Gomori's Methenanine-silver nitrate method for a coccidiod body of aspergillosis [top left], histoplasmosis (yeast form) [top right], mucoid [bottom left], and fungus [bottom right].

Additional photos of this method

FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin or frozen sections cut at 5 μm.  Smears and touch preparations may be done.

SOLUTIONS:

Modified Chromic Acid Solution

Chromium trioxide --------------------------------------------------------------                20.0 gm

Potassium dichromate ------------------------------------------------------------        20.0 gm

Distilled water --------------------------------------------------------------------        360.0 ml

Sulfuric acid ---------------------------------------------------------------------         40.0 ml

5% Silver Nitrate Solution

Silver Nitrate----------------------------------------------------------------------         1.0 gm

Distilled Water--------------------------------------------------------------------        20.0 ml

3% Methenamine Solution

Hexamethylenetetramine (methenamine)-----------------------------------------                12.0 gm

Distilled water--------------------------------------------------------------------        375.0 ml

Ethylene glycol-------------------------------------------------------------------        25.0 ml  

Methenamine-Silver Nitrate Solution, (Stock)

Silver nitrate, 5% solution ------------------------------------------------------                20.0 ml

Methenamine, 3% solution -----------------------------------------------------                400.0 ml

       This solution is good for about one month if stored in a refrigerator at 3°-6° C

3% Sodium Borate (Borax) Solution

Sodium borate --------------------------------------------------------------------        3.0 gm

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

Methenamine-Silver Nitrate Solution (Working)

Methenamine-silver nitrate solution (stock) ---------------------------------------        20.0 ml

Distilled water --------------------------------------------------------------------        20.0 ml

Sodium borate, 3% solution ----------------------------------------------------                2.0 ml

       Prepare fresh.

1% Sodium Bisulfite Solution

Sodium bisulfite ------------------------------------------------------------------        1.0 gm

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

0.2% Gold Chloride Solution

Gold chloride, 1% solution -------------------------------------------------------        20.0 ml

Distilled water --------------------------------------------------------------------        80.0 ml

2% Sodium Thiosulfate (Hypo) Solution

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

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

0.2% Fast Green Solution (Stock)

Fast green FCF, C.I. 42053 -----------------------------------------------------        0.2 gm

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

Acetic acid, glacial ---------------------------------------------------------------        0.2 ml

Fast Green Solution (Working)

Fast green (stock) ----------------------------------------------------------------        10.0 ml

Distilled water --------------------------------------------------------------------        35.0 ml

• Use control slide.

1.        Deparaffinize and hydrate to distilled water.

2.        Place in modified chromic acid solution for 5 minutes.

3.        Wash in running tap water for a few seconds.

4.        Sodium bisulfite solution for 1 minute to remove any residual chromic acid.

5.        Wash in running tap water for 15 seconds.

6.        Rinse with four changes of distilled water.

7.        Place in working methenamine-silver nitrate solution and place in a 43˚ C water bath for 2 minutes.  Transfer to a 58˚ C water bath for 19-21 minutes or until the sections turn a medium brown. Check slides after 19 minutes.

8.        Rinse in four changes of distilled water.

9.        Tone in 0.2% gold chloride solution for 1 minute.

10.        Rinse in two changes of distilled water.

11.        Remove unreduced silver with 2% sodium thiosulfate solution for 1 minute.

12.        Wash in running tap water for 15 seconds and rinse in two changes of distilled water.

13.        Counterstain with working fast green solution for 40 seconds.

14.        Wash in running tap water for a few seconds and rinse in two changes of distilled water.

15.        Dehydrate through graded alcohols.

16.        Clear in three or four changes of xylene and mount with synthetic resin.

Fungi and pneumocystis carinii -------------------------------------                sharply delineated in black

Mucin and glycogen -----------------------------------------------        ----------        rose taupe to gray

Background -----------------------------------------------------------------        green

This staining method is much more rapid than Grocotts original procedure and the results are more consistent.  The staining time in the working methenamine-silver nitrate solution may need to be varied slightly according to the age of the stock solution.  When the stock methenamine-silver nitrate solution is fresh the staining time needs to be increased by one or two minutes.  The stock methenamine-silver nitrate solution has a shelf-life of about one month when stored in a refrigerator.

There are a number of other things which the rapid GMS will stain including histiocytes, melanin, actinomyces, certain bacteria, starch, glycogen, and mucin.  Nuclei, collagen, and erythrocytes will also stain if the time in the working methenamine-silver nitrate solution is prolonged.

Silver nitrate crystals have a shelf-life of several years when stored in a refrigerator.

Ethylene glycol is added to the methenamine-silver nitrate because it helps to stabilize the solution when it is heated.  This usually prevents the formation of a mirror of silver on the slides and coplin jar.

Grocott, R.G.:  A stain for fungi in tissue sections and smears using Gomoris methenamine-silver nitrate technic.  Amer. J. Clin. Path. 25:975-979, 1955.

Churukian, C.J., and Schenk, E.A.:  Rapid Grocotts methenamine-silver nitrate method for fungi and Pneumocystis carinii.  Amer. J. Clin. Path. 68:427-428, 1977.