Kathy,
Try this:

 Sulphation - Toluidine Blue Technique For Fungi
PRINCIPLE:
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.

FIXATION:      10% buffered formalin

MICROTOMY:     5um paraffin sections

SOLUTIONS:

1.   Sulphation Solution:
Absolute Acetone 1 ml - precooled to 4oC.
Slowly add 1 ml concentrated sulphuric acid drop by
drop.  Allow to cool 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                          100ul

5.   Absolute Acetone.

CONTROLS: Fungi containing tissue.

PROCEDURE:

1.   Dewax 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.

REFERENCE:

Smith, D.J., Lowrey, T., (1986).  "A Modified
Sulfation-Toluidine Blue Technique for the
demonstration of fungi in tissue sections."  J.
Histotechn. 9(1):23-24.



--- Kathy Hill-Epperson  wrote: > Does
anyone have a rapid stain procedure for 
> pneumocystis carinii?  We are looking for a STAT
> procedure that could be done after hours or on 
> weekends (possibly performed by the pathologists)
> for bronchial cytology specimens for the rapid
> diagnosis
> of pneumocystis carinii.  Any help would be much 
> appreciated.  Thank you.
> 
> Kathy Hill-Epperson, HT
> Pathologists Assistant
> John C. Lincoln Hospital - North Mountain
> Phoenix, AZ
> 
>  

=====
Tony Henwood JP BAppSc GradDipSysAnaly CT(ASC)
Laboratory Manager
Histopathology Department
The Childrens Hospital at Westmead
Locked Bag 4001
WESTMEAD, 2145
AUSTRALIA

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