3.3.4.1. Freeze-killed brood assay: cutting brood out of comb to freeze

  1. Cut a comb section of sealed brood with purple-eyed pupae containing approximately 100 cells on each side (5 x 6 cm) from a frame and freeze it for 24 hours at -20ºC.
  2. Insert the frozen comb section into a frame of sealed brood in the colony being tested (Fig. 14). 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 (Spivak and Downey, 1998).
  3. Remove the frames no more than 24 hrs later.
  4. Record the number of sealed cells.  In addition, the number of cells that have been partially or fully uncapped and the dead pupae that have not yet been completely removed from the cells can be recorded.
  5. The tests should be repeated on the same colony at least twice
  6. A hygienic colony will have uncapped and completely removed over 95 % of the frozen brood within 24 hours on both tests. This is the most conservative (strict) assay for hygienic behaviour that should be used for breeding purposes.
  7. A less conservative measure of hygienic behaviour calculates the number of frozen pupae completely removed plus those that are in the process of being removed after 24 hours.


Fig. 14. Freeze-killed brood assay:  cutting brood out of comb to freeze. Left: Frozen section of sealed brood is carefully placed into hole cut through comb. Right: Twenty-four hours after being returned to a colony, the amount of freeze-killed brood uncapped and removed is recorded. Photos: M Spivak.

 Figure 14