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Hotchkiss’ Alcoholic Periodic Acid Schiff


Hotchkiss' Alcoholic Periodic Acid Schiff


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 acid0.8g
    Sodium acetate buffer 0.2M10mL
    Ethanol, absolute70mL
    Distilled water20mL
  • Acid reducing rinse
    Potassium iodide2g
    Sodium thiosulphate2g
    Ethanol absolute60mL
    Hydrochloric acid N/12mL
    Distilled water40mL

Tissue Sample

Presumably an alcoholic fixative should be required if glycogen were to dissolve in aqueous solutions. However, 5 µ 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

  • Oxidisable carbohydrates – red
  • Nuclei – blue

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.


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