Practical 5: Perls Prussian Blue, Congo Red, AB/PAS, Grocott's Silver

Question 1

Why might iron accumulate in organs

Answer 1

If there is excessive rbc breakdown, and in liver in haemochromatosis

Question 2

Why might we be staining for carbohydrates

Answer 2

They may be present as part of normal stores such as glycogen, mucin secretion or as part of a diseased cell phenotype

Question 3

Why might we stain for amyloid
(3)

Answer 3

Amyloid protein is deposited in blood vessel walls and extracellularly in many organs in systemic amyloidosis

It is also present in brain plaques in Alzheiner’s disease

It leads to progressive organ malfunction

Question 4

Why might we investigate fungal infections in pathology
(3)

Answer 4

We can confirm fungal infections using methanamine silver stains (and PAS stain)

These stains only identify the presence they can’t classify them

We might be able to see basic structural features to help classify

Question 5

List three types of fungal infections we might see in pathology

Answer 5

Candida

Pneumocystis jiroveci

Aspergillus

Question 6

What is perls prussian blue used for

Answer 6

Iron deposits

Question 7

What is the principle behind PPB
(4)

Answer 7

HCl releases ferric iron from haemosiderin in the form of ferric hydroxide

Potassium ferrocyanide ionizes in solution

The ferrocyanude ions bide to ferric ions in the hepatocytes to form ferriferrocyanide which is a blue chromogen

Blue chromogen forms at site of ferric iron

Question 8

What does the HCl do in PPB

Answer 8

HCl releases ferric iron from haemosiderin in the form of ferric hydroxide

Question 9

What does the ferrocyanide do in PBB
(2)

Answer 9

Potassium ferrocyanide ionizes in solution

The ferrocyanide ions binds to ferric ions in the hepatocytes to form ferriferrocyanide which is a blue chromogen

Question 10

What does ferriferrocyanide do

Answer 10

This is a blue chromogen

It forms at the site of the ferric iron in the tissue

Question 11

What is your counterstain in PPB

Answer 11

Counterstain everything pink with eosin

Question 12

What is the Alcian Blue/Period Acid Schiffs stain?
(3)

Answer 12

A histochemical technique for determining if there are any carbohydrates present

Stain for mucins

Classifies into acid mucins (blue) and neutral mucins and glycogen (pink)

Question 13

What is Alcian blue
(4)

Answer 13

A copper-containing dye which binds electrostatically to polyanions such as carboxyl and sulphate groups in mucins

All acid mucins are stained blue by this dye at pH 2.5

Alcian blue is a large molecule basic dye which binds to the acidic groups in carbs

Used to demonstrate acid mucins in normal and diseased tissues

Question 14

How does Alcian blue work
(2)

Answer 14

A copper-containing dye which binds electrostatically to polyanions such as carboxyl and sulphate groups in mucins

Alcian blue is a large molecule basic dye which binds to the acidic groups in carbs

Question 15

At what pH does AB work for all acid mucins
(2)

Answer 15

Ph 2.5

Will identify different acid mucins at different pHs

Question 16

What are the acid groups in carbs?

Answer 16

Mucins

Glycosaminoglycans

Question 17

What is the PAS reaction
(2)

Answer 17

A two-step chemical reaction

First step =
- oxidation in periodic acid

Second step=
- Schiffs reagent

Question 18

How does periodic acid of AB/PAS work?
(2)

Answer 18

oxidation in periodic acid to generate aldehyde groups from the carbs

Followed by staining with sensitive reagent for aldehydes to stain the site of the sugars a pink colour

Question 19

How does Schiffs reagent work?
(2)

Answer 19

The chromophoric groups of basic fuchsin are broken by sulphurstion to form a colourless solution

In the presence of free aldehyde groups an insoluble coloured compound is reformed from the basic fuchsin to give a visible magenta coloured product

Question 20

What is the principle behind AB/PAS colours altogether
(4)

Answer 20

Acid mucins are stained first with AB

Acid mucins also PAS positive will be blocked from reacting

The subsequent application of PAS will stain neutral mucins bright magenta

Tissues containing both Acid and neutral mucins will demonstrate a purple colour

Question 21

What is the Congo red dye
(3)

Answer 21

A direct cotton dye, acid

Highly selective for amyloid (elastic will also stain)

Will have apple green biorefringence

Question 22

How does the Congo red dye work?

Answer 22

It forms non-polar Hydrogen bonds with amyloid

Question 23

How does the Congo red dye have biorefringence

Answer 23

The planar (flat) dye molecule is arranged in a parallel fashion due to its binding to the linearly arranged amyloid fibrils

Question 24

How do you differentiate the Congo red dye

Answer 24

You want the amyloid in the glomeruli and elastin of blood vessels to be red

Take the red out of everything else - clear background

Question 25

What is grocotts silver stain

Answer 25

An argentaffin silver stain for fungal cells

Question 26

How does grocotts silver stain work
(5)

Answer 26

The mucopolysaccharide component of fungal cell walls are oxidised by chromic acid to release aldehyde groups

Aldehyde groups react with silver nitrite in hexamine silver solution, reducing it to a metallic silver, rendering them visible (argentaffin)

Post treatment with gold chloride to enhance silver deposit

Fix with sodium thiosulphate to remove any unbound ions

Counterstain with light green

Question 27

How is grocotts silver an argentaffin reaction
(2)

Answer 27

The mucopolysaccharide component of fungal cell walls are oxidised by chromic acid to release aldehyde groups

Aldehyde groups react with silver nitrite in hexamine silver solution, reducing it to a metallic silver, rendering them visible (argentaffin)