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CSMLS > HIS Special Stains > Flashcards

HIS Special Stains Flashcards

1
Q

Trichrome stains

A
  • differentiates CONNECTIVE TISSUE from other elements
  • uses three ACIDIC dyes*
  • based on porosity: smallest dye stains all tissue elements; larger dyes “evict” smaller molecules in larger pores

*NOTE: alum hematoxylin is unsuitable because it is differentiated in acidic pH

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2
Q

Which hematoxylin is used in Trichrome stains ? Why ?

A

Weigert’s (iron) hematoxylin; alum hematoxylin is differentiated by acidic pH

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3
Q

Principle: Masson Trichrome

A
  • Bouin’s fluid at 50°C for 1 hour
  • Biebrich scarlet + Acid fuchsin = STAINS all ACIDOPHILIC elements RED
  • Phosphomolybdic/tungstic = colorless dye “evicts” red stain from larger pores (ie. collagen)
  • Analine Blue = largest dye stains collagen BLUE
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4
Q

Color of cell components in Masson Trichrome

A

Collagen: Blue

Nuclei: grey to black

Cytoplasm, keratin, muscle fibre: red

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5
Q

Cause+Troubleshoot: Masson Trichrome stain all decreased

A

Cause: failure to pre-mordant with Bouin’s when using formalin-fixed tissue
Troubleshoot: pre-mordant for 1 hour at 60°C

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6
Q

What can be used in place of alanine blue for collagen staining in Masson Trichrome ?

A

Light green, SF yellowish

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7
Q

T or F: In Masson Trichrome, the nuclear stain does not have to be very dark or demonstrate fine chromatin patterns

A

TRUE; the nuclear stain is for contrast and orientation; it does not have to be very dark or demonstrate fine chromatin patterns

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8
Q

Why should little spills of picric acid from Bouin’s fluid be wiped away immediately ?

A

Dry picric acid is explosive

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9
Q

2 stains to demonstrate elastin

A
  • Verhoeff’s Van Gieson
  • Gomori’s aldehyde fuchsin
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10
Q

Principle: Verhoeff’s Van Gieson

A
  • tissue is OVER-STAINED with IODINE/ FERRIC CHLORIDE/ HEMATOXYLIN
    Ferric Chloride = mordant + oxidizer
    Iodine = oxidizes hematoxylin and elastin
  • differentiate using EXCESS FERRIC CHLORIDE = excess mordant breaks tissue-mordant complex*
  • iodine is removed by sodium thiosulfate (aka HYPO)
  • Van Gieson = counterstain

*NOTE: elastin has the highest affinity for verhoeff and would be decolorized last

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11
Q

Color of cell components in Verhoeff Van Gieson

A

Elastin: Black

Collagen: Red

Muscle: Yellow

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12
Q

In Verhoeff Van Gieson, the collagen fibres should remain __ during differentiation to avoid removal of stain from elastin fibres

A

In Verhoeff Van Gieson, the collagen fibres should remain PALE GREY during differentiation to avoid removal of stain from elastin fibres

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13
Q

What must be done after differentiating with Ferric Chloride in Verhoeff Van Gieson staining ?

A
  • Rinse in distilled water to stop differentiation
  • Slides must be checked microscopically (collagen must remain pale grey)
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14
Q

3 functions of Ferric Chloride in Verhoeff’s

A
  1. Mordant
  2. Oxidizer
  3. Differentiator
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15
Q

Verhoeff and Gomori: progressive or regressive ?

A

Verhoeff = regressive
Gomori’s Aldehyde Fuchsin = progressive

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16
Q

Principle: Gomori’s Aldehyde Fuchsin

A
  • ACIDIFIED PARALDEHYDE + alcoholic BASIC FUCHSIN = RIPEN for 2-4 DAYS before staining
  • progressive
  • (optional) Potassium permanganate = intensifies staining
  • demonstrates elastin
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17
Q

Color of cell components in Gomori’s Aldehyde Fuchsin

A

Elastin (and beta granules in Pancreas, mast cells, sulfated mucins and cartilage): Purple

Cytoplasm: Green

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18
Q

Storage temp of ripened Gomori’s aldehyde fuchsin

A

4°C

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19
Q

Although Gomori’s Aldehyde Fuchsin is intended to be used progressively, it can be differentiated with __ if desired.

A

Although Gomori’s Aldehyde Fuchsin is intended to be used progressively, it can be differentiated with 70% ALCOHOL if desired.

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20
Q

What kind of stains demonstrate Reticulin ?

A

Silver stains

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21
Q

Principle: Gordon and Sweet’s Reticulin Stain

A
  • potassium permanganate oxidizes Reticulin
  • oxalic acid bleaches discolouration from permanganate

INDUCED ARGYROPHILIA:

  • Ferric ammonium sulfate = sensitizer binds to reticulin; to be replaced by silver solution
  • formalin reduces silver to metallic form
  • gold chloride tones silver = brown to black
  • sodium thiosulfate removes any unreduced silver*
  • counterstain: nuclear fast red or eosin

*NOTE: prevents section from darkening when exposed to light

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22
Q

Color of cell components in Gordon&Sweet’s Reticulin Stain

A

Reticulin: Black

Cytoplasm: Red

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23
Q

During Gordon&Sweet’s Reticulin staining, a white precipitate forms on the walls of the jar/ solution surface. What does this mean ? How will this affect staining ?

A
  • glassware was NOT acid washed/ bleached + rinsed with dH2O
  • silver solution is CONTAMINATED
  • staining will be weak
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24
Q

Why are Reticulin silver stains usually made fresh ?

A

Ferric AMMONIUM sulfate + SILVER = EXPLOSIVE
- discard after use
- avoid sunlight

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25
Q

How are silver solutions neutralized ?

A

- add CHLORIDE ions (HCl, bleach, or saline)
- precipitate out the silver = silver chloride
- dispose in HALOGENATED waste

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26
Q

2 stains to demonstrate carbohydrates

A
  1. PAS (Periodic Acid Schiff)
  2. Alcian blue
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27
Q

Which carbohydrates are PAS positive ?

A
  • glycogens
  • NEUTRAL mucins (mucopolysaccharides)

NOTE: basement membrane, fungi (C. albicans), zymogen granules, colloid, and reticulin are also POS

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28
Q

Which carbohydrates are Alcian blue positive ?

A

(ACID mucopolysaccharides):
- carboxylated mucins (COOH)
- sulfated mucins (SO4)

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29
Q

Glycogen: PAS vs PAS-D

A

PAS = POS
PAS-D = neg

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30
Q

Principle: Periodic Acid Schiff

A
  • periodic acid oxidizes carbs = aldehydes form
  • schiff reagent (basic fuchsin + sulfurous acid*) reacts with aldehydes = bind to tissue
  • water rinses removes loose sulfurous acid groups = restores qunoid ring = BRIGHT RED

*NOTE: reduction causes quinoid ring to be lost = colorless

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31
Q

Why is periodic acid used to form aldehydes in PAS ?

A

Periodic acid is a WEAK OXIDIZER
- others will oxidize hydroxyl beyond aldehydes

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32
Q

What is Schiff reagent made of ?

A

Basic fuchsin + sulfurous acid

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33
Q

Color: PAS-positive tissue elements

A

Magenta

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34
Q

Cause+Troubleshoot: PAS Non-specific staining

A

Cause: did not rinse excess Schiff reagent

Troubleshoot: water rinse

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35
Q

In PAS, __ is important for proper color development

A

In PAS, the WATER RINSE is important for proper color development

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36
Q

PAS-D

A

Periodic Acid Schiff with Diastase (α-amylase):
- digestion step = specific for glycogen

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37
Q

Principle: PAS-D

A
  • diastase (α-amylase) breaks down glycogen into SMALLER sugars (maltose + glucose)
  • water rinse WASHES AWAY SIMPLE SUGARS
  • stained in duplicate: PAS-D vs PAS
  • INDIRECT DETECTION = when compared to PAS, absent staining on the digested slide (PAS-D) will be due to glycogen
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38
Q

Color: glycogen on PAS-D

A

UNSTAINED bc of digestion+rinse

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39
Q

Color: PAS-pos tissue elements on PAS-D

A

Magenta

NOTE: EXCEPT glycogen

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40
Q

Alcian blue

A
  • a CATIONIC dye
  • stains ACID MUCINS to differentiate adenocarcinomas
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41
Q

Principle: Alcian blue

A
  • at pH 2.5, IONICALLY binds with carboxylated and sulfated (acid) mucins
  • at pH 1.0, only sulfated acid mucins will stain
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42
Q

Color: at pH 2.5, carboxylated/ sulfated mucins in Alcian blue

A

Dark blue

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43
Q

Color: at pH 1.0, sulfated mucins in Alcian blue

A

Light blue

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44
Q

Color: Nuclei in Alcian blue

A

Red

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45
Q

T or F: Alcian blue will appear more lightly stained in a lower pH

A

TRUE; Alcian blue will appear more lightly stained in a lower pH
- sulfated mucins will be light blue in pH 1.0, but darker at pH 2.5

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46
Q

How is a low, stable pH achieved for Alcian blue staining ?

A

Acid rinse prior to staining

47
Q

Alcian Blue + PAS

A
  • Stain Alcian blue first = stains acid mucins BLUE
  • PAS = stains neutral mucins MAGENTA
  • tissue elements that contain both acid and neutral mucins = purple
48
Q

Principle: Congo red

A
  • demonstrates AMYLOID = red
  • congo red fits within β-PLEATED SHEETS of protein, stabilized by HYDROGEN BONDING
  • attaches to protein at regular intervals = APPLE-GREEN birefringence under polarized light
49
Q

Color of cell components in Congo red

A

Amyloid: Red; apple green under polarized light

Elastin: Pale pink

Nuclei: Purple

50
Q

Required thickness of tissue to observe polarized APPLE GREEN color (Amyloid) in Congo red

A

7-10 μm

51
Q

Hemosiderin in Histology

A
  • yellow-brown ENDOGENOUS pigment
  • storage form of iron
52
Q

Principle: Perl’s Prussian Blue

A
  • HISTOCHEMICAL STAIN
  • detects FERRIC iron = reacts with acid-ferrocyanide = insoluble BLUE pigment
  • ferrous iron will not react
53
Q

Color of cell components in Perl’s prussian blue

A

Hemosiderin: Prussian Blue

Cytoplasm: Pink

54
Q

Why must deionized/ distilled water be used in Perl’s Prussian blue staining ?

A

Tap water can contain iron = non-specific blue precipitate

55
Q

Why can’t decalcification be done on tissues stained for Perl’s Prussian blue ?

A

Iron can be dissolved away = false negative

56
Q

Prinicple: Oil red O

A
  • a Sudan dye; demonstrates fat/ lipids
  • uses Oil red O dissolved in isopropyl alcohol
  • SELECTIVE SOLUBILITY = Oil red O is more soluble in fat and will MIGRATE FROM SOLVENT TO FAT in tissue
57
Q

Color of cell components in Oil Red O

A

Fat: Red

Nuclei: Purple-blue

58
Q

Cause+Troubleshoot: In Oil red O, fat droplets are found throughout tissue

A

Cause: coverslipped was squished to remove air bubbles

Troubleshoot: coverslip gently and avoid heating slide with fingers

59
Q

T or F: paraffin embedded tissue can be used to demonstrate fat

A

FALSE; fat is readily soluble in alcohols and xylene so tissue cannot be processed; FROZEN SECTIONS are needed to demonstrate fat

60
Q

Fontana Masson

A
  • ARGENTAFFIN stain (binds and reduces silver)
  • demonstrates melanin and granules of enterochromaffin cells in small intestine
61
Q

Principle: Fontana Masson

A
  • silver applied to tissue
  • argentaffin substances will bind and reduce silver
  • gold chloride to tone tissue
  • sodium thiosulfate (hypo) removes any unreduced silver
  • counterstain: Nuclear-fast red or Eosin
62
Q

Color of cell components in Fontana-Masson

A

Melanin and Argentaffin granules: Black

Nuclei: Pink

63
Q

How can FM be made specific for Melanin ?

A
  • add a duplicate slide: FM + bleach (potassium permanganate OR hydrogen peroxide)
  • when compared to the regular slide, absent staining on bleached slide will be due to melanin
64
Q

T or F: do not use metallic forceps for any silver method

A

TRUE; using metallic forceps for any silver method can be a source of contamination

65
Q

What is generally a cause for non-specific staining ?

A

Contaminated glassware

66
Q

Principle: Modified Gram Stain

A
  • crystal violet stains both gram pos/ neg bacteria
  • iodine (mordant)
  • alcohol dissolves lipoprotein layer, while leaving peptidoglycan intact

Gram pos: retains crystal-violet-iodine complex due to layers of peptidoglycan
Gram neg: is decolorized
- counterstained

67
Q

Color of cell components in Modified gram stain

A

Gram pos bacteria: Purple

Gram neg bacteria: Pink

68
Q

Ziehl-Neelsten Stain

A

demonstrates acid-fast organisms (ie. Mycobacterium) = have thicker cell walls with WAXY MYCOLIC ACIDS

69
Q

Principle: Ziehl-Neelsten stain

A
  • alcoholic phenol helps basic fuchsin to PENETRATE cell wall = IONICALLY binds acid-fast bacteria = PURPLE
  • dilute acid rinse; true acid-fast bacteria RESIST acid decolorization (waxy mycolic acids on cell wall)
70
Q

Color of cell components in Ziehl-Neelsten stain

A

Acid-fast bacilli: Purple

Cytoplasm: Methylene blue

71
Q

Diff-Quik

A

Giemsa stain used to screen for Helicobacter pylori:
- cationic dye = methylene blue
- anionic dye = eosin

NOTE: any bacteria will stain blue, looking at morphology is important

72
Q

Principle: Diff-Quik

A
  • Methylene blue stains negatively-charged tissue elements (Bacteria, nuclei)
  • Eosin stains positively-charged tissue elements (Cytoplasm)
73
Q

What kind of stains are used to demonstrate Spirochetes ?

A
  • silver stains; spirochetes are argyrophilic (bind silver)
  • but NEEDS an EXTERNAL REDUCING AGENT to visualize the results (ie. hydroquinone in Warthin-Starry)
74
Q

Principle: Grocott’s Methenamine Silver stain

A
  • chromic acid oxidizes FUNGAL cell walls = FORMS ALDEHYDES
  • in warm methenamine-silver solution, silver binds to aldehydes = reduced to metallic silver (INDUCED ARGENTAFFIN)
  • gold chloride; tones silver
  • sodium thiosulfate; removes unreduced silver
  • counterstain: Light green
75
Q

Color of cell components in Grocott’s methenamine silver

A

Fungus: Black

Mucin: Grey

Cytoplasm: Light green

76
Q

You find out your aldehyde fuchsin has expired when you are ready to perform Gomori’s aldehyde fuchsin stain. Why is this an issue ?

A

It takes 72 hours for the solution to ripen; patient results will be delayed 3 days

77
Q

Purpose of toning in silver stains

A

some silver is replaced by gold ions = transforms color from dark brown to black

78
Q

Why must silver solutions be neutralized prior to disposal ?

A

Silver solutions (especially when containing ammonia) can be explosive when exposed to metal pipes
- also very toxic

79
Q

Why do tissue sections tend to fall off slides during silver staining ? How can this be prevented ?

A

Due to the alkaline nature of silver stains, using charged slides is recommended

80
Q

Best overall control for PAS stain. Why ?

A

Kidney = basement membrane contains small amounts of aldehydes after oxidation, making the control more sensitive

81
Q

Why is glutaraldehyde not an acceptable fixative for PAS staining ?

A
  • Schiff reagent will stain any aldehydes present in tissue (which we create by oxidizing polysaccharides)
  • glutaraldehyde will leave free aldehydes in tissue = binds to Schiff reagent = false pos
82
Q

Periodic Acid Schiff: How can you determine if there are pre-existing aldehydes in your tissue ?

A

Skip the oxidation step; any positivity with Schiff reagent = pre-existing aldehydes

83
Q

Why are slides rinsed in dye solvents before staining with Alcian blue ?

A
  • Alcian blue is pH-dependent
  • an acid rinse prior to staining ensures stable pH = demonstrates substances (carboxylated/ sulfated mucins) we intend to
84
Q

Why is it important to keep the Counterstain light ?

A
  • avoid masking positive results
  • counterstain only provides a morphological overview
85
Q

Why should tissue for amyloid controls be processed and embedded instead of stored in formalin until required ?

A

Amyloid staining becomes weaker with extended formalin fixation

86
Q

Staining mechanism for Perl’s Prussian blue

A

True HISTOCHEMICAL

87
Q

What type of fixatives should be avoided when fat needs to be demonstrated ?

A

Anything with alcohol will extract lipids

88
Q

What type of mounting media must be used for slides stained with Oil Red O ?

A

Aqueous mounting media; solvents in resinous media will dissolve lipids and Oil Red O

89
Q

Which fixation artifact can cause a false positive result in FM ?

A

Formalin pigment; argentaffin

90
Q

2 issues unbuffered formalin can cause when attempting to demonstrate calcium with Von Kossa’s Silver stain method

A
  1. Unbuffered formalin will have a lower pH = formalin pigment forms = false pos
  2. Low pH can dissolve calcium = false negative
91
Q

Which endogenous pigment can interfere with Von Kossa’s Silver stain

A

Melanin

92
Q

Which step is most crucial in Modified Gram staining ?

A

Differentiation/ decolorizing

93
Q

What type of tissue is used as a control in Gram staining ?

A

Tissue with both gram pos and neg bacteria
Ie. placenta seeded with organisms, infected appendix..

94
Q

If Gram control tissue is acceptable, but organisms in the patient sample are too pale, what might this indicate ?

A
  • Patient may be on antibiotics
  • Organism may be acid-fast
95
Q

Why is it important not to miss acid-fast bacilli in a tissue section ?

A
  • treatment differs from other infections (resistant to routine antibiotics)
  • Mycobacterium are level 3 organisms = severe consequences on patient
96
Q

How to fix a slide over stained with methylene blue in the Ziehl-Neelsten stain

A
  • remove methylene blue completely with acid alcohol and re-counterstain
  • organism should remain stained
97
Q

How does the Giemsa stain differ from the Wright stain ?

A
  • Romanowsky stains are very similar

Wright’s: polychromed Methylene blue and Eosin Y

Giemsa: Azure B and Eosin Y
- uses a more controlled oxidation method
- more reddish
- stain of choice for malaria

98
Q

Why is it important to know whether P. jirovecii or another fungal species is suspected ?

A

Pneumocystis jirovecii takes longer to stain than other fungal organisms

99
Q

What other organisms are silver methods used to demonstrate ?

A

Actinomyces, encapsulated bacteria, spirochetes

100
Q

What staining mechanism is involved in Alcian Blue staining ?

A. Selective solubility
B. Ionic bonding
C. Covalent bonding
D. Porosity

A

B. Ionic bonding

101
Q

When reviewing the control slide, some of the nuclei are staining with Alcian Blue. What is the most likely cause ?

A

Staining was greatly prolonged; only acid mucins should be stained

102
Q

Why may apple-green birefringence in Amyloid-positive tissue not be seen ?

A
  • sections cut too early (amyloid staining weakens with time after sections are cut)
  • extended formalin fixation
  • section too thick/ thin
103
Q

Why are saturated salt solutions used in Congo red staining ?

A

To reduce non-specific staining

104
Q

What is the recommended fixative for Congo red slides ? Why ?

A

Alcohol; formalin fixation decreases staining intensity of Amyloid

105
Q

Which staining mechanism is involved in Congo red staining ?

A

Hydrogen bonding

106
Q

What characteristic of amyloid explains its affinity for Congo red ?

A

Linear structure of Congo red fit within the secondary protein structure of Amlyloid (β-pleated sheets)

107
Q

You notice that the melanin on your Fontana Masson control is stained weak. What is the most likely cause ?

A

Excess ammonia in your silver solution

108
Q

Mechanism for Gordon and Sweet’s metallic impregnation method ?

A

Induced argyrophilia

109
Q

Your control for Gordon&Sweet’s Reticulin stain is very weak and non-continuous. What is the most likely cause ?

A
  • excess ammonia in silver solution
  • neutral buffered formalin was used as the reducing agent (NON-BUFFERED FORMALIN MUST BE USED)
110
Q

Which organism requires a longer impregnation step using Grocott’s Methenamine Silver ?

A

Pneumocystis jirovecii

111
Q

The control slide for Grocott’s Methenamine Silver have fungi stained completely black, without internal structure. What is the most likely cause of this ?

A

Overstained in silver solution

112
Q

Which characteristic of fungi is used to selectively “stain” with Grocott’s Methenamine Silver ?

A

Polysaccharide content; fungi cell wall

113
Q

Why is chromic acid the oxidizer in Grocott’s Methenamine Silver ?

A

It over-oxidizes other tissue elements beyond aldehydes, excluding fungi cell wall

114
Q

Which of the following statements apply to Oil Red O staining ? Select all that apply:

A. Demonstrates hydrophobic lipids
B. Demonstrates hydrophilic lipids
C. Avoid alcohol-containing fixatives
D. Must use aqueous mounting media
E. Heat staining solution to 65 degrees C

A

A. Demonstrates hydrophobic lipids

C. Avoid alcohol-containing fixatives

D. Must use aqueous mounting media

CSMLS (46 decks)

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