The periodic acid Schiff reaction (PAS) is used to demonstrate the presence of 1-2-glycols, including glycogen. Sometimes it is necessary for the glycogen to be removed so that a clearer picture of non-glycogen carbohydrates may be seen. Glycogen may be removed with amylase (diastase).
Dehydrate with ethanol, clear with xylene and coverslip using a resinous medium.
Glycogen – red in the undigested section, absent from the digested section.
Oxidisable carbohydrates – red
Nuclei – blue
If the intent is to remove glycogen rather than identify it,
a more convenient approach in a routine service laboratory is to dissolve the amount of α-amylase
which would cover a 1cm circle about 1mm deep in 15mL (1 test tube) of distilled water.
Shake for a few minutes, then filter onto the sections and digest at room temperature.
Glycogen is usually removed within 30 minutes to 1 hour. Wash well and continue with a regular PAS (step 7).
Please note that an older variation of the procedure in Note 1 was to collect saliva and use that for the digestion.
This is strongly deprecated. Human body fluids may contain transmissable bacterial and viral contaminants,
so anyone handling such a slide will be at risk.
Hog α-amylase is recommended as being consistent and effective. Purchase a product with a high α-amylase activity.
Glutaraldehyde fixation leaves free aldehyde groups attached to tissues,
which causes an overall positive reaction. These groups may be stopped from reacting with
an appropriate procedure such as the aniline-acetic aldehyde block.
The tap water wash at step 7 is necessary to develop the red colour.
Within limits, the longer the wash the darker the colour.
Originally, it was recommended that the Schiff's reagent be washed off with
dilute sulphurous acid (the sulphite rinses). Since water recolours Schiff's reagent,
it was believed that a water wash could lead to false positive results.
It is now known this is not the case, provided the Schiff's reagent is removed
quickly and the sections do not stay in water contaminated with it for extended periods.