[Histonet] fungal stain for frozen sections

Tony Henwood (SCHN) tony.henwood <@t> health.nsw.gov.au
Thu May 16 19:31:05 CDT 2013

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


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 Water			100ml
     Glacial Acetic Acid		0.1ml

5.   Absolute Acetone.

Controls: Fungi containing tissue.


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.

     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.0000000025.

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-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of wrglo5 <@t> aol.com
Sent: Friday, 17 May 2013 2:01 AM
To: histonet <@t> 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 H&E 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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