Direct exposure of individual larvae

  1. Prepare a spore solution of 2 x108 spores/ml in sterile distilled water. 
  2. Mix (1:1, v:v) of the solution with honey food (50 % honey : 50 % distilled sterile water)
  3. Remove sections of comb containing 5th instar larvae from the hive.
  4. Supply each of the 5th instar larvae with 5 µl of contaminated food (equals a dose of 5 x 105 spores per larvae)
  5. Place the food near the mouthpart. Use extreme care to avoid touching the larvae and confirm consumption by direct observation
  6. Immediately after feeding, store the pieces of comb for 2 hours at 25ºC
  7. Return the comb to the hive so that the bees will cap the cells containing the inoculated larvae
  8. Remove the comb a maximum of 20 hours later and store it at 25ºC and approximately 65 % humidity for 5 days. The chilling of the larvae will ensure disease development.  An open water bottle placed in the incubator will provide sufficient humidity
  9. After 5 days open all cell cappings and record the results. (See paragraph 3.1. Quantify infection degree in colonies)