H&E and Coverslipping

Question 1

What are the expected results of an H&E stain

Answer 1

nuclei, cartilage and microorganisms will stain blue-purple
collagen and elastin will stain pale pink
cytoplasm will stain shades of pink
muscle fiber will stain dark pink to red
RBCs and eosinophils will stain bright red

Question 2

Where is hematoxylin from

Answer 2

it is extracted form the heartwood of the Haematoxylum campechianum tree by boiling the wood and then precipitating it by adding urea

Question 3

What is hematein

Answer 3

the resulting dye from oxidizing hematoxylin

Question 4

What are the two ways to oxidize hematoxylin

Answer 4

natural oxidization or chemical oxidization

Question 5

Examples of naturally oxidized hematoxylin

Answer 5

Ehrlichs and Delafields

Question 6

Examples of chemically oxidized hematoxylin

Answer 6

Gills, Harris and Mayers

Question 7

What are the common mordants used in hematein

Answer 7

aluminium or iron

Question 8

What is the proper pH range for hematoxylin

Answer 8

1.0-3.0

Question 9

What is regressive staining

Answer 9

over-staining the section and then differentiating it with acid alcohol

Question 10

What is progressive staining

Answer 10

no over-staining occurs, it undergoes staining for a predetermined amount of time

Question 11

What can effect staining time

Answer 11

age of solution, formulation, progressive vs. regressive, if tissue is frozen or fixed

Question 12

What is a disadvantage of alum hematoxylins

Answer 12

they are decolourized by acidic staining solutions

Question 13

What are the ingredients in Gills Hematoxylin

Answer 13

distilled water
ethylene glycol
aluminum sulfate
sodium iodate
hematoxylin
acetic acid

Question 14

What are the ingredients in Harris hematoxylin

Answer 14

distilled water
absolute ethyl alcohol
ammonium aluminum sulfate
hematoxylin
sodium iodate

Question 15

What are the ingredients in Mayer hematoxylin

Answer 15

distilled water
sodium iodate
ammonium aluminum sulfate
hematoxylin
citric acid
chloral hydrate

Question 16

What are the ingredients in Ehrlich hematoxylin

Answer 16

distilled water
95% alcohol
glycerol
hematoxylin
ammonium aluminum sulfate
acetic acid

Question 17

What are the ingredients in delafield hematoxylin

Answer 17

distilled water
95% alcohol
glycerol
hematoxylin
ammonium aluminium sulphate

Question 18

What is the less stable hematoxylin dye

Answer 18

iron hematoxylins

Question 19

What is an example of an iron hematoxylin

Answer 19

Weigerts hematoxylin

Question 20

What is eosin

Answer 20

an acidophilic xanthene dye

Question 21

How is eosin used

Answer 21

as a 0.25-1% solution in alcohol or water

Question 22

What is the ideal pH for eosin staining

Answer 22

4.5-5

Question 23

What does the addition of phloxine to eosin do

Answer 23

produces more vivid shades of red

Question 24

What is the HPS stain

Answer 24

hematoxylin, phloxine, saffron
allows differentiation of collagen (yellow) from muscle and cytoplasm (pink)

Question 25

What is the cause of pale hematoxylin

Answer 25

insufficient stain time old stain over-differentiation

Question 26

What is the solution for pale hematoxylin

Answer 26

increase staining time replace stain with fresh solution decrease time in acid alcohol

Question 27

What is the cause of dark hematoxylin

Answer 27

too long in stain under-differentiated

Question 28

What is the solution for dark hematoxylin

Answer 28

decrease staining time increase time in acid-alcohol

Question 29

What is the cause of pale eosin

Answer 29

contamination of eosin with bluing reagent extended dehydration after eosin staining old eosin insufficient staining time

Question 30

What is the solution for pale eosin

Answer 30

check pH less time in dehydrating alcohols increase staining time

Question 31

What is the cause of dark eosin

Answer 31

pH is too low too long in eosin insufficient time in alcohols during dehydration

Question 32

What is the solution for dark eosin

Answer 32

add NaOH to increase pH decrease time in eosin increase time in 70% alcohol

Question 33

What is the cause of less than 3 shades of eosin

Answer 33

pH too high insufficient time in alcohols

Question 34

What is the solution for less than 3 shades of eosin

Answer 34

increased time in 70% alcohol check pH

Question 35

What is the cause of a blue-black amorphous artifact overlaying the sections

Answer 35

stain precipitate from hematoxylin

Question 36

What is the solution for hematoxylin pigment

Answer 36

filter the hematoxylin before use

Question 37

What is resinous mounting media

Answer 37

synthetic resins dissolved in toluene

Question 38

What is the refractive index of resinous mounting media

Answer 38

1.51-1.55

Question 39

When is aqueous mounting media used

Answer 39

when stains or tissue elements may dissolve in alcohol or xylene

Question 40

What is aqueous mounting media

Answer 40

simple syrups, gum arabic or glycerol gelatin

Question 41

What is the refractive index of aqueous mounting media

Answer 41

1.41-1.43

Question 42

What is the cause of water droplets noted in stained sections

Answer 42

insufficient dehydration clearing agent contaminated with water

Question 43

What is the solution for water droplets in stained sections

Answer 43

remove coverslip with xylene, return to absolute alcohol and clear with fresh xylene

Question 44

What is the cause of parts of the slide not being able to focus

Answer 44

mounting media on top of the cover slip

Question 45

What is the solution for mounting media on top of the coverslip

Answer 45

removed coverslip and remount with a clean one

Question 46

What is the cause of corn-flaking artifact

Answer 46

section allowed to dry prior to coverslipping mounting media too thin

Question 47

What is the solution for corn-flaking artifact

Answer 47

removed coverslip, rehydrate and soak in water for 15 minutes, dehydrate, clear and remount