RE: Freezing muscle sections/snap freeing technique

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From:jim <>
To:"'Ian Montgomery'" <>, "" <>
Date:Sun, 11 Jul 1999 22:55:12 +1000
Content-Type:text/plain; charset="us-ascii"

Ian - from what I read in your several missives about freezing technique, there 
is nothing of substance I would disagree with and that is not because of the 
possible, violent application of a "Lochhaber axe". - Is that gadget used to 
gross specimens?

For those interested in cryo, I would like to comment on a couple of points. 
Hope this will not jeopardise the prospect of some lukewarm amber, which I 
detest, but would imbibe to appease any woad attired high priest of histo.

I think that the danger of exploding propane is over-stated. Yes, I know it can 
do so very nicely, but if used within a fumehood no dangerous vapour 
concentration can occur, short of sticking a match right at the container. The 
modest size propane cylinder is only used while filling the nitrogen cooled 
I grant that isopentane is more convenient and frequently "good enough" for 
cryo fixation of "bulk" specimen. Previously and now, I am writing about 
possible remedies to poor cryo preservation. Knowing the criteria enables the 
user to make intelligent choices when modifying their methods. As previously 
noted, the larger histo specimen cannot possibly be preserved completely 
crystal free, but the means used to achieve true vitrification would minimize 
crystal interference.
One important criteria is speed of freezing. Several parameters affect speed, 
including: specimen size, lowest possible temperature of cryo agent and 
conductivity of specimen and agent.

The lowest usable temperature of propane is about 30 degree lower than 
isopentane. That is significant.
another approach is the use of slushy nitrogen. Liquid nitrogen placed under 
vacuum boils rapidly. Boiling cools a liquid (saucepan with boiling water 
remains at a hundred, despite further heat-input) and since no heat is added to 
the liq N2, boiling cools the nitrogen until reaching solidification point - it 
looks like crushed ice, but is softer. Slushy nitrogen does not quickly form 
that gas insulating layer which makes liquid nitrogen a useless cryo-agent. 
This material is used in practical cryo work. For instance, the Emitech SEM 
bolt-on preparation system includes a slushy nitrogen system. (Declare 
interest; PST is Emitech agent for Australasia) For those interested in the 
subject, PST's online has an internal link from page K4 (cryo stages) entitled 
a "technical brief  on bulk frozen hydrated specimens"
I appreciate your comment on the slammer method. This of course works well 
because it's for tiny samples only, near liq nitrogen temperature and because 
of pressure and excellent conductive properties of the polished copper anvil. 
(I'll spare you another commercial, but yes PST has a commercial interest). The 
interesting facts are, that copper is an excellent conductor at those low 
temperatures, gold and silver are slightly better, but not very practical. 
Stainless Steel conducts heat very poorly at low temperatures. I have used thin 
stainless wires to conduct electricity out of a dewar with minimal heat-gain.
Now Ian, if you really want to go to town buy a diamond anvil. (Sorry, but yes, 
I could provide that too) Diamond is the best conductor at those low 
temperatures, it takes an excellent polish and does not scratch easily. I think 
that de Beers need a break and a stampede of diamond demanding histos would 
even help the SA economy.
Ian you really should try a good Ozzie red, beats that lukewarm amber.
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

On Wednesday, July 07, 1999 8:17 PM, Ian Montgomery 
[] wrote:
> >Date: Fri, 2 Jul 1999 17:55:47 +1000
> >From: jim <>
> >Subject: FW: Freezing muscle sections/snap freeing technique
> >To: "'Tim Fairchild'" <>,
> >        HistoNet Server
> >	 <>
> >Mime-Version: 1.0
> >
> Jim,
> 	You got me when I was a wee bit nippy. Family trait, crabbit, bad
> tempered and quick to reach for the Lochaber axe. I'll buy you a pint first
> time we meet.
> >Tim - Isopentane is not the best medium to use!
> 	Have to disagree, for good muscle histochemistry, the original
> question, isopentane cooled with liquid nitrogen is essential. Ok, I have
> used talc coated specimens in the past, but that was only in an emergency.
> >Isopentane is very sticky and almost unusable when its near liq nitrogen
> >temperature.
> 	Isopentane is completely unusable at liquid nitrogen temperature,
> you have a solid block. Not much use for plunge freezing.
> 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.
> 	Ok.
> >
> >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.
> 	Small is lovely, but remember we are dealing with LM Histochemistry
> and not EM. Orientation of a flat piece of TS muscle is a bummer, 'tall and
> slim or short and squat is in'
> >
> >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.
> 	Ok, but for muscle enzyme histochemistry a wee whiff of fixatives
> is the kiss of death.
> >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).
> 	Propane is a good cryogen, but again this is muscle histochemistry
> and not EM.
> 	WITHOUT REGULATORS, in the name of the wee man, Safety Officers
> would go totally ballistic without a back flow/flame regulator.  -180
> atmospheric oxygen dissolves into the cold propane and what a combination
> that is and no regulator to stop the back flow into the propane tank. I
> agree the purity of the propane is not important, in fact impurities are
> good. I use a stirred mixture of isopentane and propane in order to depress
> the temperature and keep it away from -180 and the naughty effects of
> oxygen.
> >
> >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.
> 	Ok. Although I agree propane is very good for cryo EM, the best
> cryo-preservation I have ever obtained was with a specimen slammed on a
> cooled ultra pure copper block and freeze-substituted. I even published a
> micrograph in the Proceedings of the RMS so happy was I. Muscle
> histochemistry, isopentane cooled with liquid nitrogen is the business.
> >Hope this helps.
> >Cheers
> >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:
> >>
> >>
> >>
> >>
> >
> 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:

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