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

1% Sodium Bisulfite Solution

Sodium bisulfite -----------------------------------------------------------------        1.0 gm
Distilled water -------------------------------------------------------------------        100.0 ml

Ammoniacal Silver Solution

To 10 ml of 10% silver nitrate, add 5.0 ml of 4% lithium hydroxide, monohydrate. Then add concentrated ammonium hydroxide, drop by drop with constant shaking, until the precipitate just dissolves. Make up the solution to 1000 ml with distilled water and store in a refrigerator at 3° C. This solution is stable for about one month.

0.2% Gold Chloride Solution

Gold chloride --------------------------------------------------------------------         0.2 gm
Distilled water -------------------------------------------------------------------        100.0 ml

2% Sodium Thiosulfate Solution

Sodium thiosulfate --------------------------------------------------------------                   2.0 gm
Distilled water -------------------------------------------------------------------        100.0 ml

Fast Green, Stock Solution

Fast Green FCF C.I. 42053 ----------------------------------------------------        0.2 gm
Distilled water -------------------------------------------------------------------        100.0 ml
Acetic acid, glacial --------------------------------------------------------------        0.2 ml

Fast Green Solution, Working Solution

Fast green, stock solution ------------------------------------------------------                100.0 ml
Distilled water -------------------------------------------------------------------        35.0 ml

• Use control slide.

1.        Deparaffinize and hydrate to distilled water.
2.        Place in 45 ml of modified chromic acid solution for 5 minutes. 

3.        Rinse with three changes of distilled water.
4.        Treat with 1% sodium bisulfite for 30 seconds.
5.        Wash in running water for 30 seconds.
6.        Rinse in three changes of distilled water.
7.        Microwave 40 ml of refrigerated cold ammoniacal silver in a plastic Coplin jar at

power level 7 (420W) for I minute. Immediately place the slides in this hot solution, return to the microwave oven and microwave at power level 2 (120W) for 30 seconds. Dip the slides up and down several times and allow them to remain in the hot solution for 1-2 minutes or until the sections turn a light brown.

8.        Rinse in four changes of distilled water.
9.        Tone in 0.2% gold chloride for 30 seconds.
10.        Rinse in two changes of distilled water.
11.        Place in 2% sodium thiosulfate for 30 seconds.
12.        Rinse in four changes of distilled water.
13.        Counterstain with working fast green solution for 30 seconds.
14.        Rinse in three 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 -----------------------------------------------                black

Melanin -------------------------------------------------------------                black

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

We prefer this microwave ammoniacal silver method over the microwave methenamine silver procedure for the following reasons: 1) Dilute ammoniacal silver is much more stable at high temperatures (above 80° C) than methenamine silver. 2) When methenamine silver is heated to high temperatures the solution rapidly breaks down and sometimes produces a deposit of silver on the slides and the Coplin jar. This never occurs when dilute ammoniacal silver is heated to temperatures even above 90° C. 3) Dilute ammoniacal silver is ready for immediate use and does not need to be diluted and buffered like methenamine silver.

The volume and temperature of the ammoniacal silver used in this method is important. We remove the ammoniacal silver form the refrigerator and pour 40 ml of the cold solution into a plastic Coplin jar immediately before use. The temperature of the ammoniacal silver is 3-6° C before and about 85° C after the second exposure to microwave irradiation.

When heating a solution with microwave irradiation the top portion of the solution is warmer by 10-15° C than that near the bottom of the Coplin jar. Therefore, in order to equalize the temperature of the solutions the slides are dipped up and down in steps #2 and #7 of the staining procedure. This assures uniformity of staining throughout the tissue sections.

We have found that silver nitrate has a shelf-life of several years when stored in refrigerator. Chromic acid is a strong oxidizing agent and should be handled carefully with gloves and it is recommended that safety glasses be worn.

It is recommended that gloves and safety glasses be worn when preparing ammoniacal silver solutions because of solution instability during preparation.

Gomori, G.: Silver impregnation of reticulum in paraffin sections. Amer. J. Clin. Path. 13:993-1002, 1937.

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

Churukian, C.J. and Schenk E.A.: Dilute ammoniacal silver - a substitute for methenamine silver for demonstrating Pneumocystis carinii and fungi. Lab Med. 17:87-90, 1986.