Lillie Twort Stain

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From:Barbara Stancel <>

<html><DIV id=cdiv> <P> <DIV id=cdiv> <P> <DIV id=cdiv> <P> <DIV id=cdiv> <P> <DIV id=cdiv> <P>Dear Histonetters,   I have had several responses to this stain.....unfortunately most of them have said the some of  procedure appears to have disappeared.   I hope it makes it through this time.          Always   Barbara</P> <P><BR>Hi Kari, </P> <P>We have been using this version of the Gram-Twort called the Lillie Twort for about 35 years. We probably stain about 20-30 slides per day using the Twort. Occasionally we have the Gram negative bacteria become stubborn to stain, so we have increased the staining time over the years to 2 minutes in the Safranin O from the original 30 seconds.  As for the fast green,  we have found the older the fast green powder, the weaker it becomes <STRONG>or</STRONG> the older we get, the more intense green we prefer to see! It could go either way, since 8 of our 9 pathology members are over 40!!  Below is a copy of the Twort Modification we use along with our tips for use.     Let me know if you try it or if you have any problems, please feel free to give me a call.  7am to 4:30 pm EST.     Best of Luck!</P> <P>Histologically yours,</P> <P>Barbara Stancel</P> <P>USDA, FSIS, Eastern lab</P> <P>Athens, Georgia </P> <P>phone: (706) 546-3556</P> <P><BR><BR><U><STRONG><EM>Lillie Twort, West Modification <BR><BR></EM></STRONG></U>Purpose: To stain Gram-positive and Gram-negative bacteria <BR>Techniques: Cut paraffin sections 4 to 5 microns. Use control slides. <BR>Solutions: </P> <P><U>Crystal Violet</U>    Crystal violet (C.I. #42555) 5.0 gm <BR>                         95% Alcohol                   200.0 ml <BR>                         When solution is dissolved, add 800.0 ml of 1%ammonium oxalate. Mix well.  Filter before placing in staining dish.  Stable in staining dish for up to 2 months. <BR><BR><U>Lugol's Iodine</U>               Iodine 4.0 gm <BR>                    Potassium iodide 8.0 gm <BR>        &nb <P><U>Twort Stain - Working</U> <BR>1 part stock:2 parts distilled water. Filter <U>AFTER</U> dilution. Ready to use. <BR><BR><U>4% Acetic Alcohol</U>       Glacial acetic acid 40.0 ml <BR>                                    Absolute alcohol 960.0 ml <BR><BR><BR>Procedure: <BR><BR>1. Deparaffinize and hydrate to distilled water. <BR>2. Stain in crystal violet for 30 sec. <BR>3. Tap water rinse. <BR>4. Place in iodine for 30 sec. <BR>5. Tap water rinse. <BR>6. Dip in acetone until blue stops running from sections, approximately 10-30 sec. or 8-15 dips.<BR>7. Tap water rinse. <BR>8. Stain in safranin 0 for 1-2 minutes. <BR>9. Tap water rinse. <BR>10. Stain in working Twort solution for 5 min. <BR>11. Wash quickly in distilled water. <BR>12. Decolorize in 4% acetic alcohol until red comes out and green is cle
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