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MLSCI 250 > 350 - Special Stains > Flashcards

350 - Special Stains Flashcards

1
Q

this is used to demonstrate carboxylated and sulfated acid-mucopolysaccharides

A

alcian blue

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2
Q

alcian blue at pH 2.5

A

all acid mucosubstances are stained
- both carboxylated and sulfated

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3
Q

alcian blue at pH 1.0

A

only sulfated mucosubstances are demonstrated

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4
Q

principle of alcian blue

A
  • cationic dye; binds to acids groups of acid mucopolysaccharides
  • sialomucins also demonstrated
  • at pH 1.0 carboxyl groups are not ionized and therefore cannot react with alcian blue
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5
Q

alcian blue procedure

A
  • NBF or Bouin’s (fixative)
  • 3% acetic acid or 0.1 M HCl
    > ensures stable pH; acetic acid for 2.5 and HCl for 1.0
  • alcian blue dissolved in acid of choice ^
  • acid rinse (removes excess alcian blue)
  • nuclear fast red or eosin for counterstain
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6
Q

what is demonstrated at pH 2.5 of alcian blue

A

hyaluronic acid
acid mucosubstances
sialomucins
BLUE

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7
Q

what is demonstrated at pH 1.0 of alcian blue

A

sulfated acid mucosubstances
LIGHTER blue

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8
Q

control tissue for alcian blue

A

small intestine
appendix
colon

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9
Q

PAS-Alcian blue

A
  • PAS will demonstrate neutral polysaccharides
  • AB before PA oxidation
  • tissues with mixture of both = purple
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10
Q

what does congo red demonstrate?

A

amyloid
- pathological protein; birefringent
- birefringence is considered most specific technique for amyloid

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11
Q

principle of congo red

A
  • amyloid = linear and 2ry protein structures = B pleats
  • linear shape of congo red dye fit within the pleats very specifically and at very regular frequencies
  • dye molecule held by hydrogen bonding
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12
Q

congo red procedure

A
  • alcohol is preferred fixative
  • sections MUST BE cute 7-10 um to demonstrate birefringence under polarized light
  • hematoxylin (counterstain)
  • wash in running tap water (bluing)
  • alkaline salt solution (increases binding sites; reduced background interference)
  • congo red (stains amyloid)
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13
Q

colours in congo red

A

amyloid is deep pink to pale red under brightfield and apple green under polarized light

elastic tissue is pale pink

nuclei is purple blu

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14
Q

T or F. prolonged storage in formalin will decrease staining intensity for congo red

A

T
it’s also prone to non-specific interations so saturate with salt (ions) to prevent

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15
Q

Crohn’s disease

A

amyloid

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16
Q

this stains argentaffin substances
and is frequently used to demonstrate melanin

A

Fontana Masson

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17
Q

other argentaffin substances that may be demonstrated b FM

A

enterochromaffin cells of small intestine
some tumours
formalin pigment

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18
Q

principle of Fontana Masson

A

argentaffin reaction
- can bind silver from solution AND reduce that silver to its metallic/visible form

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19
Q

Fontana Masson procedure

A
  • fixative (avoid alcohol if demonstrating argentaffin granules of tumors or enterochromaffin cells)
  • warm ammoniacal silver solution (metal impregnation; argentaffin “stained” black)
  • gold chloride = toning agent; refines and darkens silver
  • sodium thiosulfate (hypo) = fixing agent; removes any unreduced silver
  • nuclear fast red or eosin for counterstain
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20
Q

control tissue for Fontana Masson

A

section of skin as a melanin control (darker skin = more melanin)
section of small intestine or appendix for argentaffin granules

other tisse elements = red to pink

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21
Q

Fontana Masson can be made specific for Melanin

A

adding a duplicate slide pre=treated with potassium permanganate or H2O2 = will bleach away melanin

  • compare; any staining absent on bleached slide = melanin
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22
Q

this demonstrates elastic fibers

A

Gomori’s aldehyde fuchsin

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23
Q

how is aldehyde fuchsin made?

A

by adding acidified paraldehyde to an alcoholic basic fuchsin solution
- solution must ripen for 2-4 days prior to staining

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24
Q

T or F. G Aldehyde fuchsin is a progressive elastin stain

A

T! there is no differentiator used

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25
Q

GAF procedure

A
  • avoid chromate fixatives; formalin preferred
  • aldehyde fuchsin to stain elastic fibers
  • 70% alcohol rinse to remove excess stain
  • light green SF yellowish for counterstain
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26
Q

result of GAF

A

elastic fibers are purple
other issue elements are green

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27
Q

control tissue for GAF

A

aorta embedded on edge preferred; skin or muscular artery acceptable

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28
Q

what else will aldehyde fuchsin stain?

A

beta cell granules of pancreas
sulfated mucins
mast cells
cartilage
all contain abundant negatively-charged substrates, which readily stain using cationic dyes

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29
Q

at what temp must aldehyde fuchsin be stored at?

A

4C

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30
Q

this stain demonstrates reticular fiers

A

Gordon and Sweet’s Reticulin

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31
Q

why are silver methods used to demonstrate reticulin

A

dye methods are not specific or reliable

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32
Q

principle of Gordon and Sweet Reticulin

A
  • induced argyrophilia
  • reticulin will not be demonstrated without prior oxidation/sensitization
  • oxidation with potassium perm creates aldehyde groups from hexose sugars found in reticulin
  • sensitizing metal salt binds to aldehydes => then replaced by silver from diamine silver solution
  • silver reduced to its visible metallic form by formaldehyde
33
Q

Gordon and Sweet Reticulin procedure

A
  • NBF
  • potassium perm to oxidize sugars = aldehyde formation
  • oxalic acid to remove brown discolouration by potassium perm
  • ferric ammonium sulfate = sensitizer; binds to aldehyde; provides sites for silver
  • ammoniacal silver solution = impregnation step
  • formalin = reduces silver to make it visible
  • gold chloride = toner; refines appearance of silver
  • sodium thiosulfate = fixer; removes unreduced silver
  • eosin or nuclear fast red as counterstain
34
Q

control tissue for Gordon and Sweet

A

liver

35
Q

this demonstrates fungus in tissue sections

A

Grocott Methenamine Silver

36
Q

other staining methods for fungus besides GMS

A

PAS, Gram stain, and H&E
but GMS is most selective for the widest variety of organisms

37
Q

principle of GMS

A
  • fungal cell walls = lots of polysaccharides
  • polysacchss readily oxidized into aldehydes using chromic acid (STRONG oxidizer)
  • sections placed in 60C methenamine-silver solution where silver binds aldehydes -> reduce to metallic silver
    INDUCED ARGETAFFIN
38
Q

GMS procedure

A
  • NBF
  • chromic acid = oxidizer
  • sodium bisulfite - neutralizes residual chromic acid and removes orange
  • warm meth silver = impregnation; darkens during this step = remove when tobacco brown; check mic
  • gold chloride = toner
  • sodium thiosulfate = fixer; removes any unreduced silver
  • light green SF (yellowish)
39
Q

results of GMS

A
  • fungus = black cell walls w grey interior
  • mucin is grey
  • background = light green
40
Q

control tissue for GMS

A

section w fungus
if staining for P. jirovecii, Pneumocystis control needed

41
Q

What’s so special about P. jirovecii in GMS?

A

takes longer to stain than other organisms; check microscopically after silver impregnation

also cannot be cultured

42
Q

this demonstrates connective tissue, esp. collagen

A

masson trichrome

43
Q

nuclear stain used in MT

A

iron hematoxylin b/c alum-mordanted hematoxylin would be differentiated by acidic dyes used in this procedure

44
Q

principle of MT

A

porosity is basis of staining
- fixatives react w proteins, changing their shape and formation
- connective tissue more permeable than most other components once they are fixed
- small red anionic dyes will stain components quickly
- PMA or PTA added, which bumps small red dyes from larger pores of collagen; may also act as mordants for larger dyes
- large blue/green anionic dye easily stains porous collagen

45
Q

procedure for MT

A
  • Bouin’s recommended as picric acid adds additional reactive sites to proteins = intensifies staining
  • weigert’s iron hematoxylin = nuclear stain ; wash in running tap water to blue
  • biebrich scarlet acid fuchsin = stains everything red; rinse quickly in DH2O
  • PMA/PTA = removes red from collagen
  • aniline blue or fast green FCF = collagen
  • 1% acetic acid = removes excess collagen stain
46
Q

control tissue for MT

A

uterus
small intestine
appendix
fallopian tube

47
Q

modified gram stains demonstrates…

A

many types of bacteria

48
Q

modified gram stain procedure

A

NBF
- crystal violet
- gram’s iodine (mordant)
- acetone, alcohol, or acid-alcohol
- basic fuchsin
- picric acid/acetone = differentiation/counterstain

steps used in the Brown-Brenn modification of the gram stain
red to pink nuclei and yellow tissue elements

49
Q

this demonstartes neutral lipids or fats

A
50
Q

this demonstrates neutral lipids or fats

A

oil red o

51
Q

Oil Red O special consideration

A

must be fresh or frozen; paraffin processing will remove fat from tissues

52
Q

basis of staining for Oil red O

A
  • selective solubility
  • dye is dissolved in propylene glycol or isopropyl alcohol = dye migrates from solvent into any fat present
  • as alcohol and xylene will dissolve and fat present = slides must not be dehydrated or cleared
  • Aq mounting media used
53
Q

Oil Red O procedure

A
  • oil red O = stains lipids
  • 85% propylene glycol = differentiator; use only when stain is dissolved in PG
  • hematoxylin = counterstain; wash in running tap water to blue
54
Q

PAS is used to demonstrate carbohydrate-containing structures including …

A

glycogen
neutral mucin
fungus
basement membranes

also reticulin, zymogen granules of pancreas, thyroid colloid, Russell bodies of plasma cells

55
Q

periodic acid principle

A

used to oxidize hydroxyl groups within carbohydrates to form aldehydes

Schiff reagent reacts with free aldehydes, binding it to tissue
- subsequent water rinses remove loosely bound sulfurous acid group from the central carbon atom = restores quinoid ring =bright magenta

56
Q

PAS procedure

A

NBF or Bouin’s
- periodic acid (weak oxidizer; oxidizes 1,2 glycol groups of several carbs into aldehydes

  • schiff reagent = binds to all aldehydes
  • rinse with DH2O or sodium metabisulfite = remove excess Schiff
  • wash in running tap water = restore quinoid
  • Hematoxylin = counterstain
57
Q

control tissues for PAS

A

kidney = most sensitive; little carb in basement membrane of glomeruli = easily over-oxidized

glycogen = liver or cervix

58
Q

why is it important that excess schiff removed before running water step

A

to prevent non-specific staining

59
Q

addition of diastase step in PAS

A

made specific for glycogen

  • 2 slides for patient
  • prior to oxidation w periodic acid, one slide is treated with diastase ( or salivary amylase) = hydrolyze glycogen into maltose and glucose (washed out w running water)
  • if staining present on undigested slide absent on digested slide = must be glycogen
60
Q

PASD procedure

A

fixative

  • diastase; slides not to be digested remain in DH2O
  • periodic acid
  • schiff reagent
  • rinse w DH2O or sodium metabisulfite
  • wash in running tap water = develops colour
  • hematoxylin counterstain
61
Q

control tissue for PASD

A

cervix more valuable than liver

ectocervix = glycogen which will be digested; endocervix = mucins will not be digested

62
Q

this is used to demonstrate hemosiderin

A

Perl’s Prussian Blue

  • hemosiderin = yellow-brown, endogenous, within cells made of iron (ferric hydroxide) bound to protein ‘skeleton’
63
Q

principle of PPB

A
  • true histochemical method
  • iron loosely bound will react (hemo- or myoglobin will not)
  • acid ferrocyanide used; any ferric iron present reacts to form insoluble blue pigment (ferric ferrocyanide = Prussian blue)
  • PPB is not a stain or dye; it’s a chemical rxn
64
Q

PPB procedure

A

NBF or alcohol; avoid acid-containing fixatives = may dissolve small iron deposits

  • potassium ferrocyanide/hydrochloric acid = reacts with ferric iron forming insoluble blue pigment
  • nuclear fast red or eosin for counterstain
65
Q

results of PPB

A

blue hemosiderin
pink to red background

66
Q

why do we rinse slides in distilled water when doing PPB

A

tap water may contain iron; use distilled water after hydration

67
Q

asbestos bodies

A

appear blue in PPB due to ferritin and hemmosiderin content

68
Q

Verhoeff’s elastic stain is an example of a

A

regressive stain

69
Q

Verhoeff proecure

A

NBF

  • elastic stain (hematoxylin, ferric chloride, iodine = stains everything black/dark purple
  • ferric chloride = differentiator; elastin will be last to remain stained
  • sodium thiosulfate = removes iodine staining
  • eosin or van Gieson = counterstain
70
Q

if van Gieson was used for Verhoeff counterstain = results?

A

red collagen
yellow muscle/cytoplasm

71
Q

control tissue for Verhoeff

A

aorta embedded on edge

skin or muscular artery acceptable

72
Q

this demonstrates calcium in the tissue

A

Van Kossa

73
Q

principle of the VK mehtod

A

metallic substitution

  • silver in stain reacts w carbonate and phosphate groups of calcium carbonate/phosphate
  • bright light (sunlight or UV) is used to reduce the silver to visible metallic form
  • this method detects anionic groups (not calcium) = indirect method of calcium detection
74
Q

VK procedure

A

NBF or alc fixatives

  • silver nitrate and sunlight = metal impregnation; silver reacts w carbonate and phosphate salts
  • sodium thiosulfate (hypo) = removes nureduced silver
  • nuclear-fast red or eosin for counterstain
75
Q

Alizarin Red S

A

replaced VK

direct method; dye molecules bind with calcium

76
Q

principle of ZN

A
  • AFB = mycolic acids = waxy; hydrophobic = prevents staining w most aq stains
  • warm carbol fuchsin = alcohol and phenol allow basic fuchsin to penetrate waxy coating of mycobacteria and other bacteria
  • decolourize sections with acid alc (1% HCl in 70% ethanol) which acid fast organisms resist ; other tissue elements and bacteria are decolourized
77
Q

ZN procedure

A

fixative
- carbol fuchsin
- 1% alc
- methylene blue counterstain

78
Q

why is millipore-filtered water used on water baths and during reagent prep for ZN?

A

tap water may contain non-pathological acid-fast organisms

MLSCI 250 (35 decks)

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