Quantifying hygienic behaviour via the freeze-killed
brood assay
The level of hygienic behaviour in each colony was
determined used the FKB bioassay (Spivak & Reuter,
1998a), which was carried out three times at weekly
intervals in July and August 2012. In each colony, two
patches of capped brood, located on the same area on
opposite sides of the frame, were located on a single
frame that contained brood of all stages (eggs, small
and large uncapped larvae, capped cells). A metal cylinder
(6.5 cm diameter 9 8 cm height) was pressed into
the comb until it reached the mid-rib. Approximately
300 mL of liquid nitrogen was poured into the cylinder
to kill the circle of brood inside the cylinder. After
5–10 min, the nitrogen had evaporated and the cylinder
was removed, and the other side was then treated.
Photographs of each patch and the whole frame were
made before returning the frame to the hive. After
48 h, the frame was removed from the hive and more
photographs were taken. From the photographs, we
determined the proportion of capped cells from which
the FKB had been removed.