Hi Sheila,
Here's the procedure. Our pathologist prefer it over
any other G(-) G(+) stain.
Hope it works out for you.
Have a great day.
Kathy 



STAIN:      Lillie Twort, West Modification 
            
PURPOSE:    To stain Gram-positive and Gram-negative  
bacteria
          
TECHNIQUE:  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 complete, 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.  Add remaining distilled
water.
                               
     2% Safranin 0
Safranin 0 (C.I. #50240)....................   6.0 gm 
Distilled water ............................. 300.0 ml
                               
     Modified Twort Stain - Stock
Stock:  0.2% Neutral red (C.I. #50040) .....  0.36 gm 

        0.2% Fast green (C.I. #42053) .......  0.04 gm
        Ethyl Alcohol (100%) ................200.00 ml
        (STAIN MEASUREMENTS MAY NEED TO BE ADJUSTED)  

   (We use 0.6gm neutral red and 0.2gm fast green)    
Mix as stock.  Good for 1-3 months.
                               
     Twort Stain - Working
        1 part stock: 2 parts distilled water 
        Filter AFTER dilution.  Ready to use.  
            
     4% Acetic Alcohol
Glacial acetic acid ..........................  4.0 ml

Absolute alcohol ............................. 96.0 ml

PROCEDURE:

     1.  Deparaffinize and hydrate to distilled water.

     2.  Stain in crystal violet for 30 sec.

     3.  Tap water rinse.

     4.  Place in iodine for 30 sec.

     5.  Tap water rinse.

     6.  Dip in acetone until blue discontinues   
running from sections for 10-20 sec.

     7.  Tap water rinse.

     8.  Stain in safranin 0 for 30 sec. to 5 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.(~5 DIPS)

    13.  Rinse quickly in 100% alcohol.(~5 DIPS)

    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
            
 
           
REFERENCE:  Cullin, CFA, Handbook of Histopathological
and Histochemical Techniques,
            3rd ed, 1957, pg 394

            Beltsville Laboratory, modified by Claude
West but never published


            
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 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. Too much green will
weaken the red bacteria staining.
    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 months.
    8.  The proportions of dyes given are approximate.
The optimum formula will depend on the dye content of
the powdered dye. Total dye concentration is not
critical.
            






--- Sheila Poellein <sheila_poellein@deaconess.com>
wrote:
> Kathy,
> 
> You have peaked our interest in the Twort stain. 
> Could you give us the
> whole procedure.  We have not been in the practice
> of buying kits and prefer
> to make up our own solutions.          thanks,
> sheila
> 
> 
> -----Original Message-----
> From: Kathy Wortham [mailto:kjwortham@yahoo.com]
> Sent: Thursday, February 03, 2000 9:25 AM
> To: CEJones0476@aol.com
> Cc: HistoNet Server
> Subject: Re: Twort Stain
> 
> 
> 
> --- CEJones0476@aol.com wrote:
> > Does anyone use the Twort stain by Newcomer
> Supply?
> > If you haven't heard of 
> > this procedure it is a variation of the Brown and
> > Hopps that eliminates the 
> > use of picric acid acetone. The lab I work for
> > switched to this procedure for 
> > various reasons, but we are having problems with
> > consistency and the 
> > pathologists are not satisfied. Although the gram
> > negative and gram positive 
> > bacteria are staining appropriately, the
> background
> > has never been light 
> > green as the results state. If you have any
> > suggestions or other methods that 
> > could be tried it would be really appreciated.
> > Thanks in advance,
> > Charnell Jones, HTL(ASCP)
> > Compunet Clinical Laboratories
> > Dayton, OH
> > 
> >
>  Hi Charnell,
>  We make up our own Twort for our Lillie-Twort
> modified gram stain.
> We have increased the strength of the stain from the
> original procedure(Pathologists preference) so you
> may
> need to adjust.
> 
> Modified Twort stain--Stock                original
>  Neutral red (C.I. #50040)   0.6gm         (0.36gm)
>  Fast green  (C.I. #42053)   0.2gm         (0.04gm)
>     Mix with 200ml 100% ETOH
> Procedure says stock solution is good for 1-3
> months.
> I don't agree, I make up fresh each time I need to
> make more working solution (once a month)
> 
> Twort working solution
>  1 part stock: 2 parts distilled water
> Filter AFTER dilution. Ready to use.
> 
> 
> =====
> Kathy Wortham
> USDA,Food Safety Inspection Service
> 950 College Station Road
> Athens, Ga 30605
> (706)546-3556
> kjwortham@yahoo.com
> __________________________________________________
> Do You Yahoo!?
> Talk to your friends online with Yahoo! Messenger.
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> 


=====
Kathy Wortham
USDA,Food Safety Inspection Service
950 College Station Road
Athens, Ga 30605
(706)546-3556
kjwortham@yahoo.com
__________________________________________________
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