Module 5: Carbohydrate Staining Flashcards
Name this staining technique, what are the three main steps and what is happening at each step?
PAS technique
Oxidation of the 1,2 glycols to aldehydes by the use of periodic acid, aldehydes oxidize the Schiff’s reagent back to magenta. Counterstain with Harris hematoxylin (gives a blue background). Basement membrane is being demonstrated.
Alcian blue pH 2.5 stains acid mucosubstances. What is staining dark blue and what is staining lighter blue?
Dark blue: carboxylated group
Light blue: sulphated group
This procedure stains neutral mucopolysaccharides magenta. What is the procedure called?
PAS
This carbohydrate stain can be used to demonstrate acid epithelial mucins. It uses a natural due and can also be seen with a yellow counterstain. What stain is this?
Mucicarmine
What is the most sensitive control for the Periodic Acid Schiff’s stain (ideal tissue for control)?
Kidney (basement membrane)
For what reason would we run a PAS/Diastase (Digest)? What are we trying to demonstrate? What control slide would we use for this?
Glycogen
Liver
What are the 3 pertinent reactive groups which take part in the staining and demonstration of carbohydrates?
1:2 glycols, carboxyl groups, ester sulphates
Carbohydrates can be split into what 2 functional groups?
Glycogen, mucins
List 6 places that stainable amounts of glycogen are present
Liver, cardiac muscle, skeletal muscle, endometrial glands, umbilical cord, hair follicles
What is the function of activated charcoal in the preparation of Schiff’s reagent?
It removes impurities
How should good Schiff’s reagent appear if formaldehyde is added to test it prior to staining?
Purple-red
What is the function of periodic acid in the PAS method?
Oxidizes glycol groups to aldehydes
How should Schiff’s reagent be stored?
Refridgerated in a dark container
What would be the expected results if the PAS reaction with alpha amylase is performed on a section of liver containing glycogen?
Treated slide - no glycogen staining
Untreated slide - glycogen magenta
What 2 things can be done to prevent pH from being elevated during the Alcian blue staining process?
Rinse section with stain solvent before and after staining
Blot dry rather than rinse with water
Is Schiff’s reagent a true leuco dye?
No
True or false: toluidine blue is a cationic dye
True
True or false: Alcian blue pH 2.5 demonstrates carboxyl groups
True
True or false: Malachite green is often used as a counterstain when demonstrating fungus using the PAS method
True
True or false: Diastase and alpha-amylase break glycogen down to water-soluble sugars
True
True or false: Sodium metabisulfite and HCl remove impurites from Schiff’s reagent
False
Expected results following the PAS stain
Glycogen, starch, chitin, cellulose, some epithelial mucins, thyroid colloid, fungal walls, basement membrane - magenta to rose
Background and nuclei - blue
Expected results after the mucicarmine stain
Mucins, Cryptococcus neoformans capsule - red
Nuclei - black
Other tissue elements (if counterstained) - yellow or blue; (not counterstained) - colourless to pale pink
Expected results after Alcian blue pH 1.0
Sulphated acid mucopolysaccharides - deep blue
Background - faint blue if not counterstained, pink to red if counterstained in nuclear fast red
Expected results after Alcian blue pH 2.5
Carboxylated acid mucopolysaccharides - deep blue
Sulphated acid mucopolysaccharides - lighter blue
Background - faint blue if not counterstained, pink to red if counterstained in nuclear fast red
Expected results after toluidine blue stain
Mast cells - purple
Nuclei and background - blue
Mucins and certain mucosubstances - red to reddish purple
Collagen - reddish purple
RBCs - do not stain
What stain is this? What is being demonstrated?
PAS/Alcian blue
PAS pos (neutral polysaccharides) - magenta
Acid mucopolysaccharides - dark blue
