Week 9

Question 1

What is a dye

Answer 1

-color in powder form

Question 2

What is a stain

Answer 2

-dye product in a solution with alcohol and water as a solvent

Question 3

What is staining

Answer 3

-process of colouring tissue or cellular components
for the purpose of visualization

staining is alcoholic or aqueous

Question 4

What are the characteristics of a dye

Answer 4

1. Coloured aromatic salt (organic compound)
   ◦ Coal tar or benzene derivatives
2. Exhibits resonance - delocalization of the electron
3. Imparts color to another substance when in a solution

resonance - double bonds shift constantly

Question 5

Why are dyes coloured?

Answer 5

◦ Benzene absorbs electromagnetic radiation @ 200 nm
(infrared range) → not @ visible range (400 – 700 nm range)

◦ Dyes are made up of 3 components to enhance color:
1. Chromophore- color bearing
2. Chromogen - benzene ring and chromophore
3. Auxochrome - ionizing chemical component

Question 6

What is a chromophore

Answer 6

-color bearing substance responsible for light absorption in visible range
-has chromophoric groupings (unstable electrons and double bonds): more bonds = more intensity
-double bonds are unstable
-Chromophoric groupings :Quinoid rings, Azo linkage, Nitro grouping

-easily reduced - high affinity for hydrogen
-if reduced the chromophore will be destroyed and color will be lost

how are they formed

Benzene MINUS 2 hydrogen atoms = quinoid ring -strongest chromophore
-which has two double rings outside the benzene ring
in para or ortho form

Question 7

Azo coupling and Nitro grouping

Answer 7

A benzene ring or naphthalene ring attached by N = N bond

nitro grouping - nitrogen oxide
-PHENOL + 3 NO2 = PICRIC ACID
-not allowed to dry out
-explosive when dry

Question 8

What is a Chromogen

Answer 8

-benzene derivative containing chromophoric groupings.

◦ Can impart color but it is NOT a true dye…yet.
◦ Eventually washes off the tissue upon rinsing.
◦ A true dye would have an ionizing group (auxochrome) that will link firmly to the tissue

Question 9

What is a auxochrome

Answer 9

◦ Ionizing group that converts a chromogen into a true dye that attach to tissue binding sites
◦ AUXO - “auxein” – increase CHROME - color

◦ Basic auxochrome: AMINO (–NH2)
◦ Positively charged.
◦ Attach to basophilic tissue components (e.g., nuclei-negatively charged).

◦ Acid auxochrome: SULFONIC (–SO3H ); CARBOXYL (–COOH), HYDROXYL (–OH)
◦ Negatively charged.
◦ Attach to acidophilic tissues components (e.g., RBCs, muscles, collagen fibers). positively charged

Question 10

What is “THREE in ONE”

Answer 10

Chromophore + Other groups = CHROMOGEN

Chromogen + Auxochrome = TRUE DYE

Question 11

What are Modifiers

Answer 11

◦ Chemical groups that alter the color of the dye by altering the energy of the unstable electrons
◦ They do not directly participate in the light absorption
◦ CH3, C2H5, C6H5, Cl-, Br-, I

Question 12

Acid/Basic Dyes
Anionic/Cationic Dyes

Answer 12

◦ Acid dye = anionic dye → negative charge
◦ Binds to acidophilic tissues with positive charge
◦ Stains intensely in an acidic environment

◦ Basic dye = cationic dye → positive charge
◦ Binds to basophilic tissues with negative charge
◦ Stains intensely in a basic environment

Amphoteric dyes are cationic below IEP and anionic above IEP (pH required to change charges on a chemical compound) .

Actual staining of tissue has nothing to do with pH of the dye. It’s all about charges.

Question 13

Dyes that stain lipids

Answer 13

◦ AKA Lysochromes
◦ Non-ionic (cannot bind electrolytically with the target since the target is also charge-free)
◦ No charges
◦ Neutral dyes
◦ Insoluble in water but soluble in organic reagents (e.g., alcohol)
◦ Dye is more soluble in the fat of the tissue section than it is in the solvent of the staining solution.

Note: Lipids can only be stained if tissues are fresh and cut on the cryostat (i.e., frozen section).
cant do it on processed tissue because the reagents liek xylene and alcohol are organic reagents and would dissolve out the lipids = tissue with only adipocytes and no lipids

Question 14

origin of natural dye vs synthetic

Answer 14

◦ Carmine – extract of female cochineal insect
◦ Orcein – lichens
◦ Saffron – saffron flower
◦ Hematoxylin – logwood tree heartwood

Synthetic dyes
◦ Based on organic compounds – benzene, aniline

Question 15

Factors affecting dye binding
The pH of the solution

Answer 15

◦ Acidified eosin vs alkaline eosin
◦ Acidified methylene blue vs alkaline methylene blue

Question 16

Factors affecting dye binding
Temperature

Answer 16

◦ Increase in temp = increased diffusion rate of staining
◦ Tissue swells

Question 17

Factors affecting dye binding
Concentration of the staining solution

Answer 17

◦ Increased concentration increases dye binding

Question 18

Factors affecting dye binding
Presence of salt in the staining solution

Answer 18

◦ Salts dissolved in the staining solution could increase or decrease staining intensity.
-because salt ions and dye ions compete for the same binding site

Question 19

Factors affecting dye binding
Fixatives used and length of time in them

Answer 19

Increased eosinophilia, decreased basophilia:
◦ Acidic fixatives (Zenker solution, Bouin solution, unbuffered formalin)
◦ Potassium dichromate-based fixative will decrease basophilia (nuclear staining).

Decreased eosinophilia, increased basophilia:
◦ Formaldehyde, mercuric chloride, osmium tetroxide.

Question 20

How is differentiation achieved

Answer 20

◦ Regressive staining – overstaining then removal of excess stain by differentiation

◦ Mordants – are substances or metals that act as link between the dye and the tissue- high affinity for both

◦ Mordant + dye = LAKE → act as a basic (cationic) dye.
◦ Negatively charged in action.
aluminum hematoxylin

Question 21

What are two differentiation methods

Answer 21

1. Weak acid solutions and weak alkaline solutions (solvent: water or alcohol) – used for cationic dyes and anionic dyes, - remove excess stain
   ◦ ACID DIFFERENTIATION: HCl – aluminum hematoxylin for nucleui
   ◦ ALKALINE DIFFERENTIATION: NaOH – eosin- collagen muscle rbc
2. Excess mordant as differentiator→ breaks the dye-mordant complex
   ◦ more mordant molecules in differentiator than mordant bound to the tissue.
   -tissue are stained dark, excess dye molecules must be removed by using salt of heavy metal . so using the excess mordant as a differentiator in order to diff the tissue components . The excess mordant in the differentiator will break the bond between the dye and the mordant on the tissue. Excess mordant in the diff will attract the molecules not attached to target allowing target tissue to be seen better after differentiating because there is more dye for target
   ◦ Structures that have bound the most dye molecules would be the last to lose color.

Question 22

Acid Differentiation VS Mordant Differentiation

Answer 22

ACID DIFFERENTIATION
* Use of acid alcohol for H&E stain
* Breaks the LAKE-TISSUE salt linkage
-acid alcohol

MORDANT DIFFERENTIATION
* Use of excess mordant
* Mordant in solution competes with mordant in tissue for the dye molecule
* Breaks the DYE-MORDANT bond
* Only mordant is attached to tissue

Question 23

how are oxidizers used as differentiation methods

Answer 23

-oxidize the dyes to colorless substance. When
stopped at the appropriate time, substances that retained the most dye molecules will remain stained.

chromic acid
potassimin permanganate

Question 24

how are solvents and buffers used as differentiation methods

Answer 24

solvents
-remove access dye from tissue
Eosin is differentiated in H&E stain using low grade alcohol

Buffer-acts as acidic or basic diff
-in hematology slides - wright stain

Question 25

if you have an acidic dye and basic dye
what is the charge of the dye ion , charge of the tissues stained and what are the tissues described as

Answer 25

eosin
charge of the dye ion - NEGATIVE ANIONIC
charge of tissue: BASIC CATIONIC
tissue: ACIDOPHILIC - RBC COLLAGEN MUSCLE

hematoxylin
charge of the dye ion - POSITIVE (CATIONIC)
charge of tissue: NEGATIVE ANIONIC
tissue: BASOPHILIC -NUCLEUS CHROMATIDS

Question 26

STUDY CHART

Answer 26

Proteins are the most reactive group because they have several binding sites. They react with aqueous solvents so they are hydrophilic.

Simple lipds no charges and non ionic so they are hydrophobic and insoluble in water

carbohydrates have no charges