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
Solutions:
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.
Procedure:
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.
Results:
Fungi stained purple to red against a yellow background.
Regards
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-boun...@lists.utsouthwestern.edu
[mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of TIMOTHY MALLOY
Sent: Friday, 12 December 2008 8:52 PM
To: HISTONET ...; TIM MALLOY
Subject: [Histonet] SPECIAL STAIN PNUEMOCYSTSIS
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.
tmallo...@hotmail.com
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