Module 5: Carbohydrate Staining

Question 1

Name this staining technique, what are the three main steps and what is happening at each step?

Answer 1

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.

Question 2

Alcian blue pH 2.5 stains acid mucosubstances. What is staining dark blue and what is staining lighter blue?

Answer 2

Dark blue: carboxylated group

Light blue: sulphated group

Question 3

This procedure stains neutral mucopolysaccharides magenta. What is the procedure called?

Answer 3

PAS

Question 4

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?

Answer 4

Mucicarmine

Question 5

What is the most sensitive control for the Periodic Acid Schiff’s stain (ideal tissue for control)?

Answer 5

Kidney (basement membrane)

Question 6

For what reason would we run a PAS/Diastase (Digest)? What are we trying to demonstrate? What control slide would we use for this?

Answer 6

Glycogen

Liver

Question 7

What are the 3 pertinent reactive groups which take part in the staining and demonstration of carbohydrates?

Answer 7

1:2 glycols, carboxyl groups, ester sulphates

Question 8

Carbohydrates can be split into what 2 functional groups?

Answer 8

Glycogen, mucins

Question 9

List 6 places that stainable amounts of glycogen are present

Answer 9

Liver, cardiac muscle, skeletal muscle, endometrial glands, umbilical cord, hair follicles

Question 10

What is the function of activated charcoal in the preparation of Schiff’s reagent?

Answer 10

It removes impurities

Question 11

How should good Schiff’s reagent appear if formaldehyde is added to test it prior to staining?

Answer 11

Purple-red

Question 12

What is the function of periodic acid in the PAS method?

Answer 12

Oxidizes glycol groups to aldehydes

Question 13

How should Schiff’s reagent be stored?

Answer 13

Refridgerated in a dark container

Question 14

What would be the expected results if the PAS reaction with alpha amylase is performed on a section of liver containing glycogen?

Answer 14

Treated slide - no glycogen staining

Untreated slide - glycogen magenta

Question 15

What 2 things can be done to prevent pH from being elevated during the Alcian blue staining process?

Answer 15

Rinse section with stain solvent before and after staining

Blot dry rather than rinse with water

Question 16

Is Schiff’s reagent a true leuco dye?

Answer 16

No

Question 17

True or false: toluidine blue is a cationic dye

Answer 17

True

Question 18

True or false: Alcian blue pH 2.5 demonstrates carboxyl groups

Answer 18

True

Question 19

True or false: Malachite green is often used as a counterstain when demonstrating fungus using the PAS method

Answer 19

True

Question 20

True or false: Diastase and alpha-amylase break glycogen down to water-soluble sugars

Answer 20

True

Question 21

True or false: Sodium metabisulfite and HCl remove impurites from Schiff’s reagent

Answer 21

False

Question 22

Expected results following the PAS stain

Answer 22

Glycogen, starch, chitin, cellulose, some epithelial mucins, thyroid colloid, fungal walls, basement membrane - magenta to rose

Background and nuclei - blue

Question 23

Expected results after the mucicarmine stain

Answer 23

Mucins, Cryptococcus neoformans capsule - red

Nuclei - black

Other tissue elements (if counterstained) - yellow or blue; (not counterstained) - colourless to pale pink

Question 24

Expected results after Alcian blue pH 1.0

Answer 24

Sulphated acid mucopolysaccharides - deep blue

Background - faint blue if not counterstained, pink to red if counterstained in nuclear fast red

Question 25

Expected results after Alcian blue pH 2.5

Answer 25

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

Question 26

Expected results after toluidine blue stain

Answer 26

Mast cells - purple

Nuclei and background - blue

Mucins and certain mucosubstances - red to reddish purple

Collagen - reddish purple

RBCs - do not stain

Question 27

What stain is this? What is being demonstrated?

Answer 27

PAS/Alcian blue

PAS pos (neutral polysaccharides) - magenta

Acid mucopolysaccharides - dark blue