[Histonet] RE: Churukian Silver stain for pneumocystis and fungus

[Terri Braud]

For those who have written off list for the Churukian stain procedure, I've included it here.  It is also sold in the USA in kit form by Polyscientific (no plug here, just stating)
PRINCIPLE: 
Fungi and Pneumocystis organisms are oxidized using periodic acid and then impregnated with ammoniacal silver solution.  The silver is then toned from brown to black with gold chloride.  Any unreduced silver is removed with sodium thiosulfate, leaving organisms stained black against a green counterstained background.

SPECIMENS:
Formalin fixed, paraffin embedded tissue cut at 4-5 microns or 95% alcohol fixed smears or monolayer cell preparations.

CONTROL: 
Any tissue known to contain a fungus or Pneumocystis organisms

REAGENTS:
1.	Periodic acid 1% Aqueous
2.	Ammoniacal GMS solution (Silver)
3.	Gold Chloride 0.5% Aqueous
4.	Sodium Thiosulfate 5% Aqueous
5.	Fast Green Working Solution
All working solutions are purchased from PolyScientific Kit #K101.
Individual components may be purchased separately as they are exhausted.

PROCEDURE: 
1.	Deparaffinize and hydrate to water
2.	Using a plastic Coplin jar with the lid resting upside down on top, microwave 50.ml of 1% Periodic Acid at for 30 seconds at power 5.
3.	Place slides in the hot solution, covered, for 5 minutes.
4.	Wash in 5 changes of DI Water.
5.	Using a plastic Coplin jar with the lid resting upside down on top, microwave 40.ml of Ammoniacal Silver Solution at for 30 seconds on HIGH power.
6.	Using a plastic disposable pipet, vigorously stir the solution for a 3 seconds.
7.	Place the slides in the hot silver solution
8.	Replace the lid upside down on top of the Coplin jar and microwave for an additional 20 seconds on power 5.
9.	For fungus, let the slides stand for exactly 1 minute following the last microwave step.  For pneumocystis, let the slides sit for exactly 2 minutes following the last microwave step/
10.	Wash in 5 changes of DI Water
11.	With slides lying flat, dispense enough Gold Chloride to cover the slide for 1 minute.  Rinse in DI Water.
12.	With slides lying flat, dispense enough Sodium Thiosulfate solution to cover the slide for 2 minutes.  Rinse in DI Water.
13.	With slides lying flat, dispense enough Light Green to cover the slide for 1 minute.  Rinse in DI Water.
14.	Dehydrate through graded alcohols, clear in 2 changes of Xylene and mount with a compatible mounting media.

NOTE:  Microwaves differ in power output.  This procedure is based on our current microwave.  If the power changes or the microwave is changed, then the timing of this procedure may have to be adjusted and the stain revalidated.
 
RESULTS
	Fungi – Black
	Pneumocystis – Black to blue black
	Mucin – Pale Gray
	Background - Green

REFERENCE:

Kit Procedure Insert, Rapid GMS for Fungus and Pneumocystis Carinii, Cat# k101, Poly Scientific R&D Corporation, Bay Shore NY 11706

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Holy Redeemer Hospital Laboratory
1648 Huntingdon Pike
Meadowbrook, PA 19046
Ph: 215-938-3676
Fax: 215-938-3874


**********************************

---------------------------------------------------------------------------------



CONFIDENTIALITY NOTICE:

This E-Mail is intended only for the use of the individual or entity to which
it was sent. It may contain information that is privileged and/or confidential,
and the use or disclosure of such information may also be restricted under applicable
federal and state law. If you received this communication in error, please do not
distribute any part of it or retain any copies, and delete the original E-Mail.
Please notify the sender of any error by E-Mail.

Thank you for your cooperation.