FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 7 μm.

SOLUTIONS:

Ammonia-Alcohol Solution

Alcohol, 95% -----------------------------------------------------------------                90.0 ml

Ammonium hydroxide, 28% ---------------------------------------------------                10.0 ml

Glycine-Acetic Acid (Stock Solution)

Glycine ------------------------------------------------------------------------                2.4 gm

Acetic acid, glacial -------------------------------------------------------------                0.3 ml

Distilled water -----------------------------------------------------------------                100.0 ml

       The pH of this solution is approximately 3.6.  Store in a refrigerator

       at 3-6o C.

Acetic-Acetic Acid (Working Solution)

Glycine-acetic acid (stock) ----------------------------------------------------                3.0 ml

Distilled water -----------------------------------------------------------------                300.0 ml

       The pH of this solution is approximately 3.6.

       

2% Silver Nitrate Solution

Silver nitrate -----------------------------------------------------------------                0.2 gm

Glycine-acetic acid, working solution ----------------------------------------                10.0 ml

4% Gelatin Solution

Gelatin ----------------------------------------------------------------------                1.0 gm

Glycine-acetic acid, working solution ----------------------------------------                25.0 ml

       Dissolve by placing on a magnetic stirrer and apply gentle heat.

0.2% Hydroquinone Solution

Hydroquinone ---------------------------------------------------------------                0.02 gm

Glycine-acetic acid, working -------------------------------------------------                10.0 ml

       Silver Nitrate – Gelatin – Hydroquinone Solution

Silver nitrate, 2% --------------------------------------------------------------                10.0 ml

Gelatin, 4% -------------------------------------------------------------------                25.0 ml

Hydroquinone, 0.02% --------------------------------------------------------                10.0 ml

       Heat the gelatin solution to 60o C on a hot plate.  Remove from

       heat and add the 2% silver nitrate and mix.  Then add the 0.02%

       hydroquinone.  Prepare immediately before use.

2% Sodium Thiosulfate

Sodium thiosulfate -------------------------------------------------------------                2.0 gm

Distilled water -----------------------------------------------------------------                100.0 ml

Nuclear Fast Red Solution

(See Pearl’s method for Ferric Iron)

• Use control slide.

1. Deparaffinize and hydrate to distilled water.
2. Place in ammonia-alcohol solution, to remove formalin pigment, for 10 minutes.
3. Wash well in running water.
4. Rinse in three changes of distilled water.
5. Place in freshly prepared silver nitrate-gelatin-hydroquinone solution in a plastic Coplin jar and place in a 37o C oven for 18 minutes.
6. Wash well in hot running water.
7. Rinse in two changes of distilled water.
8. Place in 2% sodium thiosulfate for 1 minute.
9. Rinse in four changes of distilled water.
10. Counterstain with nuclear fast red for 3 minutes.
11. Rinse in two changes of distilled water.
12. Dehydrate in graded alcohols.
13. Clear in three or four changes of xylene.
14. Mount with synthetic resin.

Mercury -------------------------------------------------------------        black

Calcium -------------------------------------------------------------        black

Copper --------------------------------------------------------------        black

Lipofuscin -----------------------------------------------------------        black

Neuromelanin -------------------------------------------------------        black

Nuclei ---------------------------------------------------------------        red

This new method for demonstrating mercury is a modification of the Warthin-Starry technique for spirochetes and bacteria.  It differs from the original Warthin-Starry procedure in that the tissue sections are not pre-treated with silver nitrate prior to placing them in the silver nitrate-gelatin-hydroquinone solution.  Also, the procedure is performed in a dark 37o C oven instead of a water bath or microwave oven.  It is important that the silver reaction take place in the dark in order to minimize non-specific staining.

Like most silver methods, this technique is not specific for its desired purpose.  Other metals that stain black, besides mercury, are calcium and copper, but not ferric iron.  Also, melanin, neuromelanin and lipofuscin stain black.

The type of gelatin used in this method is important in order to obtain satisfactory results.  We found that Fisher type A gelatin with 275 bloom (the higher the bloom the stronger the gel) works the best.  The presence of gelatin in the silver solution forms an emulsion which is important in that it keeps the silver ions from reacting with the hydroquinone.  This prevents the formation of a black metallic silver precipitate on the slides and tissue sections.

Danscher, G. and Moller-Madsen, B.:  Silver amplification of mercury sulfide and selenide: a histochemical method for light and electron microscopic localization of mercury in tissue.  J. Histochem. Cytochem. 33:219-228, 1985.

Moller-Madsen, B. and Danscher, G.:  Localization of mercury in CNS of the rat.  Environmental Research 41:29-43, 1986.

Churukian, C.J. and Schenk, E.A.:  A Warthin-Starry method for spirochetes and bacteria using a microwave oven.  J. Histotechnol. 11:149-151, 1988.

Churukian, C.J., Rubio, A, and Lapham, L.W.:  A simple colloidal silver method (Autometallographic technique) for demonstrating inorganic mercury in brain sections.  J. Histotechnol. 23: 337-339, 2000.