[Histonet] Isopropanol in processing
Matt Ibbs
histol <@t> rat-hole.org
Thu Apr 15 03:14:47 CDT 2004
Dear all,
Thanks for the opinions. Here in Poland we have been using industrial
methylated spirit (the mixture many of you describe as reagent alcohol) for
years. The authorities have now decided to tax that too. That's why we
need to switch to Isopropanol.
Unfortunately we are not able to install a recycling machine in our
facility as local regulations require that such machines are housed in
bomb-proof rooms - preferably in separate buildings.
In response to Gayle Callis:
Thank you! I knew it could be done and I remember doing it in another
department some 10 years ago. I tried to reproduce the processing protocol
from memory but we have some problems. Here are two protocols we've tried
so far:
Protocol 1:
In this first schedule I used three changes of 100% Isopropanol as
experiments with only two caused the xylene to go cloudy - suggesting water
remained. This problem vanished with three changes. Everything was well
for two days but shortly afterwards problems emerged with small biopsies
being too brittle.
1. 60% Isopropanol 1 hour
2. 80% Isopropanol 1 hour
3. 90% Isopropanol 1 hour
4. 100% Isopropanol 45 minutes
5. 100% Isopropanol 45 minutes
6. 100% Isopropanol 45 minutes
7. 2:1 Isopropanol:Xylene 30 minutes
8. 1:2 Isopropanol:Xylene 30 minutes
9. Xylene 1 hour
10. Xylene 1 hour
11. Paraffin 3 hours
12. Paraffin 3 hours
Protocol 2:
In order to counter the problems with small biopsies we refreshed the
paraffin and removed the third change of 100% isopropanol. The time in the
remaining two changes of 100% isopropanol was increased to 1 hour to allow
for the reduced number of changes. Mysteriously, the clouding of xylene
previously observed was no longer evident. Otherwise the schedule was
unchanged. After a day or so the 80% isopropanol (now realistically nearer
70%) started to gather a cloud of slightly viscous-looking grey "mush" in
the bottom of the container. Odd. This problem was fixed of course. At
this point we had to process whole slices of prostate alongside our routine
work - this is not uncommon so we must be able to accommodate it. The
tissue was poorly processed and fell off the slides - which is annoying
with extra-large blocks especially on the third or fourth attempt. ;o)
Next I plan to alter xylene times as you suggested. In answer to your
questions about our paraffin, it is held on the processor at around 58-60
degrees Celsius. My colleagues (a case of "we've always done it this way")
insist that a small amount of bees-wax is added to soften the wax. Ideally
I'd like to use four changes of paraffin but our processor cannot
accommodate them.
Any advice would be welcome. Now I'm going to re-search the archives as
you advised. Maybe I missed something. Thanks again everyone.
Matt.
Matthew Ibbs
Dept. of Neoplastic Pathology
Karol Marcinkowski University of Medical Sciences
Poznan
Poland
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