184.108.40.206. Direct exposure of individual larvae
- Prepare a spore solution of 2 x108 spores/ml in sterile distilled water.
- Mix (1:1, v:v) of the solution with honey food (50 % honey : 50 % distilled sterile water)
- Remove sections of comb containing 5th instar larvae from the hive.
- Supply each of the 5th instar larvae with 5 µl of contaminated food (equals a dose of 5 x 105 spores per larvae)
- Place the food near the mouthpart. Use extreme care to avoid touching the larvae and confirm consumption by direct observation
- Immediately after feeding, store the pieces of comb for 2 hours at 25ºC
- Return the comb to the hive so that the bees will cap the cells containing the inoculated larvae
- 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
- After 5 days open all cell cappings and record the results. (See paragraph 3.1. Quantify infection degree in colonies)