8: Staining Theory

Question 1

Chromophore

Answer 1

A molecular structure that absorbs visible wavelengths of light, which make objects appear to have color

Question 2

The more __ the __ are in an organic molecule, the more likely it is to absorb visible wavelengths of light.

Answer 2

The more DELOCALIZED the ELECTRONS/BONDS are in an organic molecule, the more likely it is to absorb visible wavelengths of light.

Question 3

Are triple bonds more or less delocalized than single bonds ?

Answer 3

Triple bonds are more delocalized than single bonds

Question 4

Chromogen

Answer 4

Part of the dye that contains the chromophore

Question 5

Auxochrome

Answer 5

IONIZED molecular structure that allows dyes to attach to the tissue

Question 6

T or F: categorizing dyes as acidic/ basic is misleading bc it has nothing to do with the pH of the dye solution

Answer 6

TRUE; categorizing dyes as acidic/ basic is misleading bc it has nothing to do with the pH of the dye solution

Question 7

Auxochromes with carboxyl, hydroxyl, and sulphonic groups are __ auxochromes.

Answer 7

Auxochromes with carboxyl, hydroxyl, and sulphonic groups are ANIONIC auxochromes.

COOH > COO-
OH > O-
SO3H > SO3-

Question 8

Anionic auxochromes will bind __ tissues.

Answer 8

Anionic auxochromes will bind POSITIVELY-CHARGED tissues.

Question 9

What do these dyes have in common: eosin, orange G, Biebrich scarlet, and acid fuchsin ?

Answer 9

ANIONIC dyes stain basic components

Question 10

NH2 is the __ auxochrome.

Answer 10

NH2 is the CATIONIC auxochrome.

NH2 > NH3+

Question 11

What does these dyes have in common: hematoxylin, crystal violet, methylene blue, basic fuchsin ?

Answer 11

CATIONIC dyes stain acidic components

Question 12

Consider trinitrobenzene: It contains three nitro groups (NO2). Why is it NOT a dye ?

Answer 12

It does NOT contain any auxochromes and cannot bind any tissue components

Question 13

Why is picric acid a dye, despite having a similar ring structure as trinitrobenzene ?

Answer 13

• 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

Question 14

Mordant

Answer 14

• reagent that links dye to tissue
• usually metallic salts; IRON or ALUMINUM

Question 15

Example of a mordanted dye

Answer 15

Hematoxylin; needs mordant to stain nucleus

Question 16

Dye lake

Answer 16

A dye combined with a mordant

Question 17

Metachromasia

Answer 17

• Tissue stains a different color than that of the staining solution
• Cationic dyes aggregate to negatively-charged tissue in close proximity

Question 18

Examples of metachromatic tissue

Answer 18

• MAST cells
• mucin
• amyloid

Question 19

Polychromasia

Answer 19

• 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

Question 20

Example of a polychromatic dye

Answer 20

Methylene blue = methylene blue, toluidine blue O, thionine, azure A, azure B, azure C

Question 21

Selective Solubility

Answer 21

• 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

Question 22

Porosity

Answer 22

• 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

Question 23

Principle of silver-staining methods

Answer 23

Silver nitrate solutions are easily reduced = metallic silver deposits

Question 24

Argentaffin

Answer 24

Can bind AND REDUCE silver to its metallic form
Ie. melanin, formalin pigment

Question 25

Why is formalin pigment troublesome in silver stains ?

Answer 25

Formalin pigment is a by-product of aging formaldehyde
- can be interpreted as a FALSE POSITIVE for melanoma

Question 26

Argyrophil

Answer 26

• can bind BUT CANNOT REDUCE silver to its metallic form
• chemical reducing agent is needed
  Ie. urates

Question 27

T or F: Argyrophil methods will also demonstrate argentaffin substances

Answer 27

TRUE; Argyrophil methods will also DEMONSTRATE ARGENTAFFIN SUBSTANCES (bind and reduce silver by themselves)

Question 28

Metallic Substitution

Answer 28

• 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

Question 29

What is a preferred DIRECT method over INDIRECT Metallic Substitution of calcium ?

Answer 29

Alizarin red S stain detects calcium DIRECTLY