3.3.4.2. Freeze-killed brood assay: freezing brood within comb using liquid N2

  1. Liquid nitrogen must be kept in an appropriate tank (e g. a Dewar tank) and gloves should be used when handling liquid N2 (Fig. 15)
  2. Make a 75 mm diameter tube to pour the liquid nitrogen directly on the comb. A metal vent pipe or PVC plumbing pipe can be used. A wider tube will reduce leakage of the nitrogen through empty cells along the perimeter. The tube should be at least 100 mm long.
  3. Find a section of sealed brood with purple eyed pupae to freeze.
  4. Put the frame horizontally across a support (i.e. an empty super). Press the tube down to the midrib of the comb with a twisting motion until it seals.
  5. Record the number of unsealed cells inside the cylinder.
  6. Pour 300-400 ml of liquid nitrogen into the tube. Less liquid N2 may not freeze-kill the brood. Use a 300 ml or larger polystyrene foam (coffee) cup for measuring and pouring. First pour about 5 mm of the liquid nitrogen in the tube. When it evaporates pour the rest.
  7. Wait for the liquid nitrogen to evaporate and the tube to thaw before trying to remove it (may take 10 min or more).
  8. Return the frames to the colony for 24 hours.
  9. The tests should be repeated on the same colony at least twice.
  10. 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.
  11. 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

Historically, colonies that removed freeze-killed brood within 48 hours were considered hygienic, and if they took more than a week, they were considered non-hygienic (Gilliam et al., 1983). There is, however, a better correlation between the removal of freeze-killed brood and disease resistance when only the removal of freeze-killed brood within 24 hours is considered (Spivak, unpublished data).

Fig. 15. Freeze-killed brood assay: freezing brood within comb using liquid N2. Left:  Dewar tank with valve to dispense liquid nitrogen, polystyrene foam cups for pouring liquid N2 into PVC pipes (black pipes in combs).  Right:  After 24 hours, this hygienic colony uncapped and removed >95% of the freeze-killed brood. Photos: M Spivak.

Figure 15