Tony Henwood AnthonyH <@t> chw.edu.au
Tue Dec 16 16:11:42 CST 2008

I would suggest the sulphated toluidine blue method as described below. It is quick and demonstrates PC very well (though not quite as good as well-done (or should I say under-done) methenamine silver.

Following sulphation, fungi stain metachromatically with toluidine blue. Certain hydroxyl groups are esterified by sulphuric acid to form ester sulphate groups that stain metachromatically with toluidine blue (Smith & Lowrey 1986, J. Histotechn. 9(1):23-24. ).

Fixation:      10% buffered formalin

Microtomy:     5μm paraffin sections


1.   Sulphation Solution:

Absolute Acetone 1 ml - pre-cooled to 4oC.
Slowly add 1 ml concentrated sulphuric acid drop by drop.  
Allow cooling before use.

2.   3% Acetic Acid.

3.   Toluidine Blue O Solution:

     Toluidine Blue O (CI 52040)	0.01g
     3% Acetic Acid			100ml

4.   Metanil Yellow Counterstain:

     Metanil yellow (CI 13065)		0.1g
     Distilled Water			100ml
     Glacial Acetic Acid		0.1ml

5.   Absolute Acetone.

Controls: Fungi containing tissue.


1.	Deparaffinise and bring to absolute alcohol.
2.	Dry briefly, cover with sulphation reagent, 3 minutes.
3.	Briefly wash in running water.
4.	Place in 3% acetic acid, 1 minute.
5.	Stain in Toluidine Blue O solution, 3 minutes.
6.	Rinse in 3% acetic acid, 1 minute.
7.	Differentiate in absolute acetone, 5 seconds.
8.	Rinse in distilled water, 5 seconds.
9.	Counterstain in Metanil Yellow solution, 6 seconds.
10.	Rinse in distilled water, 5 seconds.
11.	Blot, dehydrate rapidly, clear and mount.

     Fungi stained purple to red against a yellow background.


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
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 TIMOTHY MALLOY
Sent: Friday, 12 December 2008 8:52 PM

We currently use a stain called grocotts silver methenimine solution for staining pnuemocystis. We would like to use a hot plate for the procedure instead of a unvented microwave. Does anybody have any formula's the would like to share with the hot plate? 

Timothy G. Malloy, HT ( ASCP ) A.A.S.
tmalloy77 <@t> hotmail.com  
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