Re: Freezing muscle sections/snap freezing technique

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From:Tim Morken <>
Date:Fri, 02 Jul 1999 09:39:49 EDT
Content-Type:text/plain; format=flowed

The discussion in the message below points out why I use a digital 
thermometer to control the point at which I freeze tissue in isopentane. 
Isopentane (or methyl butane, if you prefer) is fine for snap freezing if 
you use it at the proper temperature: -160 deg C. Those that wait for it to 
freeze and then thaw will run into the problems of a viscous fluid not 
allowing a fast enough heat transfer. I used to have that problem but never 
have since using a thermometer.

Some good features of isopentane are that it is cheap, can be reused  and is 
easily stored.

BTW, talc is used only when freezing directly in liquid nitrogen. It is not 
clear exactly how it works (whether through dehydration of the surface or 
mechanical interference of bubble formation) but it prevents the 
slow-freezing artifact caused by LN2 bubbling around the tissue surface. 
Excellent results are obtainable with this method with practice.

Tim Morken, B.A., EMT(MSA), HTL(ASCP)
Infectious Disease Pathology
Centers for Disease Control
1600 Clifton Rd.
Atlanta, GA 30333


Phone: (404) 639-3964
FAX:  (404)639-3043

----Original Message Follows----
From: jim <>
To: "'Tim Fairchild'" <>,        HistoNet Server 
Subject: FW: Freezing muscle sections/snap freeing technique
Date: Fri, 2 Jul 1999 17:55:47 +1000

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 
reduced, because (heat transfer rates of various media aside and liquid
nitrogen is lousy, because it forms an "insulating" gas layer), the colder 
medium the better.

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

Third, specimens with lower water contents will show less damage. So, skin 
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 
Use a fumehood throughout. Prepare your cold-cup with liquid nitrogen. Have 
needle (cut off square 19 gauge or similar) inserted into a bit of tygon 
connected to the outlet valve of a small propane gas cylinder (without
regulators; purity of gas is not very important). Sit the cylinder 
so liquid is expelled (preferred but works either way).

Open the valve very slowly so gas can only just be feeled on skin. Rub 
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 
Hope this helps.
Jim Darley

ProSciTech                 Microscopy PLUS
PO Box 111, Thuringowa  QLD  4817  Australia
Ph +61 7 4774 0370  Fax:+61 7 4789 2313
Great microscopy catalogue, 500 Links, MSDS, User Notes
 > -----Original Message-----
 > From:	Tim Fairchild []
 > 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:

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