Haematoxylin And Eosin / Methenamine Silver Stain

EMS26123

For The Histological Diagnosis Of Fungi In Tissue Sections

Contains

Fungi in tissue, includes:
Chromic Acid, 10%
Sodium Bisulfite, 1%
Silver Nitrate, 5% Aqueous
Methenamine, 3% Aqueous
Sodium Borate, 5% Aqueous
Gold Chloride, 0.2%
Sodium Thiosulfate, 0.2% Aqueous
Harris’ Hematoxylin
Acetic Acid 3%
Ammonia Water, 0.25%
Eosin Y, 1% Alcoholic

Fixation

10% Buffered Neutral Formalin (NBF)

Sections

3-4µm

Staining Procedure

1. Deparaffinise 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' Haematoxylin 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

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