[Histonet] Polka dots and RE: Water bath temperature[Scanned]

Kemlo Rogerson Kemlo.Rogerson <@t> elht.nhs.uk
Tue Feb 15 02:18:34 CST 2005

I am honoured to act as Gayle's secretary, despite her getting my name wrong

-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu] 
Sent: 14 February 2005 18:58
To: Kemlo Rogerson
Subject: RE: [Histonet] Polka dots and RE: Water bath temperature[Scanned]

Kemalo ,

I unsubscribed from Histonet this a.m. for travel. You can put my reply on 
Histonet if you wish.

If the waterbath is not the problem and all rehydration agents have been 
tested or changed with timing reevaluated for good paraffin removal AND the 
paraffin is stirred before embedding, maybe the culprit is the paraffin 
itself.  Age of paraffin lot or possibly defective paraffin during 
manufacturing.   Then manufacturer could be contacted and asked about this 

At 09:01 AM 2/14/2005, you wrote:
>But why does it happen only with the 'hotter' bath? Surely the wax would
>be removed from the sections floated out on the 'cooler' bath as well? You
>would assume that it is either the effect of the 'extra' heat or that there
>is something odd about that waterbath. I assume that the bath is clean and
>we are not dealing with wax melting in the 'hotter' bath and thereby
>contaminating the sections?
>-----Original Message-----
>From: Gayle Callis [mailto:gcallis <@t> montana.edu]
>Sent: 14 February 2005 15:43
>To: JOHN PHILLIPS; Histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] Polka dots and RE: Water bath temperature[Scanned]
>I agree with John on this polka dot problem.  Also, stirring your paraffin
>redistributes polymer and other additives.  This should be done just before
>embedding EACH day insure these polymers have not settled to the bottom
>into little blobs or solid polka dots that may not be totally removed by
>xylenes.  In addition to increasing time in xylene, you can also add one
>more container of xylene or xylene substitute and change the first absolute
>alcohol after xylene more often to prevent excessive carry over of paraffin
>A quick test for rehydration solvents. Take a pasteur pipette and sample
>each alcohol change after xylene.  Squirt this sample into water.  If it
>turns milky white, then you have carry over of xylene and paraffin into the
>alcohols, and need to rotate out all changes more frequently.  If this
>happens in the 95% just before 70% and water, you really have trouble with
>paraffin residues removal.
>At 02:42 AM 2/14/2005, you wrote:
> >The problem with staining may be due to unsatisfactory removal of the wax
> >prior to staining for H&E. I think you are looking to the wrong place for
> >the solution to your problem. Try extending your dewax time in xylene
> >before you hydrate your sections on the staining run. If the problem goes
> >away PERMANANTLY then you have your answer. Good luck.
> >
> >John,
> >Wrexham,Wales.U.K.
> >
> >-----Original Message-----
> >From: Rebecca Barnhart [mailto:RBARNHART <@t> summithealth.org]
> >Sent: 11 February 2005 19:24
> >To: histonet <@t> lists.utsouthwestern.edu
> >Subject: [Histonet] Water bath temperature
> >
> >
> >We were having a problem that the tissue would not take up the
> >hematoxlyin but would take up the eosin but only in some areas, so it
> >looked like polka dots.  We started one by one changing solution trying
> >to figure out where it was coming from.  We had all fresh solutions and
> >still had polka dots.  I tried the only other thing left to change, I
> >lowered the temperature of the water bath from 45 to 38 and this worked.
> >  We currently are using ParaPlast Xtra with a melting point of 52.  We
> >have all tried figure out why the temperature in the water bath will
> >make the difference.  Several times I have cut the same tissue at
> >different temperatures ranging from 38 - 45 and every time there are
> >polka dots at the higher temperatures.  Does anyone have any idea why
> >this would be?
> >
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>Gayle Callis
>Research Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University - Bozeman
>PO Box 173610
>Bozeman MT 59717-3610
>406 994-6367 (lab with voice mail)
>406 994-4303 (FAX)
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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