Hygienic Behavior Test Using Liquid Nitrogen
Stu pouring liquid nitrogen for a Hygienic Behavior test
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Liquid Nitrogen
This method of testing involves pouring liquid nitrogen ( about 300 ml = 10 oz. ) into a tin can (3″ diameter) with both top & bottom removed which is firmly set into a patch of capped brood containing approximately 100 cells. After 5 min. the can is removed and the frame returned to the colony. The amount of brood removed after 48 hrs. is then counted and recorded. 95% or greater removal is desired. This test needs to be repeated on the colony to confirm results. 8 weeks should elapse before testing a new queen to allow for the worker turnover. Liquid nitrogen is hazardous in that it can cause freezing burns and possibly asphyxiation if in a confined area without proper ventilation. Leather gloves, tongs, and goggles are necessary safety precautions. In addition, training in this procedure is recommended if one decides to use it.
Liqid nitrogen being poured onto capped brood for a hygienic behavior test.

Non-hygienic bees after 48 hrs.

Hygienic bees after 48 hrs.

Cutting and Freezing Brood
A section of capped brood containing 100 cells can also be cut from the comb, frozen in a deep freezer for 24-48 hrs., returned to the frame, and checked 48 hrs. later for the percentage of removal. The dangers surrounding liquid nitrogen are avoided with this test; however it is more time consuming and laborous. This test should also be repeated to verify results.
Cut-out and frozen brood reinserted into the frame for a hygienic test.
The Pin Method
Straight pins are inserted into capped brood to kill the pupae. This test is not considered completely reliable and the destroyed pupae should be checked in 24 hrs. instead of 48. Test cells are often marked with a pin having a colored head.
Marker pin placed over pin-killed brood cells.
Recapping and Other Thoughts on Hygienic Behavior
The uncapping and recapping behaviors of bees are also possibly indicative of hygienic behavior. However this behavior is very hard if not impossible to quantify or measure. Therefore it does not lend itself to testing; but it is something that can be observed. Hygienic behavior itself comes in various forms mostly based on olfaction to detect disease, mites, SHB eggs, etc. Larry Connor, in ABJ, made some interesting points about resistance mechanisms being varied and all are desirable. Grooming behavior and all other resistance mechanisms used by bees should not be overlooked by concentrating solely on one particular behavior. We don’t know what other weapons bees have to keep themselves healthy and bees with as many possible means of defense are probably better bees since there is no perfect bee. This is also a good arguement for diversity as different traits are expressed by each different group of sister workers a well-mated queen will produce.