LECTURE - HT Stain Theory

Question 1

Purpose of staining

Answer 1

to provide a contrast between tissue components, achieved by:

• attaching dyes to tissue
• reacting the tissue w organic reagents to produce coloured products
• depositing metals on tissue components to produce visible insoluble black products

Question 2

classification of dyes

Answer 2

• according to their chromophores
• in reference books
• colour index number (CIN)
• according to the manufacturing process
  > natural (orcein, indigo, hematoxylin)
  > synthetic

Question 3

cationic dyes

Answer 3

• attach to anionic groups in tissue
  stain is basic; substrate basophilic, stain acidophilic
• used to stain nuclei, basophil/mast granules. cartilage matrix, cytoplasmic anion groups (RNA)
• ex: crystal violet

Question 4

anionic dyes

Answer 4

• attach to cationic groups in tissue
• stain = acidic
• substrate = acidophilic
• stain = basophilic
• used to stain cytoplasm and extracellular structures
• ex: eosin

Question 5

mordant dyes

Answer 5

• have weak auxochrome; need a ‘bridge’ or mordant
• dye lakes are highly basic and act as cationic dyes
• chief mordants are iron and alum
• stains tissues indirectly

Question 6

solvent dyes

Answer 6

• also called lysochromes
• lacks auxochrome = hydrophobic
• when applied to tissue w hydrophobic domain = only interact w parts of tissue that are also hydrophobic
  > ‘selective’ or ‘preferential’ solubility
• ex: oil red O, sudan black

Question 7

polychromatic dyes

Answer 7

• spontaneously forms other dyes in solution
• often used in rapid diagnosis of cryostat sections
• methylene blue for example
  > dye is eventually oxidized into Azure A and Azure B

Question 8

neutral stain solutions

Answer 8

• made from the interaction of anionic and cationic dyes
• ex: Romanowsky dyes
• used in hematology

Question 9

leuco dyes

Answer 9

• Leuco means colourless
• chromophores are easily reduced
• chemical rxn occurs with reactive tissue groups
• usually easily reversible

Question 10

interaction of dye with tissue involves:

Answer 10

• physical factors
• chemical
  > electrovalent, covalent, hydrogen bonds, Van der Waals forces. hydrophobic inteaction

Question 11

progressive staining

Answer 11

• in each stain until desired effect is achieved

Question 12

regressive stain

Answer 12

tissue is overstained

• ex: H&E = want to overstain nucleus; important for pathologies, need a lot of stain; some parts are slow to stain; overstain so everything has enough time to stain
• take some of stain out to provide nice detail

Question 13

selective removal of excess dye from a section

Answer 13

differentiation

methods

• washing in water, alcohol, or the solvent
• use of excess mordant
• oxidizing agents
• other dyes

Question 14

chemical group that results in colour

Answer 14

chromophores

• increased numbers = increased colour intensity
• uncoloured molecule + chromophore = chromogen

Question 15

auxchrome

Answer 15

a group of atoms attached to a chromophore which modifies the ability of that chromophore to absorb light
- attach dye to tissue

Question 16

2 classes of auxochromes

Answer 16

• anionic (acid, neg charge)

- cationic (basic, pos charge)

Question 17

chromogen + auxochrome = ?

Answer 17

stain

Question 18

metachromatic dyes

Answer 18

• dye certain components a colour different than the dye itself
• tissue components = chromotropes
• basic or cationic
• ability to stain is based on tendency to polymerize (2+ dye molecules come close together = new wavelength of light absorptivity); water necessary
• aqueous mounting media recommended
• sulfation can induce metchromasia

Question 19

physical factors of dye interaction w tissues

Answer 19

• combine by absorption
• factors:
• dimension of SA
• density
• permeability
• size of dye molecule

Question 20

chemical factors: electrovalent bonds

Answer 20

• electrostatic attraction of oppositely charged ions
• AKA ionic, electrostatic, salt linkage
• most common
• disrupted by acids, weak bases, water, alcohol, or high [electrolyte]
• dissociation = differentiation

Question 21

chemical factors: covalent bonds

Answer 21

• formed when 2 atoms share electrons
• strongest, require lots of E to disrupt
• typical of organic chemicals
• ex: Schiff’s reagent and Verhoeff’s stain

Question 22

chemical factors: hydrogen bonds

Answer 22

• due to attraction between lone pair of e- on O or N and partially exposed nucleus of H
• weaker than covalent or ionic bnods
• affect = nonaq solvents
• easy to break; not a lot use this

Question 23

chemical factors: Van der Waals

Answer 23

• between electrons of one atom and nucleus of another
• found in all staining systems to various degrees
• very short range forces
• weakest
• ex: aldehyde fuchsin

Question 24

chemical factors: hydrophobic interaction

Answer 24

• non-polar molecules’ affinity for one another
• aqueous mounting media
• dye must be more soluble in substate and the solvent

Question 25

metallic impregnation

Answer 25

• uses silver in alkaline solution readily able to precipitate
• 3 types of rxns:
  > Argentaffin: reactive groups are capable of reducing silver salts
  > Argyrophil: tissue elements are not sufficiently reactive to reduce silver ; outside reducer is required
  > metallic substitution: ion exchange

Question 26

first step in all staining

Answer 26

deparaffinization

Question 27

Hematoxylin

Answer 27

• natural dye from logwood of H. campechianum tree in Central America
• lacks a chromophore
• must be oxidized to hematein and mordanted before use

Question 28

oxidation - ripening

Answer 28

2 ways
- natural = exposure to air and light or UV
> long time (3 mos)
> long shelf life

• chemical
  > quick
  > can easily over-oxidize; must add precise amounts
  > must use immediately and discard after; oxy-hematein has no staining ability

Question 29

Alum hematoxylin

Answer 29

• most commonly used

- used progressively (differentiation = acid alcohol)

Question 30

iron hematoxylin

Answer 30

• dye-mordant lake is darker => almost black

- very resistant to differentiation (common diff = excess mordant)

Question 31

eosin

Answer 31

• anionic dye with quinoid chromophore
• different shades; yellowish or bluish
• common counterstain in many methods