Principles of Staining

Question 1

What is a typical form that dyes come in?

Answer 1

Powder form - aromatic, salt-like, crystalline solids that dissolve in water or alcoholic solutions to form ions.

Question 2

What is the pro and cons of natural plant/animal dyes?

Answer 2

Pro: Provides beautiful colours found in the environment.
Con: Expensive and time consuming to make.

Question 3

What are the pros of synthetic dyes?

Answer 3

Readily available.
Created in lab.
Large amounts can be made at one time.
Produces nice colours on tissues.

No negatives (cons) were noted about synthetic dyes.

Question 4

What wavelength and range of spectrum does benzene resonate at?

Answer 4

Benzene resonates in the UV light range (256 nm).

Question 5

What is a chromophore?

Answer 5

A chromophore is added to a benzene ring to selectively absorb light waves and confers colour to a dye. However, it can easily be reduced to be colourless because they have an unsatisfied affinity for hydrogen.

Question 6

What is the correlation between the number of chromophoric groups present and colour intensity?

Answer 6

More chromophoric groups –> more intense the colour is.

Question 7

What is an auxochrome?

Answer 7

Chemical group that attaches to the benzene ring that has a chromophore attached to it that then attaches both of those to a tissue.

Auxochromes ionize in solution forming cations and anions which attach to the tissue substrate.

They do not provide the colour.

Question 8

Name some common acidic auxochromes?

Answer 8

Carboxyl (COO-)
Sulphonic (SO3)
Hydroxyl (O-)

Question 9

What is a common basic auxochrome?

Answer 9

Amine NH2+

Question 10

What common type of salts form acidic auxochromes?

Answer 10

Sodium salts in powder form are common for acidic auxochromes.

Question 11

What common type of salts are basic auxochromes?

Answer 11

Chloride salts in powder form are common for cationic dyes.

Question 12

What are modifiers?

Answer 12

Compounds that alters the colour or intensity of a dye. Examples are methyl (CH3), aryl (C6H5) and ethyl (C2H5)

Question 13

What are some non-polar substances?

Answer 13

Xylene, Paraffin, Fats

Question 14

What is a semi-polar substance?

Answer 14

Substance where one area of the molecule can have a high concentration of electrons but all of its protons are equally distributed throughout the rest of the molecule or vice versa.

Question 15

What are some examples of semi-polar substances?

Answer 15

Ethanol and acetone.

Question 16

What type of substance are most soluble with a) non-polar b) polar and c) semi-polar?

Answer 16

a) non-polar –> most soluble with a non-polar (then semi-polar)
b) polar –> ions (me)
c) semi-polar –> polar or non-polar substances.

Question 17

What is a amphoteric dye?

Answer 17

For amphoteric: When the dye is an acid solution, it behaves as a basic dye and vice versa. Ex. Carminic acid (carmine) is amphoteric with an IEP of pH 4.5

Question 18

When acidic or basic dye dissolves in solution what are the two parts they ionize into?

Answer 18

Acid (Anionic) Dye - Negatively charged coloured dye ions and positively charged colourless sodium ions.

Basic (Cationic) Dye - Positively charged dye ions and negatively charged colourless chloride ions.

Question 19

Name some examples of natural dyes and their source?

Answer 19

Carmine – cactus insect
Orcein - Lichens
Saffron – Crocus
Hematoxylin – Tree

Question 20

What does the dye carmine required to be employed for staining to be effective.

Answer 20

Carmine must be employed with a mordant.
Example: Mayers mucicarmine procedure is used as a stain for mucin and as a stain for glycogen. The carminic acid in this procedure is linked with an aluminum mordant.

Question 21

What is litmus?

Answer 21

Litmus is a water soluble mixture of different dyes extracted from lichens, especially Roccella tinctoria. It is often absorbed onto filter paper to be used as a pH indicator.

Question 22

How is orcein created?

Answer 22

Extractions from lichens are treated with ammonia to produce orcein.

Question 23

What type of substances is orcein used for demonstrating in tissues?

Answer 23

Orcein is used for demonstrating elastic, copper, and hepatitis antigen in tissue.

Question 24

Where does saffron come from?

Answer 24

Saffron is obtained from the stamens of the Crocus sativus.

Question 25

What is saffron used to stain?

Answer 25

It is used as an alcoholic solution to stain connective tissue. It acts as an anionic dye of large molecular weight, and will stain basic tissue.

Question 26

Why is hematoxylin itself not a dye? What is done to it to make it a dye?

Answer 26

It is not a dye, lacking a chromophore. The molecule must be oxidized first to produce the typical burgundy red colour and then mordanted to allow attachment to tissue groups.

Question 27

What is hematoxylin extensively used to stain?

Answer 27

This natural dye (hematoxylin) is used extensively for staining nuclei and elastic fibres.

Question 28

What are some various quality control measures used for dyes?

Answer 28

1. Storage for stability
2. Testing for sameness.
3. Colour Index Number - to enable consistency in identifying dyes
4. Dye lot number - to assist in trouble shooting
5. Dye nomenclature - consistent description, letters or numbers following dye name indicate shade.
6. Note conditions of use.
7. Type of solvent.
8. Labelling –> name of solution, date, tech initials, WHMIS

Question 29

What are the various physical theories of staining?

Answer 29

1. Porosity –> Size of spaces
2. Adsorption –> Dye attracted to surface of tissue
3. Absorption –> Penetrates tissue
4. Solubility –> Lysochromes, coloured substances that dissolve in hydrophobic substances (e.g. lipids, etc.) but not in water. More soluble in lipid than in solvent they came from.