Re: Urea
| From: | Beckers <msadk@NYCAP.rr.com> |
John,
Wish I knew who started this urea business with staining-but I don't. You
responded to me in the past. I don't fully understand the process but the
results are gorgeous. I am probably using 1 to 2 grams of urea crytals to
1 gram toluidine blue powder dissolved in 300 ml of 200 proof ethanol and
700ml distilled water. Shelf life is indefinite-I use the 1000 ml solution
up within a 2 or 3 months. I just started adding urea to the stain and it
is so much better-contrast and cancer differentiation esp basal cell
carcinoma which shows mucinous stroma about bundles of tumor. I think the
low concentration of urea is the key if what you said is true. Perhaps
another source will add there two cents worth. There's probably an article
somewhere on the powers of urea and staining. Who know?! Thanks
Sue Becker, HTL
Albany, NY12205
USA
----- Original Message -----
From: "HistoNet Server" <histonet@pathology.swmed.edu>
To: "HistoNet Server" <histonet@pathology.swmed.edu>
Sent: Tuesday, October 03, 2000 1:01 AM
Subject: Daily Digest
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 00:15:36 -0500
> From: "J. A. Kiernan" <jkiernan@julian.uwo.ca>
> Subject: Re: Urea and Tol Blue (or other dyes?); also etc.
>
>
> I missed the original posting on this. does anyone have a reference
> for this staining method using urea + toluidine blue, or to other
> methods in which urea is mixed with a dye? It's interesting
> because urea (in quite high concentration) has been used in
> experiments on mechanisms of staining. The urea molecule can
> form up to 3 hydrogen bonds with suitable N, O or H atoms in
> a dye, a tissue, or water. It therefore competitively inhibits
> staining due to weak, non-ionic forces (which include hydrogen
> bonding and van der Waals forces) but it doesn't prevent staining
> due to attraction of oppositely charged ions. The staining of
> nuclei and mucus by a cationic thiazine dye like toluidine blue
> is mainly ionic, but non-ionic forces are involved in joining
> dye molecules to one another to produce the metachromasia (red
> colour) seen in some mucus, cartilage, mast cells etc.
>
> When staining is not due principally to simple ionic attraction
> (as in the case of collagen in Van Gieson's and similar methods, or
> pure nuclear stains such alum-haematoxylin, etc) a high concentration
> of urea prevents the staining. It seems entirely reasonable to use
> lower concentrations of urea to suppress "background" or even
> certain types of specific coloration by dyes, It would be
> interesting to know who introduced this as a practical procedure,
> in conjuction with toluidine blue or any other dye-based staining
> method.
> -----------------------------------------------
> > On Fri, 29 Sep 2000, Beckers wrote:
> >
> > > A big thank you to anyone who helped me with the question on using
urea
> > > crystals and 1% toluidine blue for demonstrating basal cell carcinoma
> > -----------------------------------------------
> An irrelevant afterthought: the only other staining method I can
> think of that has urea as a major ingredient is Ungewitter's silver
> method for axons in paraffin sections (Stain Technol. 26: 73-76,1951).
> This 2-hour method is pretty good. I used it a lot in the late 1960s
> (when young and in a hurry) but abandoned it soon after I turned 30
> and got a real job because Holmes' method (takes at least 12 hours,
> sometimes 48) was more reliable and needed much less silver nitrate.
> The cost of AgNO3 went through the roof in about 1974 because of
> some greedy American millionaire buying up all the silver bullion.
> The world price of silver soon settled again to a sane level, but
> the price of silver nitrate remains exorbitant to this day. Growl!
>
> The chemistry of Ungewitter's method was quite thoroughly
> studied by British investigators (Rowle, Brain, Gough) in the
> early 1970s. It devolves around the equilibrium between urea and
> ammonium cyanate and the low (but not too low) solubility of silver
> cyanate in water. If anyone has read this far and would like to
> know more, I'll be happy to provide references and discuss them,
> either privately or (preferably) in the open forum of HistoNet.
>
> John A. Kiernan,
> Department of Anatomy & Cell Biology,
> The University of Western Ontario,
> LONDON, Canada N6A 5C1
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 01:00:23 -0500
> From: "J. A. Kiernan" <jkiernan@julian.uwo.ca>
> Subject: Re: Vibratome (How thin?)
>
> On Fri, 29 Sep 2000, Joan Yonchek wrote:
>
> > I am interested in hearing from anyone versed in the use of a Vibratome.
> > I have been asked to obtain 10-20 micron thick sections of small pieces
(1mm
> > cubes) of formalin fixed skin.
>
> Ask the person who requested these thin sections to provide the
> necessary technical instruction (from her/his practical experience)
> or to provide references to published accounts of how it can be
> done.
>
> I've had a vibratome in my lab for about 10 years and it has been used
> by me (not enough to claim high-level skill) and by others (sometimes
> daily for weeks on end, therefore skilled). It is possible to cut
> sections of unfixed brain at 100-200 micrometres, and of fixed brain at
> 50 micrometres. I cannot believe that it would be possible to collect
> more than occasional sections with this instrument set at a nominal
> thickness of 10 or 20 micrometres. If you succeed in collecting a
> section, it is probably much fatter or deeper than its nominal thickness.
> The bumph that comes with a Vibratome tells you that the section
> thickness setting is not a true indicator of the actual number of
> micrometres. The mechanical advance of the block is not necessarily
> the thickness of the next section, any more than it is with other
> kinds of mocrotome.
>
> If you haven't used a vibrating microtome before, you need to be warned
> that this is an agonizingly slow way to cut sections. You have control
> over the speed of advance of the razor blade and the amplitude of its
> saw-like oscillation. (The frequency is that of the power supply: e.g.
> 60 Hz in N. America or 50 Hz in Britain.) A general rule of thumb for
> getting the best sections is to have a wide amplitude and a slow speed
> of advance. This can mean 30 seconds for one section.
>
> For FIXED tissue, a vibrating microtome is the best instrument to use
> if you must obtain thickish slices without freezing or embedding. Such
> slices can be used for a few specialized staining methods, including
> Golgi methods for the CNS, and they are useful for finding small areas
> that can be cut out and processed for eventual transmission electron
> microscopy. The vibrating microtome is the only instrument that will
> cut sections of UNFIXED objects, allowing their study by organ culture
> and other methods. Many cells (even adult neurons) survive and do their
> stuff in a 100 micrometre (0.1 mm) slice for several days, if lovingly
> looked after.
>
> If it's really possible to cut 10-20 micrometre sections of fixed
> objects with a Vibratome, some HistoNetter reading Joan's question
> or my answer will surely tell us how it can be done.
>
> John A. Kiernan,
> Department of Anatomy & Cell Biology,
> The University of Western Ontario,
> LONDON, Canada N6A 5C1
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 01:00:39 -0500
> From: "J. A. Kiernan" <jkiernan@julian.uwo.ca>
> Subject: Re: Urea and Tol Blue (or other?) stain
>
>
> I missed the original posting on this. does anyone have a reference
> for this staining method using urea + toluidine blue, or to other
> methods in which urea is mixed with a dye? It's interesting
> because urea (in quite high concentration) has been used in
> experiments on mechanisms of staining. The urea molecule can
> form up to 3 hydrogen bonds with suitable N, O or H atoms in
> a dye, a tissue, or water. It therefore competitively inhibits
> staining due to weak, non-ionic forces (which include hydrogen
> bonding and van der Waals forces) but it doesn't prevent staining
> due to attraction of oppositely charged ions. The staining of
> nuclei and mucus by a cationic thiazine dye like toluidine blue
> is mainly ionic, but non-ionic forces are involved in joining
> dye molecules to one another to produce the metachromasia (red
> colour) seen in some mucus, cartilage, mast cells etc.
>
> When staining is not due principally to simple ionic attraction
> (as in the case of collagen in Van Gieson's and similar methods, or
> pure nuclear stains such alum-haematoxylin, etc) a high concentration
> of urea prevents the staining. It's entirely reasonable to use lower
> concentrations of urea to reduce "background" coloration by dyes.
> It would be interesting to know who introduced this as a practical
> procedure, in conjuction with toluidine blue or any other
> dye-based staining method.
> - -----------------------------------------------
> On Fri, 29 Sep 2000, Beckers wrote:
>
> > A big thank you to anyone who helped me with the question on using urea
> > crystals and 1% toluidine blue for demonstrating basal cell carcinoma
> - -----------------------------------------------
> As an afterthought, the only other staining method I can think of
> that has urea as a major ingredient is Ungewitter's silver method
> for axons in paraffin sections (Stain Technol. 26: 73-76, 1951).
> This 2-hour method is very good. I used it a lot in the late 1960s
> (when young and in a hurry) but abandoned it soon after I turned 30
> (and got a real job) because Holmes' method (takes at least 12 hours,
> sometimes 48) was more reliable and needed much less silver nitrate.
> The cost of AgNO3 went through the roof in about 1974 because of
> some greedy American millionaire buying up all the silver bullion.
> The world price of silver soon settled again to a sane level, but
> the price of silver nitrate remains exorbitant to this day.
>
> The chemistry of Ungewitter's method was quite thoroughly
> studied by British investigators (Rowle, Brain, Gough) in the
> early 1970s. It devolves around the equilibrium between urea and
> ammonium cyanate and the low (but not too low) solubility of silver
> cyanate in water. If anyone has read this far and would like to
> know more, I'll be happy to provide references and discuss them,
> either privately or (preferably) in the open forum of HistoNet.
>
> John A. Kiernan,
> Department of Anatomy & Cell Biology,
> The University of Western Ontario,
> LONDON, Canada N6A 5C1
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 08:00:31 -0500
> From: Sinead Daly <Sinead.Daly@amnch.ie>
> Subject: unsubcribe
>
>
> - --
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 10:00:35 -0500
> From: "Dr. Ian Montgomery." <ian.montgomery@bio.gla.ac.uk>
> Subject: 2 questions
>
>
> Whipfs polychrome, anyone using the technique or, where can I
find
> it.
> NO synthase, supplier of a good antibody.
> Ian.
>
> Dr. Ian Montgomery,
> West Medical Building,
> University of Glasgow,
> Glasgow,
> G12 8QQ.
> Tel: 0141 339 8855. Extn:6602.
> Fax: 0141 330 2923
> e-mail: ian.montgomery@bio.gla.ac.uk
>
>
> ******************* NOTE *******************
> There may be important message content
> contained in the following MIME Information.
> ********************************************
>
>
> - ------------------ MIME Information follows ------------------
>
> - --=====================_31230498==_.ALT
> Content-Type: text/plain; charset="us-ascii"; format=flowed
>
> <<<<<< See above "Message Body" >>>>>>
>
> - --=====================_31230498==_.ALT
> Content-Type: text/html; charset="us-ascii"
>
> <html>
> <x-tab> </x-tab>Whipfs
> polychrome, anyone using the technique or, where can I find it. <br>
> <x-tab> </x-tab>NO
> synthase, supplier of a good antibody.<br>
> Ian.<br>
> <x-sigsep><p></x-sigsep>
> <font color="#0000FF">Dr. Ian Montgomery,<br>
> West Medical Building,<br>
> University of Glasgow,<br>
> Glasgow,<br>
> G12 8QQ.<br>
> Tel: 0141 339 8855. Extn:6602.<br>
> Fax: 0141 330 2923<br>
> e-mail: ian.montgomery@bio.gla.ac.uk</font></html>
>
> - --=====================_31230498==_.ALT--
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 10:00:52 -0500
> From: "Barry, Lilith" <Lilith.Barry@nrc.ca>
> Subject: RE: Vibratome
>
> From my experience it would be next to impossible to cut so thin
consistent
> sections with vibrotome.
> What I would recommend is to cryoprotect your tissue, embed it in O.C.T.
> compound and cut it with cryostat.
> I also think that 10-20 micron sections would be little too delicate for
> free floating work.
>
> Lilith
> - - - - - - - - - - - - - - - - - - - - -
>
> Lilith Ohannessian-Barry
> National Research Council
> Institute of Biological Sciences
> CANADA
> Tel;613-993-6460
> Fax;613-941-4475
> e-mail; lilith.barry@nrc.ca
>
>
>
>
>
>
>
>
>
> - -----Original Message-----
> From: Joan Yonchek [mailto:Jyonchek@rtitechnology.com]
> Sent: Friday, September 29, 2000 5:17 PM
> To: 'histonet@pathology.swmed.edu'
> Subject: Vibratome
>
>
>
> I am interested in hearing from anyone versed in the use of a Vibratome.
> I have been asked to obtain 10-20 micron thick sections of small pieces
(1mm
> cubes) of formalin fixed skin. Specifically I would like to know the
speed,
> amplitude and blade angle you would recommend as well as the method for
> embedding the tissue in agarose. Also, do you use a sapphire knife, a
> single edge injector type razor blade or a double edge type razor blade?
Is
> it necessary to remove the agarose from the section before staining? What
> type of
> slides are best? Do you prefer staining the free-floating section and
then
> adhering it to the slide or dry the section on the slide before staining?
>
> Other than that I got it covered!
>
> Thanks
> Joan
> RTI
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 10:01:07 -0500
> From: Gayle Callis <uvsgc@msu.oscs.montana.edu>
> Subject: Great reply, thanks B-5 substitutes
>
> Thanks, I appreciated this reply.
>
> >Date: Fri, 29 Sep 2000 19:56:54 -0400 (EDT)
> >From: RSRICHMOND@aol.com
> >Subject: Re: Entering the mercury disposal fray and what you may
encounter
> >To: HistoNet@pathology.swmed.edu
> >
> >Gayle Callis notes:
> >
> ><< True, B5 is excellent, but pathologists CAN learn to read tissues
fixed
> >with a mercury fixative substitute, since some complain about cells not
> >looking good. >>
> >
> >It's not the pathologist that loses, it's the patient. If the tissue is
> >inadequately prepared, then the diagnosis of lymphoma is compromised, and
> the
> >patient has to undergo another biopsy.
> >
> >Actually, the B-5 "substitute" we need is overnight fixation. The major
> >reason we use B-5 is that it's fast, and permits same-day processing of
> >tissue for lymphoma diagnosis. Fix overnight, and neutral buffered
formalin
> >usually does the job quite adequately. Since the full diagnosis of a
> lymphoma
> >takes several days and treatment is rarely urgent, I don't think that
this
> >one day delay compromises either patient care or Managed Care.
> >
> >About thioacetamide: it's now considered to be a bad enough carcinogen
> that I
> >don't think we're allowed to use it as a mercury precipitant any longer.
> Will
> >someone respond to my previous comment about sodium sulfide?
> >
> >Bob Richmond
> >Samurai Pathologist
> >Knoxville TN
> >
> >
> >
> Gayle Callis
> Veterinary Molecular Biology
> Montana State University
> Bozeman MT 59717-3610
> 406 994-4705
> 406 994-4303
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 10:01:22 -0500
> From: "Vinnie Della Speranza" <dellav@musc.edu>
> Subject: Freezing tissue
>
> I would welcome your opinions and feedback.
>
> What is the optimal method of freezing tissues with liquid nitrogen? with
or
> without isopentane and why?
>
> thanks,
> Vinnie
>
>
>
> Vinnie Della Speranza
> Manager for Anatomic Pathology Services
> Medical University of South Carolina
> 165 Ashley Avenue
> Suite 309
> Charleston, SC 29425
> ph: (843) 792-6353
> fax: (843) 792-8974
> email: Dellav@musc.edu
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 11:22:53 -0500
> From: "Steven E. Slap" <siksik@vgernet.net>
> Subject: job opportunities
>
> Hi Histonetters!
>
> At long last <sigh>, all job postings are finally current on "The Jobs in
> Histology Bulletin Board" (http://www.histology.to/jobs.html). If you
have
> posted a job and it has been filled, please e-mail me, and I will remove
> it. There have been many new jobs posted in September, so, if you are
> looking for a job and haven't visited the site lately, come take a look.
>
> Best regards,
> Steven Slap
> co-webmaster
>
> *********************************************
> <#161#^#248#)~~~
> http://www.geocities.com/RainForest/2966
> Radio Free Siksik, Nature's Own Broadband
> send an email to siksik-subscribe@topica.com
> ~~~(#248#^#161#>
> *********************************************
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 11:23:14 -0500
> From: "Jennifer Englin" <JLE@rice.willmar.mn.us>
> Subject: Tetrazolium
>
> Hello histonetters,
>
> One of our pathologists would like to stain sections of heart muscle fo
MI.
> Does anyone have a recipe they would share for a tetrazolium stain?
>
> I have one but it is pretty unclear as to the procudure. Any help would
be
> appreciated.
>
> Thanks in advance,
> Jennifer Englin
> Rice memorial Hospital
> Willmar MN
> jle@rice.willmar.us
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 11:23:57 -0500
> From: "Steven E. Slap" <siksik@vgernet.net>
> Subject: Vibratome
>
> Hi Histonetters
>
> Joan Yonchek asked about obtaining 10-20 micron sections of formalin fixed
> sections of skin on a Vibratome. It takes skill and patience to
> consistently get sections thinner than 30 microns. I would recommend the
> slowest possible speed and the highest possible amplitude. A sapphire
> blade would certainly help.
>
> Best regards,
> Steven Slap
>
> *********************************************
> <#161#^#248#)~~~
> http://www.geocities.com/RainForest/2966
> Radio Free Siksik, Nature's Own Broadband
> send an email to siksik-subscribe@topica.com
> ~~~(#248#^#161#>
> *********************************************
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 11:24:25 -0500
> From: E Sharon Shields <eshields@bhset.org>
> Subject: HPV typing by ISH
>
> Any experienced labs doing HPV typing by ISH willing to share information
to
> help us get started . What probes do you use (Vendor),
> have you been able to do them on thin preps as well as tissue samples,
what
> CPT code so you use and how is reimbursement?
>
> Thank you,
> E.S. Shields
> Baptist Hospital of E TN
>
>
>
>
>
>
>
>
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 11:25:02 -0500
> From: Shirley Powell <powell_sa@Mercer.EDU>
> Subject: Re: 2 questions
>
>
> Hi Ian,
> I have never heard of Whipfs polychrome, but I modified a procedure
> many, many years ago when we were unable to purchase polychrome stain
> from the supplier we were using. If you are interested my modification,
> contact me via e-mail at powell_sa@mercer.edu for the procedure.
> Shirley
>
> "Dr. Ian Montgomery." wrote:
>
> > Whipfs polychrome, anyone using the technique or, where can I find it.
> >
> > NO synthase, supplier of a good antibody.
> > Ian.
> >
> > Dr. Ian Montgomery,
> > West Medical Building,
> > University of Glasgow,
> > Glasgow,
> > G12 8QQ.
> > Tel: 0141 339 8855. Extn:6602.
> > Fax: 0141 330 2923
> > e-mail: ian.montgomery@bio.gla.ac.uk
>
>
>
> ******************* NOTE *******************
> There may be important message content
> contained in the following MIME Information.
> ********************************************
>
>
> - ------------------ MIME Information follows ------------------
>
>
> - --------------5234DC541B037FAEE692978F
> Content-Type: text/plain; charset=us-ascii
> Content-Transfer-Encoding: 7bit
>
> <<<<<< See above "Message Body" >>>>>>
>
> - --------------5234DC541B037FAEE692978F
> Content-Type: text/html; charset=us-ascii
> Content-Transfer-Encoding: 7bit
>
> <!doctype html public "-//w3c//dtd html 4.0 transitional//en">
> <html>
> Hi Ian,
> <br>I have never heard of Whipfs polychrome, but I modified a procedure
> many, many years ago when we were unable to purchase polychrome stain from
> the supplier we were using. If you are interested my modification,
> contact me via e-mail at powell_sa@mercer.edu for the procedure.
> <br>Shirley
> <p>"Dr. Ian Montgomery." wrote:
> <blockquote TYPE=CITE><x-tab></x-tab>Whipfs polychrome, anyone using the
> technique or, where can I find it.
> <br><x-tab></x-tab>NO synthase, supplier of a good antibody.
> <br>Ian.
> <br><x-sigsep>
> <p></x-sigsep><font color="#0000FF">Dr. Ian Montgomery,</font>
> <br><font color="#0000FF">West Medical Building,</font>
> <br><font color="#0000FF">University of Glasgow,</font>
> <br><font color="#0000FF">Glasgow,</font>
> <br><font color="#0000FF">G12 8QQ.</font>
> <br><font color="#0000FF">Tel: 0141 339 8855. Extn:6602.</font>
> <br><font color="#0000FF">Fax: 0141 330 2923</font>
> <br><font color="#0000FF">e-mail:
> ian.montgomery@bio.gla.ac.uk</font></blockquote>
> </html>
>
> - --------------5234DC541B037FAEE692978F--
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 11:25:23 -0500
> From: Gayle Callis <uvsgc@msu.oscs.montana.edu>
> Subject: decalcification
>
> Gayle Callis mentions "For articular cartilage in osteoarthritic knees,
> safranin O/Fast Green is commonly used, and toludine blue may also be
> useful." I think I should have qualified as "more commonly used in
> orthopedic pathology clinical situations ". I have seen them used in both
> clinical and research, clinical generally being larger institutions, often
> with orthopedic pathologists in attendance. I doubt these stains are
> routinely used in the majority of clinical labs. Neither stain is
> difficult to do, intrepretation must be something that has to be learned.
>
> Would be interested to see how many people in clinical lab use these
> stains??? And is your clinical lab small or a teaching hospital, large,
> etc??
>
> Although the majority of large bone speciemens I dealt with in my clinical
> experience were handled much as you described, hand saws, etc., Faxitrons
> wasn't available and still are few and far between, although extremely
> useful. In one lab, Femoral heads were never processed, merely grossly
> described, including fractured ones with potential for metastatic cancer.
> Another lab processed a piece of bone from every bone specimen received,
so
> policy on what was done with bone (femoral heads, knee replacement
> fragments)etc was set by that particular hospital (often as discretion of
> pathologist) or state regulation for permanent record of every specimen
> received (extracted teeth and non tissue samples the exceptions).
>
> One county clinical lab I worked in did very few special stains (on
> anything!) and if there was a question, block, slides, etc were
immediately
> sent to AFIP for consultation. Having visited AFIP, I know their battery
of
> special stains for bone and cartilage was staggering but that is highly
> specialized bone lab.
>
> I was much later I learned about fractured femoral heads/metastatic Ca
> potential. That was a scary thought that this could be missed by never
> sampling a fracture site, hoping it would never happen to me or mine. I
> applaud the pathologist who never ignores this sample.
>
>
>
>
>
>
>
> Gayle Callis
> Veterinary Molecular Biology
> Montana State University
> Bozeman MT 59717-3610
> 406 994-4705
> 406 994-4303
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 11:25:40 -0500
> From: HACKERLAB@aol.com
> Subject: Re: microwave processing
>
> Dear Wendy;
>
> You can use the H/I Milestone T/T or URM for biopsy processing. The
programs
> are factory loaded, and processing is a walk away operation - not trial
and
> error. Time is 20 minutes. Processing is accomplished using just one
reagent
> step and paraffin. (Conventional reagents may also be used). The "power"
of
> the microwave is not the relevant factor, but rather the "time at
> temperature," i.e. how long tissue is processed once temperature is
achieved.
> In the case of the Milestone systems, this is monitored and controlled by
an
> onboard computer, which keeps records and generates QA documents.
Temperature
> control and monitoring is through an infrared, non-contact system. Very
> accurate, with no margin for operator error.
>
> Please feel free to contact us with any questions.
>
> Best regards,
>
> Dorothy Murphy
> HACKER Instruments & Industries Inc.
> 1-800-4-HACKER or (973) 226-8450
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 11:26:09 -0500
> From: "Steven E. Slap" <siksik@vgernet.net>
> Subject: microwave processing
>
> Hi Histonetters!
>
> Wendy asked some basic questions about microwave processing of biopsies.
> The process is generally done in three steps (dehydration, intermedium,
> infiltration into paraffin), assuming that the biopsies are already
> properly fixed. In a 1000 watt microwave with appropriate temperature
> control, the three-step process should take between 20 and 40 minutes. I
> prefer to use 100% ethyl alcohol for the dehydration step and isopropanol
> for the intermedium. The paraffin wax must be already melted (and, the
> hotter the better), when placed in the microwave.
>
> Best regards,
> Steven Slap
>
> *********************************************
> <#161#^#248#)~~~
> http://www.geocities.com/RainForest/2966
> Radio Free Siksik, Nature's Own Broadband
> send an email to siksik-subscribe@topica.com
> ~~~(#248#^#161#>
> *********************************************
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 14:15:38 -0500
> From: CMCCOLLOUGH@dnr.state.md.us
> Subject: balsam bottle
>
> Hi Histonetters:
>
> Does anyone know of a supplier of the ground glass 'balsam bottle' that
has
> the conical dropper instead of the glass rod?
>
> Regards -
> Carol
> *****************
> Carol B. McCollough, HT(ASCP)
> Diagnostics & Histology Laboratory Manager
> Maryland Department of Natural Resources
> Fisheries Service
> Cooperative Oxford Laboratory
> 904 S. Morris Street
> Oxford, MD 21654
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 14:15:57 -0500
> From: <Kate_E_Rhodes@sbphrd.com>
> Subject: Mallory-Heidenhain's Stain
>
> HELP! Anyone have a procedure for the Mallory-Heidenhain Stain. Our
> pathologist is looking for hyaline bodies in rat kidneys. Any help would
be
> greatly appreciated.
>
> Thanks-Kate Rhodes
> SmithKline Beecham
> 610-270-7340
> FAX 610-270-7202
>
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 14:16:28 -0500
> From: maliniakrm@worldnet.att.net
> Subject: Helicobacter Pylori Stains
>
>
> We currently use the 'Genta' stain for H. Pylori. We are not satisified
> with the stain and are thinking of doing 'ALL' H.Pylori requested with
> immunohistochemistry. The questions I have been asked to answer are the
> following:
>
> 1. What is the quickest and most reliable H.Pylori stain...immuno or
> special stain? 2. What is the fastest anyone can do an immunostain for
> H.Pylori?
> 3. Are tech and reagent costs similar for 'rapid' H.Pylori via
> immunoperoxidase and special stains?
>
> Thanks in advance,
>
> Rick
>
>
> ******************* NOTE *******************
> There may be important message content
> contained in the following MIME Information.
> ********************************************
>
>
> - ------------------ MIME Information follows ------------------
>
> This is a multi-part message in MIME format.
> - --------------D3ABB50C4CF5D77879176B2E
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> Content-Transfer-Encoding: 7bit
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> begin: vcard
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> email;internet: maliniakrm@worldnet.att.net
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>
>
> - --------------D3ABB50C4CF5D77879176B2E--
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 14:16:44 -0500
> From: Philip Oshel <peoshel@facstaff.wisc.edu>
> Subject: Re: Freezing tissue
>
> There are two possibilites for plunge freezing in LN2: one is
> "normal" liquid nitrogen -- as it comes from the dewar, the other is
> slush nitrogen.
>
> If using LN2, then something like isopentane, ethane, or propane --
> ordinary cooking propane will do -- *must* be used. The caveat is
> that using these liquid gases is a serious fire and explosion hazard,
> especially since liquid oxygen forms at liquid nitrogen temperatures,
> and dissolves into the liquid hydrocarbon. These gases can be used
> safely -- I have done so -- but they take care and understanding what
> you're doing, and a safe place to dispose of the cryogen. Safe,
> keeping in mind that the isopentene, etc., is enriched in dissolved
> oxygen and heavier than air so it likes to move along floors, etc.,
> also, is a reasonably effective explosive.
>
> A simpler and much safer way is to use slush nitrogen. Slush nitrogen
> is produced by placing beaker (or whatever) full of liquid nitrogen
> in a vacuum chamber and then pulling a one atmosphere vacuum with a
> good -- meaning high-capacity -- rotary vacuum pump or the like. Pump
> on the LN2 for 10 or 30 minutes until it starts to form a slush. This
> slush can be used for plunge freezing without isopentene or any other
> agent.
>
> Plain LN2 can't be used for plunge freezing because of the
> Leidenfrost effect. What happens when water is dropped on a very hot
> skillet. Inside of immediately boiling, the water drop survives for a
> while, skittering around on the skillet. The heat of the skillet
> flash-evaporates a layer of water vapor, which then insulates the
> drop and keeps it from boiling. The same thing happens when plunging
> tissue into LN2. The relatively hot tissue flash-evaporates some LN2,
> creating an insulating layer of nitrogen gas around the specimen.
> This both slows down the rate of freezing and creates a longer
> temperature gradient over which heat must leave the specimen. This
> gives more time for ice crystals to form and grow, creating more ice
> damage. This does not happen with isopentene, ethane, etc.
>
> Then there are the slam-freezing methods, where the tissue is quick
> frozen by slamming (literally) it onto a polish metal block held at
> LN2 temperature. This is rapid and avoids the Leidenfrost effect, but
> there is the obvious potential for tissue damage. The slamming is
> done with some force, it's not a gentle act.
>
> Having said all that, plunging into LN2 would give better freezing
> than just sticking a specimen to a piece of metal and setting it in a
> cryostat.
>
> Phil
>
> >I would welcome your opinions and feedback.
> >
> >What is the optimal method of freezing tissues with liquid nitrogen?
> >with or without isopentane and why?
> >
> >thanks,
> >Vinnie
> >
> >
> >
> >Vinnie Della Speranza
> >Manager for Anatomic Pathology Services
> >Medical University of South Carolina
> >165 Ashley Avenue
> >Suite 309
> >Charleston, SC 29425
> >ph: (843) 792-6353
> >fax: (843) 792-8974
> >email: Dellav@musc.edu
>
> - --
> }}}}}}}}}}}}}}}}}{{{{{{{{{{{{{{{{{
> Philip Oshel
> AMFSC and BBPIC
> Dept. of Animal Health and Biomedical Sciences
> University of Wisconsin
> 1656 Linden Drive
> Madison, WI 53706-1581
> voice: (608) 263-4162
> fax: (608) 262-7420 (dept. fax)
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 14:17:10 -0500
> From: "Pam Marcum" <pmarcum@polysciences.com>
> Subject: RE: Vibratome
>
> I would agree as it is very difficult to stabilize the specimen and get
> consistent sections from a Vibratome. They can be useful however, one
> shouldn't expect the same type of sections as a cryostat or microtome.
The
> instrument is not designed for routine use and does require practice to
> achieve sections. Pam Marcum
>
> - -----Original Message-----
> From: Barry, Lilith [mailto:Lilith.Barry@nrc.ca]
> Sent: Monday, October 02, 2000 10:51 AM
> To: 'Joan Yonchek'; 'histonet@pathology.swmed.edu'
> Subject: RE: Vibratome
>
>
> From my experience it would be next to impossible to cut so thin
consistent
> sections with vibrotome.
> What I would recommend is to cryoprotect your tissue, embed it in O.C.T.
> compound and cut it with cryostat.
> I also think that 10-20 micron sections would be little too delicate for
> free floating work.
>
> Lilith
> - - - - - - - - - - - - - - - - - - - - -
>
> Lilith Ohannessian-Barry
> National Research Council
> Institute of Biological Sciences
> CANADA
> Tel;613-993-6460
> Fax;613-941-4475
> e-mail; lilith.barry@nrc.ca
>
>
>
>
>
>
>
>
>
> - -----Original Message-----
> From: Joan Yonchek [mailto:Jyonchek@rtitechnology.com]
> Sent: Friday, September 29, 2000 5:17 PM
> To: 'histonet@pathology.swmed.edu'
> Subject: Vibratome
>
>
>
> I am interested in hearing from anyone versed in the use of a Vibratome.
> I have been asked to obtain 10-20 micron thick sections of small pieces
(1mm
> cubes) of formalin fixed skin. Specifically I would like to know the
speed,
> amplitude and blade angle you would recommend as well as the method for
> embedding the tissue in agarose. Also, do you use a sapphire knife, a
> single edge injector type razor blade or a double edge type razor blade?
Is
> it necessary to remove the agarose from the section before staining? What
> type of
> slides are best? Do you prefer staining the free-floating section and
then
> adhering it to the slide or dry the section on the slide before staining?
>
> Other than that I got it covered!
>
> Thanks
> Joan
> RTI
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 16:23:55 -0500
> From: "Ryan.Linda" <ryan2@niehs.nih.gov>
> Subject: Demonstration of tissue Eosinophils and Mast Cells
>
> Hello,
>
> Does anyone have access to Journal of Histotechnology, June 1993,
> pp143-144? If you could fax or email the staining method, I would be
> so grateful.
>
> Thanks,
> Linda
>
> Linda Ryan, BS, HT(ASCP)HTL
> National Institute of Environmental Health Sciences
> 111 T. W. Alexander Dr.
> Bldg./rm.: 101/C-262
> P.O. Box 12233
> Research Triangle Park, NC 27709
> (919) 541-4880(Laboratory)
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 16:26:07 -0500
> From: "Clarke, Cheryl" <Cheryl_Clarke@sfhr.hnet.bc.ca>
> Subject: QA Daily H&E's
>
> We are a large histology lab and would like to implement daily QA for H&
> E's, however checking every single slide is not manageable with current
> staffing, to to have the pathologists check is unmanageable also, they are
> just as short as we are. We do approximately 750 slides a day, any good
> suggestions how to start tackling this. We have approximately five
cutters
> per day, is there an easy way to monitor all five? Thanks in advance.
Will
> wait for your replies.
>
> cc
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 16:26:30 -0500
> From: Joan Yonchek <Jyonchek@rtitechnology.com>
> Subject: Vibratome
>
> To John, Russ, Steve, Pam, Diane, and Lilith
>
> Thanks for addressing my concerns regarding the use of a vibratome for
> obtaining
> 10 to 20 micron sections of formalin-fixed skin. It was unanimously
stated
> that thin
> sections of consistent thickness are not obtainable with this piece of
> machinery.
>
> Thanks for sharing your expertise.
>
> Joan
> RTI
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 17:52:27 -0500
> From: SLHaigo@lbl.gov
> Subject: AO 860 Sliding Microtome
>
> Hi,
>
> We're looking for a good AO 860 Sliding Microtome and am wondering if
> any of you out there are selling yours. Please call back at
> (510)486-7584 and ask for me or Wayne van Deusen. Thanks!
>
> Saori Haigo
> Research Assistant
> Lawrence Berkeley National Laboratory
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 18:28:10 -0500
> From: DDittus787@aol.com
> Subject: Re: QA Daily H&E's
>
> We run a daily H&E control, small bowel and then intermittently review
bone
> marrows, cytology cell blocks, and large surgicals, the daily H& E gets
sent
> to whichever pathologist is on big specimens not biopsies for review as I
am
> sure in all histo labs , no news is good news. We also have a daily QC
sheet
> for problems, again no news is good news.
> Dana
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 18:28:36 -0500
> From: "Bill Sinai (Anatomical Pathology)" <Bills@icpmr.wsahs.nsw.gov.au>
> Subject: Re: QA Daily H&E's
>
>
>
> We are a large histology lab and would like to implement daily QA for H&
> E's, however checking every single slide is not manageable with current
> staffing, to to have the pathologists check is unmanageable also, they are
> just as short as we are. We do approximately 750 slides a day, any good
> suggestions how to start tackling this. We have approximately five
cutters
> per day, is there an easy way to monitor all five? Thanks in advance.
Will
> wait for your replies.
> ________________________________________________________
> 1. Are you attempting to QA the H&E or the cutter or both?
>
> 2. In our laboratory we have introduced a system where the cutters
> place a small identifier on the corner of the slide (obviously unique for
> each person). The person verifying the slides prior to delivery checks
the
> completeness of section, number of slides, correct tissue (just
eyeballing)
> before verifying.
>
> 3. This is the same person who checks each batch of H&E's during the
> staining procedure for adequacy of staining, correct coverslipping etc.
>
>
> Bill Sinai
> Department Manager
> Tissue Pathology
> ICPMR Westmead Hospital
> WESTMEAD NSW AUSTRALIA
> Phone 61+2+9845 7774 Fax 61+2+9687 2330
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 19:02:14 -0500
> From: Valleygal@aol.com
> Subject: Re: Vibratome
>
> Pam,
> I have recently -- very recently -- learned about cutting vibratome
sections
> and I totally agree that 10-20 micron sections would be tough. I did 40
> microns with great success on perfused brain, however.
> Before I started I called Ted Pella and was advised to practice on
> hard-boiled egg white. So off to the cafeteria I went to buy an egg and
amid
> lots of cracks about egg salad and the like I spent an afternoon
practicing
> and the next day I cut that brain like a pro! The consistency of
hard-boiled
> egg white was very similar to the perfused brain.
> Practice. Yes, do practice.
> Andi Grantham
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 19:30:14 -0500
> From: Rena Fail <RFail@charleston.net>
> Subject: Re: QA Daily H&E's
>
> We have devised a simple log sheet for QA of H&E's. A slide from a uterus
> control block is the first slide stained. One of the first Pathologist in
> reviews it and signs the sheet. Acceptable or unacceptable with room for
> comments and space to note corrective action.
> Our lab manager believes we "should own our work" The cutters are
required
> to put their initials on their slides. You would be surprised at how picky
> people get when their initials are on the slide. If any problms arise
later
> with the slide all the coordinator or manager needs to do is peel back the
> label to see who cut it.
>
> Rena Fail AS,HT(ASCP)
> Medical University of South Carolina
> Charleston, SC
>
>
>
> At 01:42 PM 10/2/00 -0700, you wrote:
> >We are a large histology lab and would like to implement daily QA for H&
> >E's, however checking every single slide is not manageable with current
> >staffing, to to have the pathologists check is unmanageable also, they
are
> >just as short as we are. We do approximately 750 slides a day, any good
> >suggestions how to start tackling this. We have approximately five
cutters
> >per day, is there an easy way to monitor all five? Thanks in advance.
Will
> >wait for your replies.
> >
> >cc
> >
> >
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 19:49:41 -0500
> From: "Pauline Burton" <pburton52@hotmail.com>
> Subject: Re: QA Daily H&E's
>
> We are cutting 600 to 800 plus slides a day. Our QC method is to make a
> block with colon, skin and umbilical cord in it. We stain one each day
> before staining the slides. This tells us several things. First, that
our
> stain set-up is working. The solutions were all placed in the right
spaces.
> You can see that everything looks good macroscopically. Second, it
tells
> us microscopically about the quality of the staining and if all tissue
> components are staining correctly. Third,our pathologists do not like the
> mucin staining the pale blue color. The umbilical cord is a quick
indicator
> to make sure that is not happening. Since we do a great deal of GI
biopsies
> and skins, those two tissues are included in the control.
> Pauline Burton
> Histology Supervisor
> Dynacare, Seattle
>
> >From: "Clarke, Cheryl" <Cheryl_Clarke@sfhr.hnet.bc.ca>
> >To: 'Histonet' <HistoNet@Pathology.swmed.edu>
> >Subject: QA Daily H&E's
> >Date: Mon, 02 Oct 2000 13:42:00 -0700
> >
> >We are a large histology lab and would like to implement daily QA for H&
> >E's, however checking every single slide is not manageable with current
> >staffing, to to have the pathologists check is unmanageable also, they
are
> >just as short as we are. We do approximately 750 slides a day, any good
> >suggestions how to start tackling this. We have approximately five
cutters
> >per day, is there an easy way to monitor all five? Thanks in advance.
> >Will
> >wait for your replies.
> >
> >cc
> >
>
> _________________________________________________________________________
> Get Your Private, Free E-mail from MSN Hotmail at http://www.hotmail.com.
>
> Share information about yourself, create your own public profile at
> http://profiles.msn.com.
>
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 19:50:03 -0500
> From: Jig1357@aol.com
> Subject: Re: balsam bottle
>
> I would also be interested in balsam bottles.
>
> Jeanne Godine
> pathology consultants
> Lynchburg, VA
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 20:24:28 -0500
> From: Jig1357@aol.com
> Subject: Re: QA Daily H&E's
>
> WE also initial our slides, and in addition, choose one slide from the
first
> batch of slides that are stained, check for quality of stain and document
on
> our "block and piece sheet", that outlines the specimen numbers with the
> number of blocks and # of pieces per block.
>
> Jeanne Godine
> Pathology Consultants
> Lynchburg, VA
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 22:30:21 -0500
> From: Debbie Pepperal <dpeppera@mail.newcastle.edu.au>
> Subject: Re: microwave processing
>
> Wendy
>
> Hi, I have been using the Mega T/T microwave from Milestone. I have done a
> series of small biopsies as well as the routine size sample for larger
> specimens. The protocol works well, I am still in the process of
optimising
> the protocol.However, a small biopsy can be fixed , processed and embedded
> and cut in 1 1/2 hours. The processing time can be reduced if the tissue
is
> well-fixed. I use neutral buffered formalin for fixation, JFC fluid for
> dehydrating and then the usual paraffin wax for impregnation. this system
> works on a "Time at Temperature" factor . feel free to contact me if you
> need any more information,
>
> Zenobia
>
> HAPS, Newcastle, Australia.At 07:17 AM 30/09/2000 -0400, you wrote:
> >Has anyone tried the microwave processing for biopsie tissue? If so what
> >procedures did you use. Length of time for final product to be put out.
What
> >was the power of the micrwave? My Pathologistsmwould like to try this as
> soon
> >as possible. So please can someone help.
>
> > Thanks,
>
> >
>
> > Wendy
> >
> >
>
>
> ----------------------------------------------------------------------
>
> Date: 2 Oct 2000 22:44:18 -0500
> From: Roger Moretz <stamptrain@yahoo.com>
> Subject: Re: Vibratome
>
> Since I am officially "on vacation" this week, I am
> rather late about getting to reading any email. So,
> even tho' I am nowhere near that dear vibratome, a
> couple of additional comments. Granted that 10 to 20
> microns is very difficult. 40 microns is easy with
> fixed tissue (such as the perfused brain, or just NBF
> fixed liver--about 2mm thick sections collected at
> necropsy). I don't do this a lot, so I cannot
> remember knife angle or speed or amplitude. The
> critical parameters for me have always been to ensure
> that the bath remains chilled (mine has a Peltier
> cooler that requires a high capacity chiller for a
> cold water source, and even then I routinely add ice
> to the bath. For brain, liver and kidney I prefer a
> #22 scalpel blade. The blade can be mounted to use
> the straightest portion of the cutting edge. I find
> these to be far superior to single or double edge
> blades. Haven't tried sapphires--too expensive for
> the limited use we have. Finally, the practice is so
> important. Due to the long times between use, I find
> that I spend 45min to an hour just re-familiarizing
> myself, and then getting into a consistent rhythm. I
> have yet to get decent sections of skin off the bloody
> thing. Tried for nearly two months. Not even
> reasonable results at anything less than 75 microns.
> Just about everything else can be done. Best of luck!
>
> Roger Moretz
> Dept of Toxicology
> Boehringer Ingelheim Pharmaceuticals
>
>
>
> - --- Valleygal@aol.com wrote:
> > Pam,
> > I have recently -- very recently -- learned about
> > cutting vibratome sections
> > and I totally agree that 10-20 micron sections would
> > be tough. I did 40
> > microns with great success on perfused brain,
> > however.
> > Before I started I called Ted Pella and was advised
> > to practice on
> > hard-boiled egg white. So off to the cafeteria I
> > went to buy an egg and amid
> > lots of cracks about egg salad and the like I spent
> > an afternoon practicing
> > and the next day I cut that brain like a pro! The
> > consistency of hard-boiled
> > egg white was very similar to the perfused brain.
> > Practice. Yes, do practice.
> > Andi Grantham
> >
>
>
> __________________________________________________
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