> Tim, Ignore this posting. In my opinion, for the most part this is
> rubbish, full of flaws and not written by a muscle histochemist.
> Ian.
 Dr. Ian Montgomery,
> West Medical Building,
> University of Glasgow,
> Glasgow,
> G12 8QQ,
> Scotland.
> Tel: 0141 339 8855 Extn. 6602.
> Fax: 0141 330 4100.
> e-mail: ian.montgomery@bio.gla.ac.uk
>
		Dear Ian - 	Why flame? Why so bitter. Where is you objectivity?
The thread concerned poor structural preservation, not histochemistry.
As it happens I have worked in cryo- preservation of fine structures for many 
years.
I used Isopentane for about five years before Freons and then Propane were 
proposed for cryo fixation about 25 years ago.

True, histologist using larger samples cannot aspire to achieve true 
vitrification and must live with some ice-crystal damage. This I made clear in 
a second submission on this thread, which is also appended to this email.
The original query concerned poor preservation and deserved a reasoned reply 
(not just " that's rubbish").  I suggest that the answers to poor cryo fixation 
are contained in my two contributions. Adapt these for your situation and your 
cryo preservation will improve.
Cheers
Jim Darley
ProSciTech                 Microscopy PLUS
PO Box 111, Thuringowa  QLD  4817  Australia
Ph +61 7 4774 0370  Fax:+61 7 4789 2313  service@proscitech.com.au
Great microscopy catalogue, 500 Links, MSDS, User Notes
                      www.proscitech.com.au

On Monday, July 05, 1999 8:22 PM, Ian Montgomery 
[SMTP:ian.montgomery@bio.gla.ac.uk] wrote:
> >Date: Fri, 2 Jul 1999 17:55:47 +1000
> >From: jim <jim@proscitech.com.au>
> >Subject: FW: Freezing muscle sections/snap freeing technique
> >To: "'Tim Fairchild'" <timf@cyllene.uwa.edu.au>,
> >        HistoNet Server
> >	 <HistoNet@pathology.swmed.edu>
> >Mime-Version: 1.0
>
> >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
> >isopentane.
> >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
> >rapid.
> >Hope this helps.
> >Cheers
> >Jim Darley
> >
2nd contribution on 3 July

Bernice -
Although your point was not in reply to my previous contribution to this
discussion, it does impinge on it.
I had described requirements essential to achieve vitrification: glasslike
freezing without ice-crystals. This methods is essential for high resolution
TEM and would be preferable for much light microscopy.
For SEM and some light microscopy a slower freezing method which results in
some fine crystals is often acceptable and very commonly practiced.

Now to your point: A cryoprotectant which only surrounds and not infiltrates
the specimen can only be useful for the second method, since such a medium
contributes to the bulk of the specimen and does not lower the freezing point
of the specimen itself. To be effective during vitrification the cryoprotectant 
must infiltrate the specimen.

> >ProSciTech                 Microscopy PLUS
> >PO Box 111, Thuringowa  QLD  4817  Australia
> >Ph +61 7 4774 0370  Fax:+61 7 4789 2313  service@proscitech.com.au
> >Great microscopy catalogue, 500 Links, MSDS, User Notes
> >                      www.proscitech.com.au
> >> -----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