RE: Pneumocystis staining

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From:"Kellar, Eric" <kellarec@MSX.UPMC.EDU>
To:"' '" <>, "' '" <>

I have been using this method daily for 20 years and have found it to be
very faithful. It provides reproducible, consistent staining results with
less background staining than with some microwave modifications. Preparation
and staining time is approximately 30 minutes. 


1. 10% aqueous chromic acid (chromium trioxide)
2. 5% aqueous silver nitrate
3. 3% aqueous methenamine (hexamethylenetetramine)
4. 5% aqueous borax (sodium borate)
5. 1% aqueous gold chloride
6. 1% aqueous sodium metabisulfite
7. 5% aqueous sodium thiosulfate
8. 0.2% aqueous light green

To 40 ml of 3% methenamine in a graduated cylinder add 2 ml of 5% silver
nitrate.  A white precipitate will form but disappears on shaking.  Add 3 ml
of 5% borax and then add DH20 up to a final total of 80 ml. 
This solution should then be heated in a beaker on a hotplate until the
solution becomes a deep golden brown (approximately 85 C) and then poured
immediately on the slides. (Care must be taken so that overheating will not
precipitate all the available silver onto the sides of the beaker).

The use of acid-cleaned glassware is recommended. Use plus
treated slides.
1. Deparaffinize sections and hydrate to DH20.
2. Oxidize slides into 10% chromic acid for ten minutes.
3. Wash slides in tap water for a few seconds to remove excess
chromic acid.
4. Place in 1% sodium metabisulfite for one minute.
5. Rinse in hot tap water for one minute or until working
methenamine silver is prepared.
6. When the working methenamine silver is ready and heated, pour
it immediately on slides, cover and time for three minutes.  It
may take longer if a large amount of slides are to be stained at
one time.
7. Rinse in hot tap water and cool gradually with cooler tap
water to avoid cracking the glassware.
8. Remove and rinse in DH20.
9. Tone in 1% gold chloride, approximately ten seconds.
10. Rinse in distilled water.
11. Place in 5% sodium thiosulfate for three minutes.
12. Wash thoroughly in tap water.
13. Counterstain in light green for 1 minute.
14. Dehydrate, clear in xylene and mount.

Dark brown to black staining of Pneumocystis carinii, Histoplasma capsulatum
var. capsulatum and fungi, including Aspergillus, Candida, Zygomycetes,
Monosporium apiospermum and Torulopsis glabrata.

REFERENCES: Arch. Pathol. Lab Med - Vol. 102, July 1978.
Lillie & Fullmer, Histopathological Technic and Practical
Histochemistry, McGraw-Hill, Inc., 1976.

Eric C. Kellar
University of Pittsburgh Medical Center

Sent: 	Wednesday, April 12, 2000 2:59 PM
Subject: 	pneumocystis staining

We have been having trouble with our  stain off and on.  Our routine fungus
stain does not work so well on pmeumocystsis.  We purchased controls and
follow their procedure, still no getting consistent staining. Also, it is a
long stain.
Does anyone have a quick procedure that has been  "faithful"

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