Staining

Question 1

5 factors affecting staining

Answer 1

pH

Concentration of inorganic salt

Electrolyte concentration

Rates of reagent uptake and loss

Rate of reaction

Question 2

Progressive staining

Answer 2

Dye left for right amount of time to stain particular molecule.

If left too long unwanted molecules stain

Question 3

Regressive staining

Answer 3

Over stain tissue, wash out with buffer (acid/alcohol/salt solution)

Question 4

How does pH affect dye staining

Answer 4

Acid/Base solutions affect dye-target interactions

pH also used in differentiation - selectively wash out dye

Question 5

How does size affect staining

Answer 5

Different size diffuse at different speeds

Large slow dyes only stain rapidly staining structures

Small fast dyes uniformly stain

Question 6

Differentiation

Answer 6

Non-selective due to overstrain
Retracted selectively lost with buffer
Removed from permeable structures and retained by impermeable structures

Question 7

Metachromasia

Answer 7

Dye can stain different tissue elements different colours by absorbing light at different wavelengths

Question 8

Basophilic

Answer 8

Cationic (+ve) she has affinity for basic (-ve) molecules (DNA)

Question 9

Acidophilic

Answer 9

Anionic (-ve) dyes have affinity for acidic molecules/structures

E.g. eosin - eosinophilic

Question 10

How does tissue geometry effect staining

Answer 10

Thickness - thin sections stain faster. Variation in thickness = different staining intensities

Surfaces - irregular surfaces stain faster than smooth

Question 11

Aims of staining

Answer 11

Make cell structure visible

Show structure variation

Indicate pre-molecular makeup of tissue (present of nuclei)

Question 12

Why do tissue take up stains

Answer 12

Dye-tissue affinity

Question 13

Coloumbic attractions

Answer 13

Electrostatic reaction between ions in dye and ions in tissue structures (DNA/RNA due to phosphate group)

Question 14

3 cationic dyes

Answer 14

Haematoxylin

Geimsa

Alvina Blue

Question 15

What are the 2 staining mechanisms

Answer 15

Direct attachment - dye binds target molecule via coloumbic, vdw or covalent bond

Indirect attachment - uncharged dye binds via mordant = dye lake

Question 16

How is haematoxylin dye lake formed?

Answer 16

Haematein = uncharged

Must bind to mordant (metal salt -alm/iron)

Gives +ve charge to bind to -ve DNA

Question 17

How to break dye lake for differential staining

Answer 17

Differentiate with acid/alcohol to remove stain - BETTER

Flood tissue with different mordants to act as competitors. It’s cell specific so breaks lake

Question 18

How is alcohol differentiation method used in gram +ve/-ve staining

Answer 18

Crystal violet and iodine = dye lake stains peptidoglycan

Gram +ve p wall more layers than Gram -ve

When dye washed with alcohol, Gram +ve remains stained because thicker wall. Gram -ve = colourless