[Histonet] Thank you for reply: Manipulation time of human muscle biopsies and formation of freezing artifacts

From:"Mauricio S. Morais"

Hello Histonet community.

I want to thank you all for sending suggestions to help me walking the
pathways of the "Histo World".
Special thanks to George Cole for sending the DVDs. We look forward to
watch and learn.
My apologies for delaying this Thanks mail.

Below I add all replies I had to help others in future search through
the Histonet Archives.

Thank you very much.

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

Replies received:
Of the thousands of muscle biopsies performed in my lab, I never saw ice
crystals formed by quick searches through their containers in  the
freezer.  They were kept about 70 0r 80 per box in a -70C freezer. I
kept an empty box in the freezer.  When searching for a case, I would
look at the biopsies one at a time in their small individual plastic zip
lock bags, ID'd by heavy lead pencil on 1.5 inch pieces of tongue
blade----any paper or cardboard will curl and become hard to read---I
would read the tag on the biopsy while in the freezer, putting them in
to the cold empty box in the freezer until the one I was looking for
showed up. I would then return the bags I had just looked at to their
storage box. Put the box back where it was, and carry the bag with the
biopsy I had just found 30 paces, holding it in the air, to my lab and
put it in the cryostat. I would do all I had to do to the biopsy in the
cryostat. The Leitz cryostat had a metal kind of shield in the bottom
which I promptly took out so I could keep a 100 slide box in the botom.
I cut sections onto slides and put them directly into slide  boxes in
the bottom of the cryostat.  When I had to do anything with a block, I
would put it in the cold box in the bottom of the cryostat.  Never, ever
did I see ice crystals added to a biopsy handled this way.  Freezing
artifact is the province of initial freezing of the muscle. My packet of
3 DVD's and 18 pages of procedures shows the entire orientation,
freezing and cutting of muscle,  I will be glad to send you one, if you
wish me to. There is no charge for the packet.  The DVD's are crude, but
they do manage to show how it's done.

I can give you some advice on how to keep things from thawing but my
experience comes from a frozen brain bank.  I was given the job of
cataloging every piece on frozen tissue from dissected human AD brains
normal brains as well.  It was a very very large job and I am not sure
muscle but brain thaws pretty fast.   You are not even supposed to
them with gloved hands for more than a few seconds.
What I used to do was get a styrofoam box of dry ice and anything that I
took out of the freezer for moving around or cataloging purposes I put
the dry ice.   That way it never thawed.   I used a large box and lots
dry ice.  I hope this helps you a little. 
Good luck.  I had four large -80 freezers packed to the brim.  It took
almost a year. 

"Gehan, Loralee" 


We have found it helpful, and best for specimen preservation to do the
organization with the specimens setting in an open cooler on a bed of
ice, only removing from the freezer the amount you can work with in this
space.  Another alternative is to work inside a cryostat.

Good luck!  It sounds like you have quite a challenge ahead of you!

Bonnie Whitaker
Technical Director, Histology Laboratory
Department of Pathology and Laboratory Medicine
UT Med School
6431 Fannin
MSB 2.231
Houston, TX  77030

My advice would be to work with the boxes in the cryostat that is
usually set at -20C.  Maybe a couple of boxes of tissue at one time
until you can catalog their contents and then begin rearranging your
frozen tissues to the appropriate boxes.  Again a couple of boxes at a
time.  I would only remove the whole rack just long enough to remove the
needed boxes.

I work with volumes of muscle samples held at -70C.  When I need to pull
a particular case out I will remove a box from the -70C and work very
quickly, maybe for 1-2 minutes maximum.  It all begins to thaw very

I don't know if there is a proven study that deals with this type of
problem - but if anyone responds to you quoting a reference I would
appreciate learning that information also.  Hope some of this helps

Jean Mitchell
University of Wisconsin Hospital & Clinics
Madison, WI 
"Mitchell (Jean A.)" 


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
or a large plastic cooler (like those used for camping) containing dry
Once in the walk-in freezer you would be able to work with the specimens
some time without having them thaw out (handle with gloves to insulate
hands from the cold and the specimens from your body heat.  However,
walk-in freezers (-20 C) are not as cold as the cryofreezers commonly
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
or damage.

Good luck,

Joe Galbraith
"Galbraith, Joe" 

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
mutiple smaller dry ice chunks and still be able to handle the vials
sorting. Use good insulation of hands and forceps (I have some LONG
forceps with large rounded serrated tips - shoved into dry ice to keep
or long, grippy hemostats to pick up vials. Vials can be laid on or
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
can be worked with at RT for as long as you need.  You could still work
a cold room with dry ice, however close quarters could be a problem -
very last comment.  

If possible, try to recycle some dry ice pellets (Sigma et al use to
biologicals, antibodies, etc in styrofoam containers and if possible
stockpile pellets in -80C freezer in a styrofoam box, some means for
purpose (hope you have some space!) We do this for snap freezing
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
in bottom of a dewar.  Be sure you have good ventilation, you don't want
be found passed out on the floor due to oxygen depletion by CO2 or N2

Good luck  

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610

After removal of boxes from the cryo freezer, take it to your cryostat
and work there, Make sure you maintain your cryostat temp at -24
or higher. You can work with your regular  gloves but try not to hold
the tissue ,hold it on the cork or use forceps that have been freeze
from  the liquid nitrogen. After each tissue checked or organized.
freeze it to liquid nitrogen to prevent ice crsytal formation and to
the integrity of you tissue. I hope this can help you with your problem
on organizing your freezer works.
I'm working on muscle tissue and i don't have problems. i may have few
problems only when the tissue was not properly freezed.
Good luck.
reuel cornelia
Reuel Cornelia 

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