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FIXATION:        Unfixed specimens on slides are briefly heat fixed.

TECHNIQUE:        Cytologic preparations of lung aspirates, bronchial washings, sputum

specimens, and other cytologic preparations.

SOLUTIONS:

Modified Chromic Acid Solution

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

Potassium dichromate --------------------------------------------------------------     20.0 gm
Distilled water ---------------------------------------------------------------------        360.0 ml

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

                                                              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 at least one month.

0.2% Gold Chloride

Gold chloride --------------------------------------------------------------------        0.2 gm

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

2% Sodium Thiosulfate

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 -----------------------------------------------------------------------        0.2 ml

Fast Green, Working Solution

Fast green, stock solution ------------------------------------------------------           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.        Rinse in three changes of distilled water.

4.        Treat with 1% sodium bisulfite for 30 seconds.

5.        Wash in running water for 30 second

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 (420 W) for 1

           minute. Immediately place the slides in this hot solution, return to the microwave oven and microwave at power

           level 2 (120 W) 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 40 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 carinli --------------------------------------        sharply delineated in black

If more than three slides are being stained, it may be necessary to prolong the time in the hot ammoniacal silver solution in order to achieve satisfactory staining results. When the material appears a yellow-brown, it is adequately impregnated. It is recommended that gloves and safety glasses be worn when preparing ammoniacal silver solutions because of solution instability during preparation.

The modified chromic acid solution is a strong oxidizing agent and should be handled carefully with gloves and that safety glasses be worn.

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

Churukian, C.J.: A more stable ammoniacal silver using lithium hydroxide. Lab. Med. 17:260, 1986.

Brinn, N.T., and Churukian, C.J.: Chromic acid and arcing. Lab. Med 17:384, 1986.

Churukian, C.J., and Schenk, E.A.: Staining pneumocystis carinii and fungi in unfixed specimens with ammoniacal silver using a microwave oven. J. Histotechnol. I 1:19-21, 1988.