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AMMONIACAL SILVER METHOD FOR FUNGI
IN UNDECALCIFIED BONE MARROW SECTIONS
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FIXATION: 10% buffered neutral formalin.
TECHNIQUE: Undecalcified glycol methacrylate embedded bone marrow sections cut at 4 µm.
SOLUTIONS:
4% Periodic Acid
Periodic acid, H5IO6 ------------------------------------------------------------ 4.0 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
3-6° C. This solution is stable for about 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 ------------------------------------------------------------------- 10.0 ml
Acetic acid ----------------------------------------------------------------------- 0.2 ml
Fast Green, Working Solution
Fast green, stock solution ------------------------------------------------------ 10.0 ml
Distilled water ------------------------------------------------------------------- 35.0 ml
1. Place in 4% periodic acid for 5 minutes.
2. Rinse in five changes of distilled water.
3. Pour 40 ml of ammoniacal silver in a glass Coplin jar and allow to warm to near
room temperature. Place slides in the solution and set the Coplin jar in a 43° C waterbath for 3 minutes, then transfer to a 58° C waterbath for 25-35 minutes or until the sections appear light brown. Check the slides after 25 minutes.
prevent the loss of sections in the proceeding steps.
6. Tone in 0.2% gold chloride for 30 seconds.
7. Rinse in two changes of distilled water.
8. Place in 2% sodium thiosulfate for 30 seconds.
9. Rinse in four changes of distilled water.
10. Counterstain with working fast green solution for 3 minutes.
11. Rinse in three changes of distilled water.
12. Dehydrate through graded alcohols.
13. Clear in three or four changes of xylene and mount with synthetic resin.
Fungi----------------------------------------------------------------- sharply delineated in grey to black
Background --------------------------------------------------------- green
In the staining method of Castro and Maynard for plastic embedded sections chromic acid is used as the oxidizing agent. We found that this causes the plastic to stain varying shades of grey and brown but, even so, the fungi stain a rather intense greyish brown. However, because the plastic is stained the fungi are somewhat difficult to locate. Oxidizing the sections with periodic acid prevents the plastic from staining, but the fungi are less intensely stained than when they are oxidized with chromic acid. Even so, the fungi are stained well enough to be easily located and identified.
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.
Churukian, C.J., and Schenk, E.A.: Staining pneumocystis carinii and fungi in unfixed specimens with ammoniacal silver using a microwave oven. J. H~stotechnol. I 1:19-21, 1988.
Castro, M.D., and Maynard, J.H.: Routine and special staining techniques in glycol methacrylate. Presented at the 1988 NSH Symposium/Convention in Louisville, Kentucky.
Churukian, C.J: Ammoniacal silver method for fungi in undecalcified bone marrow sections. Histologic 20:163-164, 1990.
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