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Chromic Acid-Schiff Reaction

Gridley’s Stain for Fungi

By Aldehydes, Chromic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

Gridley's Stain

for Fungi

15
steps
7
materials
print

Materials

  • Schiff’s reagent
  • Aldehyde fuchsin
  • Chromic acid
    MaterialAmount
    Chromium trioxide5g
    Distilled water500mL
  • Bleach
    MaterialAmount
    Sodium metabisulfite5g
    Distilled water500mL
  • Metanil yellow
    MaterialAmount
    Metanil yellow1g
    Distilled water400mL
    Acetic acid, glacial2drops

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in chromic acid for 1 hour.
  3. Wash well with tap water.
  4. Treat with the metabisulfite bleach for 1 minute.
  5. Wash well with tap water.
  6. Rinse with distilled water.
  7. Place in Schiff’s reagent for 20 minutes.
  8. Wash well with tap water.
  9. Rinse with 70% ethanol.
  10. Place in aldehyde fuchsin 30 minutes.
  11. Rinse off excess with 95% ethanol.
  12. Wash well with tap water.
  13. Counterstain with metanil yellow for 1 minute.
  14. Rinse well with distilled water.
  15. Dehydrate, clear and mount in a resinous medium.

Expected Results

  • Fungi  –  purple
  • Background  –  yellow

Notes

  • At step 2, a 10% solution of chromic acid applied for 10 minutes will give similar results.

Safety Note

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

References

  1. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
  2. Humason, G. L., (1967)
    Animal tissue techniques, Ed. 2
    W. H. Freeman and Company, San Francisco, Ca, USA
March 30, 2023
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Bauer Reaction for Carbohydrates

By Aldehydes, Chromic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

Bauer Reaction

for Carbohydrates

9
steps
7
materials
print

Materials

  • A Schiff reagent
  • A progressive hemalum, such as Mayer
  • Chromic acid
    MaterialAmount
    Chromium trioxide4g
    Distilled water100mL
  • Sulfurous acid
    MaterialAmount
    Sodium metabisulfite, 10% aqu.6mL
    Hydrochloric acid, 1N5mL
    Distilled water100mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidize in chromic acid for 40-60 minutes.
  3. Rinse with tap, then distilled water.
  4. Place into Schiff’s reagent for 15 minutes.
  5. Place into sulfurous acid rinses, 3 changes of 2 minutes each.
  6. Wash with running tap water.
  7. Counterstain with hemalum for 1 minute, and blue
  8. Dehydrate with ethanols.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Glycogen, mucin  –  red
  • Fungi  –  red
  • Nuclei  –  blue

Notes

  • Modern practice is to leave out the sulfite rinses and wash with large amounts of tap water.
  • A progressive hemalum should be used as counterstain because regressive hemalums sometimes stain mucin.
  • Mucins are not usually as dark as with a PAS.
  • Applying chromic acid for too long weakens staining due to continued oxidation of the aldehydes first produced.

Safety Note

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

References

  1. McManus, J. F. A. and Mowry, R. W., (1960)
    Staining Methods Histologic and Histochemical
    Harper & Row, New York, NY, USA.
March 30, 2023
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Fluorescent Schiff

By Aldehydes, Chromic Acid-Schiff Reaction, Protocols, Schiff's Reagent Reactions, Stain Target, Stain Type
Protocol

Fluorescent Schiff

11
steps
7
materials
print

This method demonstrates fungi, and may also be called a Fluorescent Gridley or CAFS.

Materials

  • Fluorescent Schiff reagent
  • Solution A
    MaterialAmount
    Chromium trioxide50g
    Distilled water500mL
  • Solution B
    MaterialAmount
    Sodium sulfite5g
    Distilled water500mL
  • Solution C
    MaterialAmount
    Ethanol, 70%495mL
    Hydrochloric acid5mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in solution A for 10 minutes.
  3. Wash well with tap water.
  4. Bleach with solution B for 1 minute.
  5. Wash well with tap water.
  6. Rinse with distilled water.
  7. Place in fluorescent Schiff reagent for 20 minutes.
  8. Wash well with tap water.
  9. Place in solution C, two changes, 5 minutes each.
  10. Wash well with tap water.
  11. Dehydrate, clear and mount in a non fluorescent resinous medium.

Expected Results

Using a BG 12 exciter filter, and OG 4 (yellow) and/or OG5 (orange) barrier filter, fungi fluoresce yellow with acriflavine and green-red with acridine orange.

Notes

  • This is most useful as a screening method.
  • If background fluorescence is too bright for fungi to be distinguished,it may be quenched with an alum hematoxylin for 1 minute,or 0.5% aqueous potassium permanganate for 1 minute.Quench immediately before the final dehydration step.This should be done with caution as it may reduce fungal fluorescence.

Safety Note

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

References

No specific reference is given for this method. A similar technique using periodic acid as the oxidant is found in:

  1. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
March 30, 2023
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