour are PAS positive substances
magenta
What colour are other tissue elements
dependant on counterstain
What causes the positive control to not stain
old/expired Schiff reagent
Schiff not stored in the fridge
old/expired periodic acid
What causes non-specific staining
failure to rinse the excess Schiff reagent prior to colour developer
What causes weak stain
old/overused Schiff reagent
What is the theory of PAS-D
diastase acts on glycogen breaking it down into smaller sugar units which are washed out of the section. Slides are run in duplicate. If staining on the non-digested slide is absent in the digested slide it must be from glycogen
What colour is glycogen in PAS-D
unstained
What colour are other PAS substances
magenta
What is alcian blue
a large cationic dye used to stain acid mucins
What is the theory of alcian blue
when used at 2.5pH alcian blue ionically bonds with different tissues at different pHs
What causes weak staining with alcian blue
old/expired alcian blue
tissue or control is autolyzed
staining at pH 1.0
What causes nuclei stain with alcian blue
prolonged staining time
What causes the slide to be cloudy after cover slipping with alcian blue
insufficient washing after application of nuclear fast red counterstain
What is AA amyloid
composed of serum amyloid A proteins. Associated with inflammation and chronic disease
What is AL amyloid
composed of immunoglobulin light chains. Associated with B-cell diseases
What is the theory of congo red
it is a linear dye molecule known to react with amyloid in a specific way. It fits with the B-pleats and is held in place by hydrogen bonds which causes a birefringence pattern
What colour is amyloid tissue in congo red
pale to deep red, bright green under polarized light
What colour is elastic tissue in congo red
pale pink
What colour are nuclei in congo red
purple
What causes no birefringence under polarized light in congo red
old sections
extended formalin fixation
section too thick or thin
What causes non-specific staining in congo red
older methods of staining
What is hemosiderin
yellow-brown endogenous pigment normally found within cells
What tissue is hemosiderin normally in
bone marrow and spleen
What is the theory of perls prussian blue
iron that is loosely bound to protein in the ferric oxidization state will react
What colour is hemosiderin in perls prussian blue
prussian blue
What colour is the background in perls prussian blue
pink
What causes staining solution to turn blue or non-specific blue precipitate in perls prussian blue
contaminated glassware
diffusion of iron from tissue
What causes cloudy slides after coverslipping with perls prussian blue
nuclear fast red not adequately rinsed before dehydration
What is the family of dyes used in fats
sudan family
What is oil red O used to stain
neutral lipids
What is the theory of oil red O
oil red O is dissolved in isopropyl alcohol or propylene glycol and then migrates from the solvent to the areas of fat
What colour is fat in oil red O
red
What colour are nuclei in oil red O
purple-blue
What causes fat droplets through out the tissue with oil red O
the coverslip was squished to remove air bubbles
What is the fontana-masson stain
an argentaffin silver stain used to demonstrate melanin and granules of argentaffin cells in the small intestine
What is the theory of the fontana-masson stain
a diamine silver solution is added and argentaffin tissue components will bind the silver and reduce it to metallic silver. The section is then toned with gold chloride and sodium thiosulfate to removes unreduced silver
What colour is melanin in fontana-masson stain
black
What colour is argentaffin granules in fontana-masson stain
black
What colour are nuclei in fontana-masson stain
pink
What causes non-specific staining in fontana-masson stain
overheated silver solution
contaminated glassware
What are the two stains used to dye calcium
von kossas silver stain and alizarin red s
What is von kossas silver stain
an indirect method of demonstrating calcium, silver ions replace calcium and are reduced to visable silver
What is alizarin red s
a direct stain that binds to calcium in tissue
What is the colour of calcium in von kossas silver stain
black
What is the colour of the background in von kossas silver stain
pink
what causes non-specific staining with von kossas silver stain
contaminated glassware
What causes brown staining instead of black staining with con kossas silver stain
insufficient light
What causes control organisms to all stain gram positive with gram staining
insufficient decolourization
What causes control organisms to all stain gram negative with gram staining
prolonged decolourization
What causes organisms to stain weakly with gram staining
expected result for spirochetes or acid-fast organisms
What is the ziehl neelsen stain
used to demonstrate acid fast organisms in tissue sections
What is the theory of the ziehl neelsen stain
organisms are stained using basic fuchsin dissolved in alcoholic phenol solutions and then decolourized
What colour are acid fast bacilli with the ziehl neelsen stain
pink
What colour is the background with the ziehl neelsen stain
blue
What causes methylene blue counterstain to be weak with ziehl neelsen stain
failure to fully remove acid alcohol following dehydration step
What causes methylene blue counterstain to be too dark with ziehl neelsen stain
left in methylene blue too long
What causes control organisms to fail to stain with ziehl neelsen stain
over-decolourized
What is diff-quik
a brand of giemsa stain used to screen slides for H. pylori
What is the theory of diff-quik
solution 1 stains positively charged tissue components and solution 2 stains negatively charged tissue components
What colour are bacteria and nuclei using diff-quik
blue
What colour is cytoplasm using diff-quik
pink
What causes weak staining with diff-quik
prolonged distilled water rinse or dehydration
insufficient time in staining solution
How do spirochete stains work
spirochetes are argyrophilic
What are the two common spirochete stains
warthin-starry and steiner and steiner
What does the Grocotts methenamine silver stain stain
fungus
What is the theory of Grocotts method
fungal walls are oxidized by chromic acid to produce aldehydes then sections are placed in warm methenamine-silver solution where the silver binds the aldehydes and is reduced to metallic silver
What colour is fungus in Grocotts method
black
What colour is mucin in Grocotts method
grey
What colour is the background in Grocotts method
light green
What causes lots of background staining in Grocotts method
failure to use chromic acid
old/expired chromic acid
What causes non-specific staining in Grocotts method
overheated silver solution
contaminated glassware
What causes no interior detail within organisms in Grocotts method
overstained with silver
