LECTURE - HT Stain Theory Flashcards
Purpose of staining
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
classification of dyes
- according to their chromophores
- in reference books
- colour index number (CIN)
- according to the manufacturing process
> natural (orcein, indigo, hematoxylin)
> synthetic
cationic dyes
- 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
anionic dyes
- attach to cationic groups in tissue
- stain = acidic
- substrate = acidophilic
- stain = basophilic
- used to stain cytoplasm and extracellular structures
- ex: eosin
mordant dyes
- 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
solvent dyes
- 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
polychromatic dyes
- 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
neutral stain solutions
- made from the interaction of anionic and cationic dyes
- ex: Romanowsky dyes
- used in hematology
leuco dyes
- Leuco means colourless
- chromophores are easily reduced
- chemical rxn occurs with reactive tissue groups
- usually easily reversible
interaction of dye with tissue involves:
- physical factors
- chemical
> electrovalent, covalent, hydrogen bonds, Van der Waals forces. hydrophobic inteaction
progressive staining
- in each stain until desired effect is achieved
regressive stain
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
selective removal of excess dye from a section
differentiation
methods
- washing in water, alcohol, or the solvent
- use of excess mordant
- oxidizing agents
- other dyes
chemical group that results in colour
chromophores
- increased numbers = increased colour intensity
- uncoloured molecule + chromophore = chromogen
auxchrome
a group of atoms attached to a chromophore which modifies the ability of that chromophore to absorb light
- attach dye to tissue
2 classes of auxochromes
- anionic (acid, neg charge)
- cationic (basic, pos charge)
chromogen + auxochrome = ?
stain
metachromatic dyes
- 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
physical factors of dye interaction w tissues
- combine by absorption
- factors:
- dimension of SA
- density
- permeability
- size of dye molecule
chemical factors: electrovalent bonds
- 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
chemical factors: covalent bonds
- 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
chemical factors: hydrogen bonds
- 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
chemical factors: Van der Waals
- 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
chemical factors: hydrophobic interaction
- non-polar molecules’ affinity for one another
- aqueous mounting media
- dye must be more soluble in substate and the solvent
metallic impregnation
- 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
first step in all staining
deparaffinization
Hematoxylin
- natural dye from logwood of H. campechianum tree in Central America
- lacks a chromophore
- must be oxidized to hematein and mordanted before use
oxidation - ripening
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
Alum hematoxylin
- most commonly used
- used progressively (differentiation = acid alcohol)
iron hematoxylin
- dye-mordant lake is darker => almost black
- very resistant to differentiation (common diff = excess mordant)
eosin
- anionic dye with quinoid chromophore
- different shades; yellowish or bluish
- common counterstain in many methods
