The Sulphation Toluidine Blue stain should work a treat and very quick to do:
Sulphation - Toluidine Blue Technique for Fungi
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,
Henwood et al 2013).
Fixation: 10% buffered formalin
Microtomy: 5μm paraffin sections
Solutions:
1. Sulphation Reagent:
For the sulphation reagent, 45 ml of glacial acetic acid is poured into a
Coplin jar which has been placed into a plastic tub filled with cool tap water
(not below 10°C). A 15-ml portion of concentrated sulfuric acid is slowly added
with a glass pipette, being careful not to produce splashing. The solution is
gently mixed with a glass rod. The Coplin jar is then sealed with petroleum
jelly. The sulphation reagent is kept at room temperature and can be used for 1
week (Gosey et al 1985).
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 Water100ml
Glacial Acetic Acid0.1ml
5. Absolute Acetone.
Controls: Fungi containing tissue.
Procedure:
1. Cut frozen sections, air dry, defat in methanol 1 minute, rinse in water
2. Dry briefly, cover with sulphation reagent, 10 minutes.
3. Wash in running water, 5 minutes
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, dry and mount.
Results:
Fungi stained purple to red against a yellow background.
Smith DJ, Lowrey T, (1986) A Modified Sulfation-Toluidine Blue Technique for
the demonstration of fungi in tissue sections J Histotechnol. 9(1):23-24.
Gosey LL, Howard RM, Witebsky FG, Ognibene FP et al (1985) Advantages of a
Modified Toluidine Blue 0 Stain and Bronchoalveolar Lavage for the Diagnosis of
Pneumocystis carinii Pneumonia J Clin Microbiol 22(5):803-807.
Henwood, A. F., Prasad, L., Bourke, V. M. (2013). The application of heated
detergent dewaxing and rehydration to techniques for the demonstration of
fungi: a comparison to routine xylene-alcohol dewaxing J Histotechnol, early
online DOI 10.1179/2046023613Y.25.
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
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 wrg...@aol.com
Sent: Friday, 17 May 2013 2:01 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] fungal stain for frozen sections
Hi, I work in a Surgical Pathology frozen section lab outside the OR rooms.
Normally we only perform an HE on the frozens. Now we are looking for a fast
and inexpensive stain for fungus on our frozen sections. Fast is the operative
word because we have to report diagnoses back to ORs in a 20 minute timeperiod.
Any ideas to help? Thanks!
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