[Histonet] Reply #3 Manipulation time of human muscle biopsies and formation of freezing artifacts.

[Gayle Callis]

To sustain a lengthy sort of biopsies, buy a big block(s) of dry ice and
pulverize it so you can lay things on top of ice and/or surround boxes with
mutiple smaller dry ice chunks and still be able to handle the vials while
sorting. Use good insulation of hands and forceps (I have some LONG Russian
forceps with large rounded serrated tips - shoved into dry ice to keep cold
or long, grippy hemostats to pick up vials. Vials can be laid on or shoved
into pulverized dry ice and certainly stay out of a freezer for more than 5
minutes as long as they are surrounded by dry ice (hmmm approx -70 to
-90C). BE sure to use a styrofoam container to hold the dry ice, open boxes
can be worked with at RT for as long as you need.  You could still work in
a cold room with dry ice, however close quarters could be a problem - see
very last comment.  

If possible, try to recycle some dry ice pellets (Sigma et al use to ship
biologicals, antibodies, etc in styrofoam containers and if possible
stockpile pellets in -80C freezer in a styrofoam box, some means for this
purpose (hope you have some space!) We do this for snap freezing purposes
and rarely buy dry ice blocks.   

We sort large 50 ml tubes w/frozen tissue blocks using dry ice method
frequently and the biggest problem is cold hands, take care to insulate
your fingers from snap freezing! I have seen people use thinner silk
glove/liners from Winter Silks inside latex gloves and forceps to handle
(juggle?) tubes. 

We find the dry ice easier to work with than colder, fog creating liquid
nitrogen where I end up going on a frustrating fishing for tubes somewhere
in bottom of a dewar.  Be sure you have good ventilation, you don't want to
be found passed out on the floor due to oxygen depletion by CO2 or N2 fumes. 

Good luck  

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610


 At 04:17 PM 9/12/2003 -0500, you wrote:
>Mauricio:
>
>If you have access to a walk-in freezer as we do, you may be able to
>transfer the racks/boxes to the walk-in using styrofoam shipping containers
>or a large plastic cooler (like those used for camping) containing dry ice.
>Once in the walk-in freezer you would be able to work with the specimens for
>some time without having them thaw out (handle with gloves to insulate your
>hands from the cold and the specimens from your body heat.  However, most
>walk-in freezers (-20 C) are not as cold as the cryofreezers commonly used
>here to store muscles (-70 C).  But the closet-sized sized freezer might
>offer enough working time to allow you to sort and label without much loss
>or damage.
>
>Good luck,
>
>Joe Galbraith
>
>-----Original Message-----
>From: Mauricio S. Morais [mailto:Mauricio.Morais <@t> tufts.edu]
>Sent: Thursday, September 11, 2003 6:25 PM
>To: Histonet
>Subject: [Histonet] Manipulation time of human muscle biopsies and
>formation of freezing artifacts.
>
>
>Hello Histonet community.
>
>Because I'm new into the histology "word" I've been facing some
>unexpected (to me) challenges in my actual Laboratory work.
>My background is originally from Organic and Analytical Chemistry,
>Teaching, and a bit of Biochemistry (my MSc degree).
>I'm kind of new in the country too (been here less than 2 years) and
>English is not my primary language, so please accept my apologies for
>any errors within this text.
>
>I'm assign to reorganize and analyze human muscle biopsies, stored in
>boxes distributed among 4 racks inside a Cryoplus cryofreezer.
>Seeking for information on how to work safely and at the same time not
>compromise the integrity of the stored samples, I found the Histonet
>Histosearch archives.
>They were very helpful providing answer about most of the common issues
>related with formation of freezing artifacts and possible solutions to
>the problem at different stages the samples can generate it, from the
>biopsy to the cryostat slice machine.
>
>Because of the enormous amount of samples that'll need to be handle at
>one time during the reorganization of our cryofreezer, an issue about
>"time"  became critical (in my opinion) raising some questions:
>
>1-How long can a box (full of biopsies), or the hole rack be kept out of
>the cryofreezer, while I'm updating data and re-arranging boxes between
>racks, without compromise the biopsies and generate (and/or enhancing
>the chances of creating) freezing artifacts?
>
>2-Does any damage may occur if I have several biopsies floating inside a
>dewar container while I'm making notes or grouping them into other dewar
>containers to a new order? 
>
>A website information (from www.asymptote.co.uk) says that tissue inside
>straws immersed in liquid Nitrogen when exposed to air at room
>temperature have their temperature raised to -50C in 40 seconds. Thawing
>may start around -10C or early.
>
>I was told they (racks, boxes) could be out for about 5 minutes before
>some damage can start to affect the biopsies inside it. None of them
>been hold by hands during all this time, but on the bench top (boxes) or
>floor (racks) while the work is done.
>I was also told that 5 minutes is away too long...
>
>For me it seems impossible to safely remove a rack (containing a dozen
>boxes) from the cryofreezer, make notes or, for example, remove one box
>from the rack to add a new sample and return it to the cryofreezer in 40
>seconds. Reminding that I'm working with cryogloves over my lab.
>gloves... I was never able to open a box without remove the cryogloves
>first.
>Maybe I need more training...
>
>I would greatly appreciate any comments from this expert community to
>help me solve my immediate problems (above) and improve my knowledge and
>safety at work place.
> 
>
>Thank you all.
>
>
>Mauricio S. Morais
>Senior Research Technician
>Nutrition Exercise Physiology and Sarcopenia Laboratory (NEPS)
>TUFTS University
>Jean Mayer USDA Human Nutrition Research Center on Aging
>711 Washington Street, Rm 436
>Boston, MA 02111
>(617) 556-3226
>
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>