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

[Tony Henwood (SCHN)]

Hi Terri,

I agree, Churukian's Ammoniacal silver method is very good.

I assume you are referring to the method given in Churukian, C. J., & Schenk, E. A. (1988). Staining Pneumocystis carinii and fungi in unfixed specimens with ammoniacal silver using a microwave oven. Journal of Histotechnology, 11(1), 19-21.

In their discussion they note:
"Paraffin sections can also be stained with this method with only minor changes. In place of 5% periodic acid use 5% chromic acid"
Though the reason given is that paraffin sections tend to detach from slides.

Charles repeats this recommendation in his later article: Churukian, C. J. (1996). Consistent silver methods for demonstrating basement membranes, reticulum, and fungi. Journal of histotechnology, 19(3), 211-217.

I follow Charles recommendations for paraffin sections (ie chromic acid).


Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist, the Children’s Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Terri Braud
Sent: Friday, 6 June 2014 11:00 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: Churukian Silver stain for pneumocystis and fungus

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


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