Allen Dick posted a nice synopsis of the fast-freeze liquid nitrogen method
used to assay honey bee colonies for hygienic behavior.

We developed this procedure under funding from the U.S. EPA and posted it
to our web pages several years ago.

Marla Spivak later wrote an article in Bee Culture describing our procedure
and how it compared to the one described by Steve Tabor.

Steve and others cut out bits of comb, freeze them overnight in a freezer,
and then return them to the hive.  We found this to be too time consuming
and the act of cutting the comb often induced vigorouse house cleaning
activities.  We were never able to get consistent results with this method.

With the liquid nitrogen procedure and a sharpened tube (we used metal
flashing rather than a soup can), you get a consistent amount of damage to
the comb; the liquid nitrogen thoroughly kills the brood, and you don't
have to try to re-insert a piece of cut-out comb.

Pat Heitkam, a queen producer in California, was the first to try our
method in a commercial application mode.  I told him about the method at
breakfast at an ABF meeting in Portland.  He had been working with Marla on
breeding for this trait.  Marla thought he would have trouble getting the
nitrogen, but Pat was able to borrow a Dewar from a local veterinarian, who
used it in his artificial insemination of livestock.  Pat quickly became
convinced that it was a fast and practical method, and he was able to get a
Dewar and find a local supplier was happy to sell him the nitrogen (the
same one supply the nitrogen to the vet).  (In the U.S., you can usually
buy or rent Dewars - check with the companies that sell gases for medical
or industrial purposes).

You don't need to freeze a very large area - in fact the test is a bit more
sensitive if you freeze small areas of brood.  But you must thoroughly
freeze the brood.  Medhat was using a double treatment with liquid nitrogen
to be sure that the brood was frozen.  We usually just checked the opposite
side of the comb.  If the brood is frozen solid on the underside of the
comb, in addition to the area where you poured the nitrogen, you can be
sure the brood has been killed.  But don't use so much nitrogen that it
pours over the sides of the tube and kills most of the surrounding brood -
you want a nice, clean delineation as illustrated by Allen and on our pages.

And one more caution, if you don't think liquid nitrogen is cold, drop a
rubber ball into it (on a string).  Pull it out and toss it onto a concrete
sidewalk.  It will shatter.

One word of caution - as stated on Allen's site, liquid nitrogen can cause
serious burns.  Protective clothing includes gloves, BUT NOT JUST ANY OLD
GLOVES.  Cloth gloves may be worse than no gloves at all, because the
liquid will pass right through the fabric and the gloves will hold it
against your skin.  Be sure that the gloves are made of a material that
will not pass liquids through.

Jerry
Jerry J. Bromenshenk, Ph.D.
Director, DOE/EPSCoR & Montana Organization for Research in Energy
The University of Montana-Missoula
Missoula, MT  59812-1002
E-Mail: [log in to unmask]
Tel:  406-243-5648
Fax:  406-243-4184
http://www.umt.edu/biology/more
http://www.umt.edu/biology/bees