Genta, Robason and Graham Stain for Simultaneous Visualisation of Helicobacter Pylori and Gastric Morphology

EMS26116

Components:

Uranium Nitrate

Silver Nitrate 1% Aqueous

Gum Mastic 2.5%

Hydroquinone 2%

Silver Nitrate 0.04%

Alcian Blue, pH 2.5

Harris Hematoxylin

Acid Alcohol 1%

Ammonia Water 0.25%

Eosin Y 1%

Fixation:

Formalin fixed

Sections:

Paraffin @ 4µm sections

Staining:

1. Deparaffinise and hydrate sections to distilled water.
2. Sensitise sections by placing them in Uranium Nitrate. 1%, Aqueous at room temperature for 3 minutes and transfer them to distilled water.
3. Rinse slides in distilled water until the possibility of cross contamination is eliminated.
4. Place sections in Silver Nitrate, 1% at room temperature and then heat then in a microwave oven to just below the boiling point. DO NOT BOIL!
5. Rinse slides in 3 changes of distilled water.
6. Rinse in 2 changes of 95% alcohol.
7. Rinse in 2 changes of 100% alcohol.
8. Place slides in 2.5% Gum Mastic for 5 minutes.
9. Air dry sections for 1 minute
10. Rinse in 2 changes of distilled water.
11. Reduce in reducing solution in a 450°C water bath for 10 to 15 minutes or until sections have developed satisfactorily with black or dark brown H-Pylori and a light yellow background. Check periodically under microscope.
    To prepare reducing solution
    - Gum Mastic, 2.5 % - 10 ml
    - Hydroquinone, 2% - 25 ml
    - Absolute Alcohol - 5 ml
    - Silver Nitrate, 0.04% - 2.5 ml
     Mix together - DO NOT FILTER!!
12. Rinse sections in water to stop reduction.
13. Stain in Alcian Blue, pH 2.5 for 10 minutes.
14. Rinse in running tap water.
15. Stain sections in Harris' Haematoxylin for 5 minutes.
16. Rinse in running tap water.
17. Quickly dip in Acid Alcohol.
18. Rinse in running tap water.
19. Rinse in Ammonia Water, 0.25% until blue.
20. Rinse in running tap water.                            
21. Stain sections in Eosin Y, 1% for 5 minutes.
22. Quickly rinse in tap water.
23. Rinse twice in 95% alcohol.
24. Rinse three times in absolute alcohol.
25. Rinse three times in Xylene and then coverslip.

Staining Results:

H-pylori are easily recognised in the mucus, on the cell surface, and within the gastric pits.

References:

Genta PM., Robason GO, Graham DY- Simultaneous Visualization of Helicobactor Pylon and Gastric Morphology A New Stain Hum Pathol 25: 22 1-226, 1994.