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Histotechnology > Special Stains > Flashcards

Special Stains Flashcards

1
Q

What do the trichrome stains demonstrate

A

connective tissue

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2
Q

What is the basis for trichrome staining

A

porosity

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3
Q

what is the pH of trichrome stains

A

1-3

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4
Q

What type of hematoxylin is normally used with a trichrome stain

A

Weigerts iron hematoxylin

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5
Q

What should be done for best results of masson trichrome staining

A

fixed in bouins fluid or premordanted in bouins at 50-60C for an hour

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6
Q

What is the purpose of a masson trichrome stain

A

differentiate between smooth muscle and collagen in tumors and demonstrate increased collagen in disease states such as cirrhosis

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7
Q

What do biebrich scarlet and acid fuchsin do in masson trichrome stain

A

stain all acidophilic tissue components red

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8
Q

What does phosphomolybdic acid do in masson trichrome stain

A

bumps red out of the larger tissue pores and attract aniline blue

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9
Q

What does aniline blue do in masson trichrome stain

A

stains collagen and other larger pore tissues blue

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10
Q

What colour are nuclei in mason trichrome

A

grey to black

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11
Q

What colour are cytoplasm, keratin, muscle fibers and erythrocytes in mason trichrome

A

red

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12
Q

what colour is collagen in masson trichrome

A

blue

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13
Q

What causes decreased red staining in masson trichrome

A

old or overused biebrich/scarlet acid solution

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14
Q

What causes decreased blue staining in masson trichrome

A

prolonged rinse following PMA
old/overused aniline blue
over-differentiation with acetic acid

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15
Q

What cause all staining to be decreased in a masson trichrome

A

failure to premordant with bouins when using formalin fixed tissue

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16
Q

What are the two common elastin stains

A

verhoeffs and gomori’s aldehyde fuchsin

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17
Q

What is the theory of elastin stains

A

elastin contains disulfide bridges which may be oxidized to form sulfonic acid derivatives which are strongly basophilic

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18
Q

What is the theory of verhoeffs elastin stain

A

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

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19
Q

What colour are elastic fibers in verhoeffs elastic stain

A

black

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20
Q

What colour is collagen in verhoeffs elastic stain

A

red

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21
Q

What colour is muscle in verhoeffs elastic stain

A

yellow

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22
Q

What is the theory of gomoris aldehyde fuchsin

A

acidified paraldehyde added to an alcoholic basic fuchsin solution forms aldehyde fuchsin which is used progressively

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23
Q

What colour are elastin fibers in gomoris aldehyde fuchsin

A

purple

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24
Q

What colour are other tissue elements in gomoris aldehyde fuchsin

A

green

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25
Q

What is the cause of elastin fibers being poorly stained with gomoris aldehyde fuchsin

A

old or improperly stored aldehyde fuchsin
paraldehyde is old
slides cut too far in advance

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26
Q

What techniques are used to demonstrate reticular fibers

A

silver stains

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27
Q

What is the preffered method for silver stains

A

induced argyrophilia

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28
Q

What is the theory of gordon and sweets silver stain

A

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

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29
Q

What colour is reticulin with gordon and sweets

A

black

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30
Q

What colour are other tissues with gordon and sweets

A

red

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31
Q

What causes weak reticular staining with gordon and sweets

A

excess ammonia in silver solution
dirty glassware
use of buffered formalin in reduction step

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32
Q

What causes nuclei stain with silver

A

oxidization of nuclear material with potassium permanganate

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33
Q

What are common carbohydrate stains

A

periodic acid schiff or alcian blue

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34
Q

What tissues are positive in alcian blue at pH 2.5

A

carboxylated mucins and sulfated mucins

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35
Q

What tissues are positive in alcian blue at pH 1.0

A

sulfated mucin

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36
Q

What tissues are positive in PAS

A

glycogen and neutral mucin

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37
Q

What tissues are positive in PAS with diastase digestion

A

neutral mucin

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38
Q

What is the PAS used for

A

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

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39
Q

What is the theory of PAS

A

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

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40
Q

What colour are PAS positive substances

A

magenta

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41
Q

What colour are other tissue elements

A

dependant on counterstain

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42
Q

What causes the positive control to not stain

A

old/expired Schiff reagent
Schiff not stored in the fridge
old/expired periodic acid

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43
Q

What causes non-specific staining

A

failure to rinse the excess Schiff reagent prior to colour developer

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44
Q

What causes weak stain

A

old/overused Schiff reagent

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45
Q

What is the theory of PAS-D

A

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

46
Q

What colour is glycogen in PAS-D

A

unstained

47
Q

What colour are other PAS substances

A

magenta

48
Q

What is alcian blue

A

a large cationic dye used to stain acid mucins

49
Q

What is the theory of alcian blue

A

when used at 2.5pH alcian blue ionically bonds with different tissues at different pHs

50
Q

What causes weak staining with alcian blue

A

old/expired alcian blue
tissue or control is autolyzed
staining at pH 1.0

51
Q

What causes nuclei stain with alcian blue

A

prolonged staining time

52
Q

What causes the slide to be cloudy after cover slipping with alcian blue

A

insufficient washing after application of nuclear fast red counterstain

53
Q

What is AA amyloid

A

composed of serum amyloid A proteins. Associated with inflammation and chronic disease

54
Q

What is AL amyloid

A

composed of immunoglobulin light chains. Associated with B-cell diseases

55
Q

What is the theory of congo red

A

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

56
Q

What colour is amyloid tissue in congo red

A

pale to deep red, bright green under polarized light

57
Q

What colour is elastic tissue in congo red

A

pale pink

58
Q

What colour are nuclei in congo red

A

purple

59
Q

What causes no birefringence under polarized light in congo red

A

old sections
extended formalin fixation
section too thick or thin

60
Q

What causes non-specific staining in congo red

A

older methods of staining

61
Q

What is hemosiderin

A

yellow-brown endogenous pigment normally found within cells

62
Q

What tissue is hemosiderin normally in

A

bone marrow and spleen

63
Q

What is the theory of perls prussian blue

A

iron that is loosely bound to protein in the ferric oxidization state will react

64
Q

What colour is hemosiderin in perls prussian blue

A

prussian blue

65
Q

What colour is the background in perls prussian blue

A

pink

66
Q

What causes staining solution to turn blue or non-specific blue precipitate in perls prussian blue

A

contaminated glassware
diffusion of iron from tissue

67
Q

What causes cloudy slides after coverslipping with perls prussian blue

A

nuclear fast red not adequately rinsed before dehydration

68
Q

What is the family of dyes used in fats

A

sudan family

69
Q

What is oil red O used to stain

A

neutral lipids

70
Q

What is the theory of oil red O

A

oil red O is dissolved in isopropyl alcohol or propylene glycol and then migrates from the solvent to the areas of fat

71
Q

What colour is fat in oil red O

A

red

72
Q

What colour are nuclei in oil red O

A

purple-blue

73
Q

What causes fat droplets through out the tissue with oil red O

A

the coverslip was squished to remove air bubbles

74
Q

What is the fontana-masson stain

A

an argentaffin silver stain used to demonstrate melanin and granules of argentaffin cells in the small intestine

75
Q

What is the theory of the fontana-masson stain

A

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

76
Q

What colour is melanin in fontana-masson stain

A

black

77
Q

What colour is argentaffin granules in fontana-masson stain

A

black

78
Q

What colour are nuclei in fontana-masson stain

A

pink

79
Q

What causes non-specific staining in fontana-masson stain

A

overheated silver solution
contaminated glassware

80
Q

What are the two stains used to dye calcium

A

von kossas silver stain and alizarin red s

81
Q

What is von kossas silver stain

A

an indirect method of demonstrating calcium, silver ions replace calcium and are reduced to visable silver

82
Q

What is alizarin red s

A

a direct stain that binds to calcium in tissue

83
Q

What is the colour of calcium in von kossas silver stain

A

black

84
Q

What is the colour of the background in von kossas silver stain

A

pink

85
Q

what causes non-specific staining with von kossas silver stain

A

contaminated glassware

86
Q

What causes brown staining instead of black staining with con kossas silver stain

A

insufficient light

87
Q

What causes control organisms to all stain gram positive with gram staining

A

insufficient decolourization

88
Q

What causes control organisms to all stain gram negative with gram staining

A

prolonged decolourization

89
Q

What causes organisms to stain weakly with gram staining

A

expected result for spirochetes or acid-fast organisms

90
Q

What is the ziehl neelsen stain

A

used to demonstrate acid fast organisms in tissue sections

91
Q

What is the theory of the ziehl neelsen stain

A

organisms are stained using basic fuchsin dissolved in alcoholic phenol solutions and then decolourized

92
Q

What colour are acid fast bacilli with the ziehl neelsen stain

A

pink

93
Q

What colour is the background with the ziehl neelsen stain

A

blue

94
Q

What causes methylene blue counterstain to be weak with ziehl neelsen stain

A

failure to fully remove acid alcohol following dehydration step

95
Q

What causes methylene blue counterstain to be too dark with ziehl neelsen stain

A

left in methylene blue too long

96
Q

What causes control organisms to fail to stain with ziehl neelsen stain

A

over-decolourized

97
Q

What is diff-quik

A

a brand of giemsa stain used to screen slides for H. pylori

98
Q

What is the theory of diff-quik

A

solution 1 stains positively charged tissue components and solution 2 stains negatively charged tissue components

99
Q

What colour are bacteria and nuclei using diff-quik

A

blue

100
Q

What colour is cytoplasm using diff-quik

A

pink

101
Q

What causes weak staining with diff-quik

A

prolonged distilled water rinse or dehydration
insufficient time in staining solution

102
Q

How do spirochete stains work

A

spirochetes are argyrophilic

103
Q

What are the two common spirochete stains

A

warthin-starry and steiner and steiner

104
Q

What does the Grocotts methenamine silver stain stain

A

fungus

105
Q

What is the theory of Grocotts method

A

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

106
Q

What colour is fungus in Grocotts method

A

black

107
Q

What colour is mucin in Grocotts method

A

grey

108
Q

What colour is the background in Grocotts method

A

light green

109
Q

What causes lots of background staining in Grocotts method

A

failure to use chromic acid
old/expired chromic acid

110
Q

What causes non-specific staining in Grocotts method

A

overheated silver solution
contaminated glassware

111
Q

What causes no interior detail within organisms in Grocotts method

A

overstained with silver

Histotechnology (10 decks)

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