[Histonet] liquid nitrogen

[Karen Weidenheim]

Hi Manal, 
The group has been discussing this topic lately.  Our lab does
classical muscle biopsy freezing with liquid nitrogen and isopentane. 
(I am happy to hear that you are successful with the cryostat and will
have to check this out, but anyway, here is a little synopsis):
1.  Liquid nitrogen comes in a large pressurized tank that one orders
from a company that provides compressed gases.
2.  For freezing,  We fill a 2 liter Dewar flask , Cat # 2119, Lab Line
Instruments Inc, Melrose Park, IL about 75% full of liquid nitrogen and
we suspend our 250 ml stainless steel, very clean beaker that is around
65% full of isopentane in it using a home-made wire contraption made
from coat hangers (!) that encircles the beaker and allows us to lower
it slowly in to the isopentane (this whole procedure requires 2 people,
1 to handle the isopentane and 1 to do the specimen.)
If you are alone, say, on call at night, you can use a ring stand to
hold the beaker suspended by the wires.  Or you can bend the wire
contraption so it hooks on the side of the beaker.  
When ordering your equipment, you have to be sure, by measuring the
internal diameter of the Dewar flask and the external diameter of your
stainless beaker, that you can suspend the beaker in the flask
comfortably.  You have to lower the beaker of isopentane slowly into the
liquid nitrogen to avoid the liquid nitrogen boiling into the isopentane
and making a mess, at which point you have to start over.
3.  We freeze a 0.8 x 0.5 cm portion of oriented skeletal muscle 25
seconds in syrupy isopentane that has ice all around the sides and the
bottom.  THe textbooks say 5-10 seconds, but we find we need 25 seconds.
 I think lots of this method is empiric and depends on your location,
altitude and humidity.  
We never reuse the isopentane which, by the way, has to be kept in a
flammable cabinet according to regulations.  I might do 4 blocks in one
aliquot of isopentane but I change it frequently and I think this helps
me avoid artifacts.
As we cannot put the liquid nitrogen back in the tank, it is not reused
either, though if we have several biopsies in a day we use the same
liquid nitrogen.
Frozen biopsies are stored in a little white box (EM sciences, I
think)labeled with patient name and number, in the ultrafreezer.
Best of luck with your lab.  If your pathologist is satisfied with the
method you use now you should stick to it, as I said, I will investigate
it with our cryostats here.  
Karen



Karen M. Weidenheim, M.D.
Professor of Pathology, Clinical Neurology and Clinical Neurosurgery
Albert Einstein College of Medicine
Chief, Division of Neuropathology
Montefiore Medical Center
111 East 210th Street
Bronx, NY   10467 
(718) 920-4446
FAX (718) 653-3409
Beeper (917) 556 3696
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>>> manal galal <galalmkh <@t> yahoo.com> 7/16/2005 8:06:46 AM >>>
Hi all, 
 
  I wanted to ask about how to use liquid nitogen in freezing muscle
biopsies. I mean what does it come in? How much do I use? Do I discard
it after I use it? What kind of container do I use? How long do I put
the biopsy in it? How do I store it? 
  By the way I freeze muscle in the quick freezing chamber of the
cryostat, and am getting very little to no artifacts. But anyone that
learns of how I freeeze biopsies says that it will give me disasterous
resuls. Do you think liquid nitrogen will be superior to my old method.
 
Thanks in advance
manal

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