FW: Freezing muscle sections/snap freeing technique

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From:jim <jim@proscitech.com.au>
To:"'Tim Fairchild'" <timf@cyllene.uwa.edu.au>, HistoNet Server <HistoNet@Pathology.swmed.edu>
Date:Fri, 2 Jul 1999 17:55:47 +1000
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Tim - Isopentane is not the best medium to use!
Isopentane is very sticky and almost unusable when its near liq nitrogen 
temperature. Warmed up (with a metal rod) the cooling rate of the specimen is 
reduced, because (heat transfer rates of various media aside and liquid 
nitrogen is lousy, because it forms an "insulating" gas layer), the colder the 
medium the better.

The second rule is that smaller specimens freeze better and the out-most part 
of the specimen will be best preserved. Flat specimens are suitable.

Third, specimens with lower water contents will show less damage. So, skin (or 
cork!) will be less affected by ice crystal formation than liver.
 Ice crystal damage can be reduced by fixing and then infusing with say 20% 
glycerin, but this may defeat your reasons for cryo.

A very good freezing medium is liquid propane, cooled by liq nitrogen like your 
Use a fumehood throughout. Prepare your cold-cup with liquid nitrogen. Have a 
needle (cut off square 19 gauge or similar) inserted into a bit of tygon tubing 
connected to the outlet valve of a small propane gas cylinder (without 
regulators; purity of gas is not very important). Sit the cylinder up-side-down 
so liquid is expelled (preferred but works either way).

Open the valve very slowly so gas can only just be feeled on skin. Rub needle 
gently in the cold cup. The emitted gas will turn into liquid.
Snap freeze specimens by very rapid immersion movement. The best freezing 
happens in a fraction of a second and largely depends on the temperature 
differential between specimen and the medium; since the interface warms 
rapidly, the rapid movement is required.
Also assure that the transfer into liquid nitrogen for specimen storage is very 
Hope this helps.
Jim Darley

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> -----Original Message-----
> From:	Tim Fairchild [SMTP:timf@cyllene.uwa.edu.au]
> Sent:	Thursday, July 01, 1999 3:14 AM
> To:	HistoNet Server
> Subject:	Freezing muscle sections
> We have recently undertaken a project which required a portion of muscle
> to be analysed for fibre type and oxidative capacity.  The technique we
> adopted to freeze the muscle (human muscle), was to mount the muscle on
> cork using 'gum tragacanth', and then freezing this in isopentane cooled
> in liquid nitrogen.  The trouble we're having is that every 5th sample
> (roughly speaking) has ice crystal artifact through it.  I am
> attributing this to the isopentan not being cold enough.  I guess my
> questions therefore are:
> 1. Is there a way to protect the muscle from the freezing process, i.e.
> putting O.C.T. over the muscle?
> 2. If the muscle has to be frozen in isopentane, what 'set up' has
> worked for other people (i.e. we put the isopentane in a long metal
> cylindrical container, inserted in a larger container holding liquid
> nitrogen) and what techniques have you found useful (e.g. hold in
> isopentane for 20 seconds)?
> Any help (or small tips) would be very much appreciated!
> Thanks in advance,
> Tim Fairchild.
> -----------------------------------------------------------------
> Timothy J. Fairchild B.Sc. (Hons)
> PhD Candidate
> Co-ordinator for Centre of Athletic Testing
> Department of Human Movement and Exercise Science
> Nedlands, Western Australia 6907
> Telephone: (+61 8) 9380 2793
> Facsimile:  (+61 8) 9380 1039
> Email: timf@cyllene.uwa.edu.au
> http://www.general.uwa.edu.au/~hmweb/index.htm

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