8: Staining Theory Flashcards
Chromophore
A molecular structure that absorbs visible wavelengths of light, which make objects appear to have color
The more __ the __ are in an organic molecule, the more likely it is to absorb visible wavelengths of light.
The more DELOCALIZED the ELECTRONS/BONDS are in an organic molecule, the more likely it is to absorb visible wavelengths of light.
Are triple bonds more or less delocalized than single bonds ?
Triple bonds are more delocalized than single bonds
Chromogen
Part of the dye that contains the chromophore
Auxochrome
IONIZED molecular structure that allows dyes to attach to the tissue
T or F: categorizing dyes as acidic/ basic is misleading bc it has nothing to do with the pH of the dye solution
TRUE; categorizing dyes as acidic/ basic is misleading bc it has nothing to do with the pH of the dye solution
Auxochromes with carboxyl, hydroxyl, and sulphonic groups are __ auxochromes.
Auxochromes with carboxyl, hydroxyl, and sulphonic groups are ANIONIC auxochromes.
COOH > COO-
OH > O-
SO3H > SO3-
Anionic auxochromes will bind __ tissues.
Anionic auxochromes will bind POSITIVELY-CHARGED tissues.
What do these dyes have in common: eosin, orange G, Biebrich scarlet, and acid fuchsin ?
ANIONIC dyes stain basic components
NH2 is the __ auxochrome.
NH2 is the CATIONIC auxochrome.
NH2 > NH3+
What does these dyes have in common: hematoxylin, crystal violet, methylene blue, basic fuchsin ?
CATIONIC dyes stain acidic components
Consider trinitrobenzene: It contains three nitro groups (NO2). Why is it NOT a dye ?
It does NOT contain any auxochromes and cannot bind any tissue components
Why is picric acid a dye, despite having a similar ring structure as trinitrobenzene ?
- has a ring of nitro groups AND A HYDROXYL GROUP (OH)
- OH has a ability to ionize and react with positively-charged tissue components
- is a chromophore AND an auxochrome = ANIONIC DYE
Mordant
- reagent that links dye to tissue
- usually metallic salts; IRON or ALUMINUM
Example of a mordanted dye
Hematoxylin; needs mordant to stain nucleus
Dye lake
A dye combined with a mordant
Metachromasia
- Tissue stains a different color than that of the staining solution
- Cationic dyes aggregate to negatively-charged tissue in close proximity
Examples of metachromatic tissue
- MAST cells
- mucin
- amyloid
Polychromasia
- staining solution is a mixture of multiple dyes (IMPURE)
- different shades of (blue) due to tissue elements selectively binding to different dye components in the solution
Example of a polychromatic dye
Methylene blue = methylene blue, toluidine blue O, thionine, azure A, azure B, azure C
Selective Solubility
- staining method for LIPIDS based on PHYSICAL PROPERTIES (solubility in solvent vs in lipid)
- dye migrates from solvent to lipids of tissue
Ie. Oil Red O, Sudan Black B
Porosity
- combination of a physical + chemical staining method
- multiple dye solutions with varying sizes of dye molecules are used
- smallest dye molecules applied first > “bumped” out by larger dye molecules
- held in place in pores by ionic bonds
Ie. Masson Trichrome
Principle of silver-staining methods
Silver nitrate solutions are easily reduced = metallic silver deposits
Argentaffin
Can bind AND REDUCE silver to its metallic form
Ie. melanin, formalin pigment
Why is formalin pigment troublesome in silver stains ?
Formalin pigment is a by-product of aging formaldehyde
- can be interpreted as a FALSE POSITIVE for melanoma
Argyrophil
- can bind BUT CANNOT REDUCE silver to its metallic form
- chemical reducing agent is needed
Ie. urates
T or F: Argyrophil methods will also demonstrate argentaffin substances
TRUE; Argyrophil methods will also DEMONSTRATE ARGENTAFFIN SUBSTANCES (bind and reduce silver by themselves)
Metallic Substitution
- outdated, INDIRECT method to detect calcium
- “Von Kossa” method: silver replaces carbonate and phosphate anions of calcium salts > bright light used to reduce silver to metallic form
What is a preferred DIRECT method over INDIRECT Metallic Substitution of calcium ?
Alizarin red S stain detects calcium DIRECTLY
