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

9: H&E Staining & Coverslipping Flashcards

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

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

Disadvantage of Alum hematoxylin

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 ?

A
  • Gill
  • Mayer
22
Q

Which hematoxylin stains are naturally oxidized ?

A
  • Ehrlich
  • Delafield
23
Q

Which hematoxylin stain is often used progressively AND regressively ?

A

Mayer

24
Q

Which hematoxylin stain is often used progressively ?

A
  • Gill
25
Q

Which hematoxylin stains are often used regressively ?

A
  • Harris
  • Ehrlich
  • Delafield
26
Q

Alum hematoxylin recipe

A

Hematoxylin + alum-containing mordant + solvent
- may contain: oxidizer, preservative, an acid

27
Q

Iron hematoxylin use ferric iron as both the __ and the __.

A

Iron hematoxylin use ferric iron as both the MORDANT and the OXIDIZER.

28
Q

Iron hematoxylin

A
  • 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
Q

Most frequently used iron hematoxylin formulation

A

Weigert’s hematoxylin:

A= hematoxylin dye + 95% alcohol
B= aqueous ferric chloride + HCl + dH2O

30
Q

What is added to prevent microbial growth in Eosin stain ?

A

Thymol

31
Q

Form of Eosin mainly used in histology

A

Eosin Y (yellowish)

32
Q

Optimal pH for Eosin stain. What can be added to adjust pH ?

A

Acetic acid = (pH 4.5 - 5.0)

33
Q

When properly differentiated, eosin alone can stain tissues at least __ shades of __.

A

When properly differentiated, eosin alone can stain tissues at least THREE shades of PINK to RED).

34
Q

Why may laboratories use phloxine B followed by saffron instead of Eosin ?

A

Phloxine B = more vivid shades of red
Saffron = differentiates collagen (yellow) from muscle and cytoplasm (pink to red)

35
Q

General staining procedure of H&E

A
  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
Q

Cause+Troubleshoot: Hematoxylin is too pale

A

Cause: insufficient staining time, expired stain, over-differentiation, forgot to blue

Troubleshoot: increase staining time, use fresh stain, decrease time in acid alcohol

37
Q

Cause+Troubleshoot: Hematoxylin too dark; cytoplasm is blueish

A

Cause: prolonged staining time, under-differentiation

Troubleshoot: decrease staining time, increase time in acid alcohol

38
Q

Cause+Troubleshoot: Eosin is too pale

A

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
Q

Cause+Troubleshoot: Eosin too dark

A

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
Q

Cause+Troubleshoot: Eosin does not have a 3-shade gradient

A

Cause: insufficient time in dehydrating alcohols, pH too high/low

Troubleshoot: increase dehydrating time in 70% alcohol, check pH and alter as necessary

41
Q

Cause+Troubleshoot: Blue-black amorphous artifacts

A

Cause: stain precipitate from Hematoxylin

Troubleshoot: filter stain before use (especially Harris)

42
Q

Why may aqueous mounting media be used instead of resinous mounting media ?

A

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
Q

Why may resinous mounting media be used over aqueous mounting media ?

A

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
Q

Cause+troubleshoot: Water droplets noted in stained sections after coverslipping

A

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
Q

Cause+Troubleshoot: Some parts of the slide cannot be brought into focus after coverslipping

A

Cause: Mounting media on top of the coverslip

Troubleshoot: remove coverslip by soaking in xylene and re-mount with a new coverslip

46
Q

Cause+Troubleshoot: corn-flaking artifact obscures section after coverslipping

A

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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