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Electron Microscopy Sciences

Technical Data Sheets

A Combined Hematoxylin And Eosin / Methenamine Silver Stain

EMS Catalog # 26123

For The Histological Diagnosis Of Fungi In Tissue Sections

Fixation

10% Buffered Neutral Formalin (NBF)

Sections

3-4 um

Staining Procedure

  1. Deparaffinize and hydrate to distilled water.
  2. Oxidize in Chromic Acid, 10% for 10 minutes.
  3. Rinse with 3-4 changes of tap water.
  4. Rinse in Sodium Bisulfite, 1% for 1 minute to remove any residual Chromic Acid.
  5. Wash in running water, 5-10 minutes.
  6. Rinse with 3-4 changes of distilled water.
  7. Place in freshly mixed working methenamine Silver Nitrate Solution in a water bath @ 58-60 °C, for 15-25 minutes or until sections turn yellowish brown.
    Methenamine Silver Nitrate Stock:

    •  Silver Nitrate, 5% Aq. . . . 5 ml
    •  Methenamine, 3% Aq. . . . 100 ml

    Use chemically clean glassware. Shake well to dissolve precipitate. Store in refrigerator .
    Then to prepare Methenamine Silver Nitrate Working solution:

    •  Methenamine Silver Nitrate Stock Solution . . . 25 ml
    •  Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25 ml
    •  Sodium Borate, 5% Aq. . . . . . . . . . . . . . . . . . . . . . 4 ml
  8. Rinse in 6 changes of distilled water.
  9. Tone in Gold Chloride, 0.2% for 2-5 minutes.
  10. Rinse in 3-4 changes of distilled water.
  11. Remove unreduced Silver with Sodium Thiosulfate, 2% for 2-5 minutes.
  12. Wash thoroughly in tap water for 10 minutes.
  13. Place in Harris ' Hematoxylin for 10 minutes.
  14. Rinse in 3-4 changes of tap water.
  15. Place in Acetic Acid, 3%) for 1 minute.
  16. Rinse in 2 changes tap water.
  17. Place in Ammonia Water, 0.25% 10 minutes.
  18. Rinse in 2 changes tap water.
  19. Place in 95% alcohol, 1 minute.
  20. Place in Eosin Y, 1% Alcoholic for 5 minutes.
  21. Dehydrate in 95% alcohol , absolute alcohol and clear in xylene, 2 changes each.
  22. Mount with Permount.

Stain Results

Organisms Blue-Black
Background Rose

References

Lamps, Laura W. et al A Combined Hematoxylin and Eosin/ M ethenamine Silver Stain for the Histological Diagnosis of Fungi in Tissue Sections. , J. of Histotechnology 23:4 p. 341-345

Product Information

Chromic Acid
Silver Nitrate
Methenamine
Sodium Borate
Gold Chloride
Sodium Thiosulfate
Harris’ Hematoxylin
Acetic Acid
Ammonia Water
Eosin Y
Alcoholic

Combined Hematoxylin And Eosin / Methenamine Silver Stain