PAS WITH AND WITHOUT DIASTASE

              PAS WITH AND WITHOUT DIASTASE

Principle

Section must be oxidized with periodic acid to convert 1:2 glycol group to          di-aldehyde in order that they will react with Schiff reagent to produce a colored end product. Glycogen is destroyed by the enzyme diastase which may be obtained from saliva or Malt

Solutions

Periodic Acid                 -           500mg

Distilled Water               -           100 ml

The solution is colourless

All the glass wares should be properly cleaned and rinsed thoroughly in distilled Water.

Procedure

1)   Add 1gm of basic fuchsin into 200 ml of hot distilled  water in a conical flask.

2)   Bring it to the boiling point, while shaking it well.

3)   Cool it 50°C and add 2 gm of potassium meta bisulphate.

4)   Bring it to room temperature and add 10 ml of 1N HCl,

5)   Plug it with cotton and keep it in dark for 24 hrs.

6)   The next day, add 2 gm of activated charcoal, to stop the bleaching action and filter.

7)   The solution should be straw yellow in color

8)   Store it in dark brown bottle and keep it in fridge.

Test to check the Schiff's Solution:

The Solution if properly prepared should turn into pink immediately, when added to formalin Solution.

Procedure

1)   Deparaffinise the section in xylene for 10 mnts.

2)   Bring the section to water through descending-series of alcohol.

3)   Add 0.5% periodic acid for 5-10 mnts.

4)   Rinse the section well with distilled water

5)   Treat with Schiff reagent for 15-20 mnts.

1)   6)Keep it in running tap water for 5-10 mts for the development of pink color.

6)   Counter stain with Haenatoxylin for 1-2 mnts

7)   Differentiate with 1% acid alcohol

8)   Keep in running tap water for 5-10 mts

9)   Dehydrate clear and mount .

PAS diastase

Treat the section with saliva for 20-30 mnts before adding periodic acid and continue the same procedure…

 Result

Glycogen, other periodate reactive carbohydrates, fungi                  -           Magența

Nuclei                                                                                                 -          Blue

In diastase treated sections, glycogen will not stain.