Microtechniques 1 - Staining

Question 1

4 different staining mechanisms?

Answer 1

1. Acid-base reactions
2. Metal impregnation e.g. gold
3. Elective solubility e.g. fat stains
4. Vital dyes e.g. trypan blue

Question 2

What is the CHROMOGEN?

Answer 2

Whole dye molecule

Question 3

What is the CHROMOPHORE?

Answer 3

Part responsible for colour

NO2 or azo -N=N-

Question 4

What is the AUXOCHROME?

Answer 4

Part that binds to tissue - ionisable to either acid or base.
COO- or NH3+

Question 5

What is the MODIFIER?

Answer 5

Part responsible for altering shade or tone of colour

-CH^3 (a methyl - the more of them the bluer something is)

Question 6

What is special about picric acid?

Answer 6

It is a fixative as well as a dye. Some of its side groups act as chromophores and auxochromes.

Question 7

Basic tissue components = _____philic

Answer 7

Acidophilic e.g. haemoglobin

Question 8

Acid tissue components = _____philic

Answer 8

Basophilic e.g. DNA, RNA

Question 9

What are the 2 ways in which a mordant can act?

Answer 9

1. Bind dye to tissue permanently e.g. sulphates

2. Stabilise dye and render it insoluble in the tissue e.g. picric acid, mercury

Question 10

What does a typical mordant look like?

Answer 10

They are usually metal ions e.g. Al3+ or Fe3+

Question 11

____ - ____ - _____ = LAKE

Answer 11

DYE, MORDANT, TISSUE

Question 12

Difference between PROGRESSIVE and REGRESSIVE staining?

Answer 12

P: stain for long enough until desired colour is obtained
R: overstain and then remove dye until desired colour is obtained - this gradual removal is known as DIFFERENTIATION

Question 13

What is the ability of a dye to stain different parts of a tissue different colours called?
What is an example of a dye that does this?

Answer 13

METACHROMASIA

e.g. toluidine blue

Question 14

What 7 things affect how well a dye binds to tissue?

Answer 14

1. The charge (ionisation) of the dye and tissue
2. Porosity of the tissue
3. Size of the dye particle
4. Concentration of dye solution
5. Solvent - either water or alcohol
6. Temperature
7. Time

Question 15

What does CI stand for? Who came up with it? What are 2 benefits of it?

Answer 15

Colour Index, US Commission of Standardisation of Biological Stains, allows standardisation and consistency between batches.

Question 16

2 varieties of MOUNTING MEDIA?

Answer 16

1. Natural resins e.g. Canada balsam
2. Synthetic e.g. xylene - good because RI is v close to glass and we want the same RI
   Good MM should be: durable, won’t corrode stain, adhesive

Question 17

What are the 2 ways to prepare calcified tissues?

Answer 17

1. DECALCIFICATION: using acid e.g. HCl (faster) or chelating agents e.g. EDTA (binds Ca2+ preferentially, slower, gentler) - removes calcified components but soft tissue elements retained
2. GROUND SECTIONS: section sliced with a saw and then polished (ground) until thin enough to transmit light - calcified components remain but soft tissue elements damaged