Hotchkiss' Alcoholic PAS

It was initially thought that the periodic acid Schiff reaction could result in less glycogen being demonstrated than was actually present because it might dissolve in aqueous reagents. It is now known this is not a concern. Hotchkiss recommended an alcoholic method to ensure it did not take place. This method is now redundant.

Schiff's reagent
Mayer's hemalum

Alcoholic periodic acid
Periodic acid 0.8 g
Sodium acetate buffer 0.2M 10 mL
Ethanol, absolute 70 mL
Distilled water 20 mL
Acid reducing rinse
Potassium iodide 2 g
Sodium thiosulphate 2 g
Ethanol absolute 60 mL
Hydrochloric acid N/1 2 mL
Distilled water 40 mL

Tissue sample
Presumably an alcoholic fixative should be required if glycogen were to dissolve in aqueous solutions. However, 5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are usually satisfactory, although glutaraldehyde should be avoided.


  1. Bring sections to water via xylene and ethanol.
  2. Place into periodic acid for 10 minutes.
  3. Rinse with 70% ethanol.
  4. Place in acid reducing rinse for 1 minute.
  5. Rinse with 70% ethanol.
  6. Wash with running water to remove ethanol.
  7. Rinse with distilled water.
  8. Place in Schiff's reagent for 10-30 minutes.
  9. Wash off with distilled water.
  10. Wash well with tap water for about 10 minutes.
  11. Counterstain with Mayer's hemalum for 1 minutes.
  12. Wash well with tap water until hemalum is blued.
  13. Dehydrate with ethanol, clear with xylene and coverslip using a resinous medium.

Expected results


  1. 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.
  2. The tap water wash at step 10 is necessary to develop the red colour. Within limits, the longer the wash the darker the colour.


Culling C.F.A., (1963)
Handbook of histopathological and histochemical techniques Ed. 2
Butterworth, London, UK.




Translate in
Google Translate