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

    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 . . . 25ml
    •  Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25ml
    •  Sodium Borate, 5% Aq. . . . . . . . . . . . . . . . . . . . . . 4ml
  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

Online Ordering

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

Combined Hematoxylin And Eosin / Methenamine Silver Stain are available online from the EMS Catalog. For ordering or product information, click here.