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Photo B. A comb section
of sealed brood containing approximately 100 cells on each side
was cut out of the comb, frozen for at least 24 hours, and replaced
in the hole left in the comb. |
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Testing Honey Bee Colonies for Hygienic
Behavior
It is relatively easy to determine if a colony of bees displays
hygienic behavior by testing them using one of the methods described
below [5, 6].
Two best methods to test for hygienic
behavior
1. The freeze killed brood assay: In this assay,
a comb section of sealed brood containing approximately 100 cells
on each side (2 x 2.5 inches) is cut from a frame and frozen for
24 hours at -100 F. The frozen comb section is inserted
into a frame of sealed brood in the colony being tested (Photo B).
Tests have shown that it does not matter if the frozen section comes
from the same colony from which it was removed or from a different
colony. The frame with the freeze-killed brood insert is placed
in the center of the brood nest. Two days (48 hours) later the frame
is removed and the number of sealed cells remaining is recorded.
A hygienic colony will have uncapped and removed over 95% of the
frozen brood within 48 hours. A non-hygienic colony will take over
six days to completely remove the frozen brood. The speed with which
a colony removed dead brood is correlated with its ability to remove
diseased and parasitized brood.
2. Liquid nitrogen: Freezing the brood with liquid
nitrogen is more efficient and less destructive to the combs than
cutting, freezing and replacing comb inserts. Liquid N2
is relatively inexpensive and easy to obtain; check with your local
gas and welding suppliers, veterinary practice, or livestock artificial
inseminating firm. There are no laws in any state restricting the
use of industrial grade liquid N2 by individuals. It
must be kept in an appropriate tank (e.g., a Dewar tank, which can
be purchased through gas and welding supply houses), and the tank
should be securely fastened to the truck during travel to avoid
spillage.
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Photo C. A 3" PVC tube
is twisted into the comb down to the midrib. The liquid N2
is slowly poured into the tube. |
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Common sense and several precautions must be used when handling
liquid nitrogen. It has a boiling temperature of -320°F, which
means that it is extremely cold and will kill skin (causing severe
frostbite) on contact. We recommend that users read the Material
Safety Data Sheet on liquid N2 from the supplier.
You will need to construct (or find) a hollow cylinder into which
you will pour the liquid N2 to freeze a circular section
of sealed brood. We have been using a 3-inch diameter PVC pipe.
The cylinder must be at least 4 inches long because the nitrogen
will boil on contact with the brood.
A minimum of 10 oz of liquid N2 is needed to freeze-kill
all the brood (approximately 160 cells) within a 3-inch diameter
cylinder. A smaller amount will not kill all of the brood, leading
to erroneous results. Use a 10-oz or larger polystyrene foam coffee
cup for measuring and pouring. Other materials will shatter on contact
with the liquid N2.
Select a frame with at least a 3-inch diameter circle of sealed
brood containing fewer than 30 unsealed cells within the circle.
Lay the frame horizontally across a support (i.e. an empty super).
Twist the cylinder into the sealed brood until it reaches the midrib.
Record the number of unsealed cells inside the cylinder. Pour 1.5
to 2 oz. of the liquid N2 into the cylinder and wait
for it to freeze the edges or evaporate. Then pour the remainder
of the liquid N2 into the cylinder. Wait to remove the
cylinder until it thaws, which may take three to five minutes (Photo
C). If you have additional cylinders, you can start the next test
while you are waiting for previous ones to thaw. We put a drawing
pin (thumbtack) in the top of the frame to mark the frame and the
location of the test on the frame. Some hygienic colonies clean
and repair the comb so quickly that it is hard to locate the test
when you return. Place the frame in the center of the brood nest
(Photo D).
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Photo D. Honey bees bred
for resistance and certain behaviors can prevent diseases and
parasitic mites. |
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Remove the frame containing the frozen brood 48 hours later, and
record the number of sealed cells remaining within the circle. When
testing a colony that has been re-queened, six to eight weeks must
elapse after requeening for the bees in the colony to be daughters
of the new queen (Photos E-1 and E-2).
Important note: Both of these tests should be repeated on the same
colony, and it will be noticed that the results between tests may
vary. For example, a colony may remove 95% of the frozen brood on
the first test, but only 50% on the second. This colony is not hygienic!
It is very important that colonies be considered hygienic only if
they remove >95% of the brood on two consecutive tests. The speed
with which a colony removed dead brood is correlated with its ability
to remove diseased and parasitized brood.
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