Practical 5: Perls Prussian Blue, Congo Red, AB/PAS, Grocott's Silver Flashcards
Why might iron accumulate in organs
If there is excessive rbc breakdown, and in liver in haemochromatosis
Why might we be staining for carbohydrates
They may be present as part of normal stores such as glycogen, mucin secretion or as part of a diseased cell phenotype
Why might we stain for amyloid
(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
Why might we investigate fungal infections in pathology
(3)
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
List three types of fungal infections we might see in pathology
Candida
Pneumocystis jiroveci
Aspergillus
What is perls prussian blue used for
Iron deposits
What is the principle behind PPB
(4)
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
What does the HCl do in PPB
HCl releases ferric iron from haemosiderin in the form of ferric hydroxide
What does the ferrocyanide do in PBB
(2)
Potassium ferrocyanide ionizes in solution
The ferrocyanide ions binds to ferric ions in the hepatocytes to form ferriferrocyanide which is a blue chromogen
What does ferriferrocyanide do
This is a blue chromogen
It forms at the site of the ferric iron in the tissue
What is your counterstain in PPB
Counterstain everything pink with eosin
What is the Alcian Blue/Period Acid Schiffs stain?
(3)
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)
What is Alcian blue
(4)
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
How does Alcian blue work
(2)
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
At what pH does AB work for all acid mucins
(2)
Ph 2.5
Will identify different acid mucins at different pHs
What are the acid groups in carbs?
Mucins
Glycosaminoglycans
What is the PAS reaction
(2)
A two-step chemical reaction
First step =
- oxidation in periodic acid
Second step=
- Schiffs reagent
How does periodic acid of AB/PAS work?
(2)
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
How does Schiffs reagent work?
(2)
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
What is the principle behind AB/PAS colours altogether
(4)
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
What is the Congo red dye
(3)
A direct cotton dye, acid
Highly selective for amyloid (elastic will also stain)
Will have apple green biorefringence
How does the Congo red dye work?
It forms non-polar Hydrogen bonds with amyloid
How does the Congo red dye have biorefringence
The planar (flat) dye molecule is arranged in a parallel fashion due to its binding to the linearly arranged amyloid fibrils
How do you differentiate the Congo red dye
You want the amyloid in the glomeruli and elastin of blood vessels to be red
Take the red out of everything else - clear background
What is grocotts silver stain
An argentaffin silver stain for fungal cells
How does grocotts silver stain work
(5)
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
How is grocotts silver an argentaffin reaction
(2)
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)
