Procedure for quantifying the level of colony infection

  1. Remove one brood comb from the hive containing 5th instar larvae that have not yet been sealed (i.e., the larvae are close to being capped with wax)
  2. Mark on a plastic transparency the area with these unsealed larvae (this step is only important if the frame also contains capped brood)
  3. Return the brood comb to the colony
  4. Remove the brood comb from the hive a maximum of 20 hours later
  5. Cut a piece of comb containing at least 100 recently capped cells (capped within the last 20 hours). These larvae can be identified using the plastic transparency. (It is best to remove unsealed brood from the comb before introducing into incubator)
  6. Place one or more pieces of comb with capped cells in an incubator at 25ºC at approximately 65 % humidity for 5 days. The chilling will ensure disease development. An open water bottle placed in the incubator will provide sufficient humidity
  7. After 5 days, open all capped cells and record the results
  8. Repeat the experiment three times to obtain individuals from enough