[Histonet] Isopropanol in processing

[Matt Ibbs]

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