350 - Topic 9 (H&E and Coverslipping)

Question 1

the dye responsible for characteristic purple colour associated with hematoxylin

Answer 1

hematein
- via oxidation

Question 2

natural oxidation of hematoxylin

Answer 2

light/sunlught and air to oxidize hematoxylin to hematein
- may take 2-4 mos
- solution will stay stable for years
- Ehrlich’s and Delafield’s

Question 3

chemical oxidation of hematoxylin

Answer 3

uses sodium iodate (or another oxidizer)
- converts hematoxylin to hematein instantly
- can be used immediately
- but shorter shelf life
( maybe bc continue to oxidize beyond hematein to colourless compounds )

• Gill’s, Harris’, Mayer’s

Question 4

what is a mordant?

Answer 4

a chemical that serves as a link between the dye and the substrate. The result is an insoluble compound that helps adhere the dye to the cells.

Question 5

the choice of mordant will influence…

Answer 5

• which tissue components are stained
• what the resulting colour will be

Question 6

T or F. All hematoxylin formulations are used at a high pH

Answer 6

F! low pH to suppress non-nuclear staining

Question 7

which hematoxylin resists differentiation?

Answer 7

iron! strong covalent bonding
therefore only alum hematoxylins may be used progressively or regressively

Question 8

most routine hematoxylin solutions are …

Answer 8

alum-mordanred
- potash or ammonium
- stains brick red then converted blue-purple by bluing agent

Question 9

exmples of bluing agents

Answer 9

lithium carbonate
dilute ammonia
Scott’s tap water sub (mag sulfate, sod bicarb, water)

Question 10

T or F. Alum hematoxylin may be used progressively or regressively

Answer 10

T!

Question 11

Progressive vs. Regressive staining

Answer 11

Progressive: no diff step’ section stained for a pre-determined amount of time in order to stain nucleus without staining other tissue components

Regressive: over-staining; followed by diff step with acid alc which removes stain from less desirable tissue elements

Question 12

what is stainig time depdent on ?

Answer 12

• age of solution
• formulation
• prog vs regressive
• whether tissue is fresh-frozen or fixed

Question 13

one major disadvantage of alum hematoxylins:

Answer 13

they are decolourized by acidic staining solutions
so unsuitable for special staining techniques using acidified stains, such as MT

Question 14

T or F. Hematoxylin is cationic

Answer 14

T! it is a basic dye; positively charged
stains neg charged tissue elements (nucleus)

Question 15

most frequently used iron hematoxylin

Answer 15

Weigert’s hematoxylin
(sol’n A = hematoxylin dye + 95% alc solvent; sol’n B = aq ferric chloride mordant/ox, HCl acidifier, distileld water solvent)

Question 16

why do we only combine sol’n A and B of Weigert’s hematoxylin immediately before use

Answer 16

it is less stable than alum mordanted hematoxylin
= can become over-oxidized within days so stored as two separate reagents

Question 17

iron hematoxylns are not used routinely, but are frequently used in …

Answer 17

special staining (acidic stainig solutions spedcifically)
- MT
- VVG

Question 18

an acidophilic xanthene dye

Answer 18

eosin

Question 19

this may be added to eosin to control microbial growth

Answer 19

thymol

Question 20

this is added to eosin to adjust pH

Answer 20

acetic acid; best staining = 4.5 -5.0

Question 21

phloxine B

Answer 21

some labs use eosin AND phloxine B as counterstain
- more vivid shades of red
- other labs omit eosin and use phloxine B and saffron (HPS); this can differentiate collagen (yellow) from muscle + cytoplasm (shades of pink to red)

Question 22

general staining procedure of H&E

Answer 22

1. bring to water (hydrate) - remove paraffin via xylene, then absolute alcohol THEN hydrated and removing xylene through graduated alcohols and running water
2. hematoxylin (5-45mins)
3. water wash
4. differentiate (OPTIONAL)
5. bluing (DO NOT AGITATE)
6. water wash
7. eosin
8. dehydrate, clear and mount - remove water by graduated alcohols then absolute alcohol(here the eosin is differentiated to different shades as well); xylene to remove alcohol and stay there until ready for moiunting

Question 23

hematoxylin pale; poor contrast with eosin

Answer 23

• insuff. staining
• old stain
• over-diff
  = forgot to blue (unlikely)

Question 24

hematoxylin very dark; no chromatin details; cytoplasm bluish

Answer 24

• too long in solution
• under diff

Question 25

eosin pale

Answer 25

• contaminated eosin with bluing agent
• extended dehydration after eosin
• old eosin (not likely)
• insuff staining time

Question 26

what do we do when eosin is contaminated w bluing agent?

Answer 26

check pH and if above 5.5, change solution or re-acidify w acetic acid

Question 27

eosin too dark; obscures cellular details

Answer 27

• pH of eosin too low
• too long in eosin
• insuff time in dilute alcohols during dehydration

Question 28

less than 3 shades of eosin

Answer 28

• insuff time in dilute alcohols during dehydration
• pH too high or low

Question 29

blue-black amorphous artifact noted overlying section

Answer 29

stain precipitate from hematoxylin => filter hematoxylin before use! Harris very prone to this

Question 30

which hematoxylin demonstrates mucins

Answer 30

Gill’s stains goblet cells purple

Question 31

which hematoxylins are slow and which are fast?

Answer 31

slow = Mayer’s
fast = Harris

Question 32

gentle and alcohol-free hematoxylin

Answer 32

Mayer’s
can use for counterstain of oil red O b/c no alcs to dissolve lipids

Question 33

routine mounting media

Answer 33

resinous
- transparent slides (easy to focus)
- once dry = permanent

Question 34

what mounting media do we use if tissue elements can dissolve in alcohol or xylene

Answer 34

aqueous mounting media is used
- water-based
- don’t need dehydration or clearing
- simple syrups
- not as high of a refractive index as resinous mounting media (makes it difficult to focus on high-power)
- not permanent
- bleeding of stains can be a problem (can remedy by adding sugars; this can also raise refractive index slightly)

Question 35

“corn flaking”

Answer 35

when slides dry before coverslipping
- disruptive brown artifact forms
- greatly reduces diagnostic potential
- nuclear drying artifact may also appear; obscures chromatin patterns

Question 36

water droplets noted in stained section

Answer 36

• insuff dehydration
• clearing agent contaminated with water

Question 37

some parts of slide cannot be brought into focus

Answer 37

mounting media on top of coverslip

Question 38

cornflake artifact obscures the section

Answer 38

• section allowed to dry prior to coverslipping
• mounting media too thin