Dear Jerry, We have been using the Lillie Twort for over 40 years. Below is 
a copy of the procedure.  If the copy doesn't come through well enough for 
you please let me know.

Histologically yours,


Barbara H. Stancel, HTL(ASCP)HT
USDA, FSIS, OPHS, Eastern Laboratory, Pathology
RRC, 950 College Station Road
Athens, Georgia  30604
phone: (706) 546-3556
fax: (706) 546-3589


Name:   Lillie Twort, West Modification
Purpose:  To stain Gram-positive and Gram-negative bacteria
Techniques:   Cut paraffin sections 4 to 5 microns.  Use control slides.
Solutions:
     Crystal Violet
        Crystal violet (C.I. #42555)	  5.0 gm
        95% Alcohol			200.0 ml
        When solution is dissolved, add 800.0 ml of 1% ammonium oxalate.  
Mix well, filter before use.

     Lugol's Iodine
        Iodine 				   4.0 gm
        Potassium iodide 		   8.0 gm
        Distilled water 		 400.0 ml
        Dissolve potassium iodide in 20 ml of distilled water; add iodine 
until dissolved; then add remaining water.

     2.0% Safranin 0
        Safranin 0 (C.I. #50240)	   6.0 gm
        Distilled water 		 300.0 ml

     Modified Twort Stain
        Neutral red (C.I. #50040)	   1.36 gm
        Fast green (C.I. #42053)	   0.04 gm
        Absolute alcohol 		 200.00 ml
        (amount of dye may vary due to dye content and intensity    
staining.
     Twort Stain - Working
        1 part stock:2 parts distilled water.
        Filter AFTER dilution.  Ready to use.

     4% Acetic Alcohol
        Glacial acetic acid 		  40.0 ml
        Absolute alcohol 		 960.0 ml


Procedure:

     1.  Deparaffinize and hydrate to distilled water.
     2.  Stain in crystal violet for 25-35 sec.
     3.  Tap water rinse.
     4.  Place in iodine for 25-30 sec.
     5.  Tap water rinse.
     6.  Dip in acetone until blue stops running from sections, 
approximately 10-30 sec.
     7.  Tap water rinse.
     8.  Stain in safranin 0 for 1-2 minutes.
     9.  Tap water rinse.
    10.  Stain in working Twort solution for 5 min.
    11.  Wash quickly in distilled water.
    12.  Decolorize in 4% acetic alcohol until red comes out and green is 
clear and definite.
    13.  Rinse quickly in 100% alcohol.
    14. Clear in xylene and coverslip with synthetic mounting medium.


Results:   Gram-positive bacteria .....................blue
               Gram-negative bacteria .....................red
               Cell nuclei ........................................red
               Cytoplasm, fibrin, collagen ...............pink

References:   Beltsville Laboratory, Unpublished manuscript by Claude West

Comments and Notes:

1.  Should tissues remain blue or brown after the acetone rinse, the problem 
is usually in the iodine.
    Change to fresh iodine.
2.  If G+ fails to stain, check the crystal violet and iodine.
3.  If G- fails to stain, check the Safranin O and Twort.
4.  If the green background fails to be bright and clear, make fresh Twort 
stain.
5.  If the fresh Twort stain is still faded and weak, check the fast green 
powder.  Fast green powder can weaken rapidly with age. You may need to 
double or triple the amount of fast green powder in the Twort to get a 
bright, clear green background.
6.  Fresh water should be used.  Some of us in the lab prefer to use running 
tap water in steps 3, 5, 7, and 9.
7.  Crystal violet, and Safranin 0 are fairly stable at room temperature and 
may be made in 300-1000 ml quantities and used as needed.  Twort solutions 
should be made fresh at least every 2 - 3 months.



Fellow histonetters,

I am looking for a stain to replace our current B and B to avoid the se of 
ether.  If you could help me out it would be appreciated.

Thank You,
Jerry Duncan HT(ASCP)








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