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MLSCI 350 > 9: H&E Staining & Coverslipping > Flashcards

9: H&E Staining & Coverslipping Flashcards

(46 cards)

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1
Q

Color of Nuclei, cartilage, microorganisms in H&E

A

Blue-purple to black

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2
Q

Color of collagen and elastin in H&E

A

Pale pink

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3
Q

Color of cytoplasm in H&E

A

Shades of pink

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4
Q

Color of muscle fibres in H&E

A

Dark pink to red

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5
Q

Color of RBCs and Eosinophils in H&E

A

Bright red

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6
Q

Source of hematoxylin dye

A
  • from heartwood of logwood tree (Haematoxylum campechianum)
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7
Q

How is hematoxylin extracted from heartwood ?

A

wood is boiled + urea is added

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8
Q

T or F: Hematoxylin is a dye

A

FALSE; hematoxylin is NOT a dye—it is oxidized to form hematin, the dye responsible for its purple color

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9
Q

Natural oxidation

A
  • uses light and air to oxidize hematoxylin to hematin
  • 2-4 month process
  • stable for years
    Ie. Ehrlich’s and Delafield’s hematoxylin
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10
Q

Chemical oxidation

A
  • uses sodium iodate to oxidize hematoxylin to hematin
  • formed instantly
  • shorter shelf-life; continues to oxidize hematin to colorless compounds
    Ie. Gill’s, Harris’, and Mayer’s hematoxylin
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11
Q

Hematin requires a __ to stain predictably

A

Hematin requires a MORDANT to stain predictably

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12
Q

What compounds are used as a mordant for Hematin?

A

Aluminum or iron compounds
- also chrome, tungsten, molybdenum compounds

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13
Q

What is the optimal pH for hematoxylin ?

A

Low/ acidic pH (1.0 - 3.0)

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14
Q

Why is hematoxylin used at a low pH ?

A

To suppress non-nuclear staining

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15
Q

Which mordant for hematoxylin is most routinely used ? Why ?

A

Potassium ALUMINUM sulfate or ammonium ALUMINUM sulfate; produce good nuclear staining, quick and easy to use

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16
Q

Alum Hematoxylin

A
  • potassium aluminum sulfate OR ammonium aluminum sulfate
  • stains nucleus brick red
  • bluing (alkaline) solution = blue-purple
  • used progressively or regressively
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17
Q

List bluing agents

A
  • Lithium carbonate
  • dilute ammonia
    -Scott’s tap water substitute (magnesium sulfate, sodium bicarbonate, and water)
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18
Q

Regressive staining vs Progressive

A

Regressive: Over-staining the tissue, followed by differentiation with acid alcohol (removes stain from less desirable tissue elements)

Progressive: No differentiation; uses pre-determined time to stain nucleus without staining other tissue elements

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19
Q

List some factors that determine staining time

Study These Flashcards
A
  • age of solution
  • formulation
  • progressive vs regressive staining
  • fresh/ frozen or fixed tissue
20
Q

Disadvantage of Alum hematoxylin

Study These Flashcards
A

Decolonized by acidic stain solutions = CANNOT be used for special staining techniques using acidified stains (Masson trichrome)

21
Q

Which hematoxylin stains are alcohol free ?

22
Q

Which hematoxylin stains are naturally oxidized ?

Study These Flashcards
A
  • Ehrlich
  • Delafield
23
Q

Which hematoxylin stain is often used progressively AND regressively ?

24
Q

Which hematoxylin stain is often used progressively ?

25
Which hematoxylin stains are often used regressively ?
- Harris - Ehrlich - Delafield
26
Alum hematoxylin recipe
Hematoxylin + alum-containing mordant + solvent - may contain: oxidizer, preservative, an acid
27
Iron hematoxylin use ferric iron as both the __ and the __.
Iron hematoxylin use ferric iron as both the MORDANT and the OXIDIZER.
28
Iron hematoxylin
- uses ferric iron as the mordant + oxidizer - less stable; over-oxidized within a day = hematoxylin and iron are stored separately and mixed immediately before use - mostly used progressively - prolonged staining times
29
Most frequently used iron hematoxylin formulation
Weigert’s hematoxylin: A= hematoxylin dye + 95% alcohol B= aqueous ferric chloride + HCl + dH2O
30
What is added to prevent microbial growth in Eosin stain ?
Thymol
31
Form of Eosin mainly used in histology
Eosin Y (yellowish)
32
Optimal pH for Eosin stain. What can be added to adjust pH ?
Acetic acid = (pH 4.5 - 5.0)
33
When properly differentiated, eosin alone can stain tissues at least __ shades of __.
When properly differentiated, eosin alone can stain tissues at least THREE shades of PINK to RED).
34
Why may laboratories use phloxine B followed by saffron instead of Eosin ?
Phloxine B = more vivid shades of red Saffron = differentiates collagen (yellow) from muscle and cytoplasm (pink to red)
35
General staining procedure of H&E
1. Bring slides to water (xylene>100%alcohol>95%alcohol>70%alcohol) 2. Hematoxylin 3. If regressive: differentiate w/ 0.5% acid alcohol 4. Bluing w/ lithium carbonate 5. Wash 6. Eosin 7. Dehydrate + clear: 70%alcohol>95%alcohol>100%alcohol>xylene) 8. Coverslip
36
Cause+Troubleshoot: Hematoxylin is too pale
Cause: insufficient staining time, expired stain, over-differentiation, forgot to blue Troubleshoot: increase staining time, use fresh stain, decrease time in acid alcohol
37
Cause+Troubleshoot: Hematoxylin too dark; cytoplasm is blueish
Cause: prolonged staining time, under-differentiation Troubleshoot: decrease staining time, increase time in acid alcohol
38
Cause+Troubleshoot: Eosin is too pale
Cause: - contamination with bluing reagent = increased pH decreases strength of anionic dye - extended dehydration after eosin - insufficient staining time - expired eosin Troubleshoot: - check pH of eosin (4.6 - 5.0) = if >5.5, then replace eosin - decrease time in dehydrating alcohols - increase staining time
39
Cause+Troubleshoot: Eosin too dark
Cause: - pH of eosin is too low - prolonged eosin time - insufficient dehydration time in alcohols Troubleshoot: - add NaOH to increase pH - decrease time in eosin - increase time in dehydration alcohols
40
Cause+Troubleshoot: Eosin does not have a 3-shade gradient
Cause: insufficient time in dehydrating alcohols, pH too high/low Troubleshoot: increase dehydrating time in 70% alcohol, check pH and alter as necessary
41
Cause+Troubleshoot: Blue-black amorphous artifacts
Cause: stain precipitate from Hematoxylin Troubleshoot: filter stain before use (especially Harris)
42
Why may aqueous mounting media be used instead of resinous mounting media ?
Resinous mounting media = tissue must be dehydrated/ cleared prior to mounting - crystal violet is alcohol-soluble, and fat in tissues will dissolve in xylene Aqueous mounting media = can be used immediately after staining, without dehydrating/ clearing
43
Why may resinous mounting media be used over aqueous mounting media ?
Resinous mounting media= higher refractive index (1.5); easier to focus on - permanent, and stains will not fade Aqueous mounting media= lower refractive index (1.4) - NOT permanent, and stains can bleed
44
Cause+troubleshoot: Water droplets noted in stained sections after coverslipping
Cause: insufficient dehydration, clearing agent contaminated with water Troubleshoot: remove coverslip by soaking in xylene, dehydrate and clear with fresh alcohol/ xylene before coverslipping
45
Cause+Troubleshoot: Some parts of the slide cannot be brought into focus after coverslipping
Cause: Mounting media on top of the coverslip Troubleshoot: remove coverslip by soaking in xylene and re-mount with a new coverslip
46
Cause+Troubleshoot: corn-flaking artifact obscures section after coverslipping
Cause: section allowed to dry prior to coverslipping, mounting media was too thin Troubleshoot: remove coverslip by soaking in xylene, re-hydrate section (soak in water for 15 min), dehydrate, clear, and re-mount

MLSCI 350 (6 decks)

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