Re: Freezing tissue

From:Philip Oshel <>

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 

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 


>I would welcome your opinions and feedback.
>What is the optimal method of freezing tissues with liquid nitrogen? 
>with or without isopentane and why?
>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

Philip Oshel
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)

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