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Double Oxidation Schiff

Double Oxidation Schiff


Double oxidation may be used with any procedure which demonstrates aldehydes, including Schiff’s reagent and methenamine silver reduction. Its use is not necessarily confined to staining fungi and it may be found useful when demonstrating other structures and the background stains too darkly.


  • Periodic acid – 1% aqueous
  • Analine-acetic block
    Acetic acid, glacial5mL
    Aniline oil45mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.


  1. Bring sections to water via xylene and ethanol.
  2. Oxidize with periodic acid for 10 minutes.
  3. Rinse well with tap water.
  4. Treat with aniline-acetic for 30 minutes.
  5. Rinse well with tap water.
  6. Re-oxidize with periodic acid for 20 minutes.
  7. Rinse well with tap water.
  8. Rinse with distilled water.
  9. Place in Schiff’s reagent for 10-30 minutes.
  10. Wash off with distilled water.
  11. Wash well with tap water for about 10 minutes.
  12. Counterstain with Mayer’s hemalum for 2 minutes.
  13. Wash well with tap water until hemalum is blued.
  14. Dehydrate with ethanol, clear with xylene and coverslip using a resinous medium.

Expected Results

  • Oxidizable carbohydrates  –  red
  • Fungi  –  red
  • Nuclei & background  –  blue


  • The time periodic acid is applied in the first oxidation determines the amount of background staining eliminated. If there is only a small amount of this material, then all of it may be blocked
  • The first oxidation will also oxidize some of the target material, and this staining will also be inhibited. If the first oxidation is applied for too long, the depth of staining of the target material may be affected. The 10 minutes specified is usually enough.
  • The time periodic acid is applied in the second oxidation determines the depth of staining for the target material. 20 minutes is usually adequate, but extending it may give darker staining. At some point, extending the time in periodic acid will cease to produce darker staining as all available carbohydrate will have been oxidized.

Safety Note

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


  1. Personal observation, B. Llewellyn,