Re: Rapid stain for pneumocystis carinii


Try this:

 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.

FIXATION:      10% buffered formalin

MICROTOMY:     5um paraffin sections


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.


1.   Dewax and bring to absolute alcohol.

2.   Dry briefly, cover with sulphation reagent, 3

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

10.  Rinse in distilled water, 5 seconds.

11.  Blot, dehydrate rapidly, clear and mount.


     Fungi stained purple to red against a yellow


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
- It's My Yahoo! Get your own!

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