Tim,

This problem sounds as though the specimen is not completely frozen.  Also,
make sure the specimen is not too large which would effect quick freezing.

 Avoid placing the muscle directly in saline. We would have the muscle sent
fresh to the lab when they were inhouse (it was treated like a frozen).
And sent in a dampened saline gauze when received from outside labs.  I
guess my point is to avoid extra moisture when possible.

We never used anything else but O.C.T. for freezing muscle.  However, it is
important to apply enough to the cork just to cover the muscle and give it
support (avoid big goobs). Try sprinkling talc powder over the specimen
before freezing.  This will help eliminate artifact.

The lab used a dewar's flask with liquid nitrogen.  For the isopentane, try
a metal beaker (that will fit into the dewar's flask) 1/4 filled with
isopentane.  Freeze the isopentane in the liquid nitrogen until if forms a
cloudy miniscus.  The center should appear liquid.  If it over freezes.
simply let it thaw a bit.  Snap the specimen into the isoentane in one
quick motion, specimen side down.  It only needs 30 seconds to freeze.

Hope this helps.

Rande Kline, HT (ASCP)





From: Tim Fairchild <timf@cyllene.uwa.edu.au> AT INTERNET-MAIL on 07/01/99
      04:13 PM

To:   HistoNet Server <HistoNet@Pathology.swmed.edu> AT
      INTERNET-MAIL@ccMail
cc:    (bcc: Rande Kline/EMI/Merck)
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