Tim,

Your method sounds good for the most part.

I've had consistently good results with your method and with OCT. With OCT I 
found the best way is to put the muscle in a metal paraffin mold and then 
cover partially with OCT. That way the metal will conduct the cold right 
into the muscle.

As far as temperature of the isopentane goes, the best way to ensure the 
correct temperature is to use a digital thermometer. We used a Fluka 
thermometer with a low temp probe able to measure down to -200 deg C. We 
cooled the isopentane to -160 before plunging the muscle in for 25-30 
seconds. I would say getting the temperature right is the most critical part 
of the method.

Tim Morken, B.A., EMT(MSA), HTL(ASCP)
Infectious Disease Pathology
Centers for Disease Control
MS-G32
1600 Clifton Rd.
Atlanta, GA 30333
USA

email: tim9@cdc.gov
       timcdc@hotmail.com

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


----Original Message Follows----
From: Tim Fairchild <timf@cyllene.uwa.edu.au>
To: HistoNet Server <HistoNet@Pathology.swmed.edu>
Subject: Freezing muscle sections
Date: Thu, 01 Jul 1999 16:13:38 +0800

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











_______________________________________________________________
Get Free Email and Do More On The Web. Visit http://www.msn.com