5.3.1. The rearing method

The rearing method used for this test is detailed in Crailsheim et al. (2013), summarized in Fig. 9, and outlined in the steps below.

1) For one replicate, larvae are collected preferably from a unique colony. If two colonies are necessary, larvae originated from both colonies must be distributed in two samples of equal size (24 larvae) in each plate. The colonies have to be healthy and must not show any visible clinical symptoms of pests, pathogens (see BEEBOOK Volume II) and/or toxin stress.

2) Tests are performed with summer larvae during a period from the middle spring to the middle autumn (the exact time of year varies by location).

3) In case of sanitary treatment (i.e. products added to the hive for purposes of disease/pest control), the date of application and the kind of product has to be noted. No treatment should be applied within the 4 weeks preceding the beginning of experiments.

4) The queen is confined in its own colony in an excluder cage containing a comb with emerging worker brood and empty cells for less than 30 hours in order to obtain a large number of fresh laid eggs. According to queen vigour, the queen’s isolation time can be reduced in order to minimize variability in larval size (age).

5) To ensure one obtains enough larvae, it is recommended to isolate the queens in 2 or 3 colonies in the eventuality that one queen lays few or no eggs.

6) The queen is removed from the cage and the caged comb is left in the hive for 3 days until the larvae hatch.

7) At day 1 (D1, Fig. 9), the comb containing fresh laid eggs is carried from the hive to the laboratory (regulated at a constant temperature of 25°C if possible), in a special wooden container in order to avoid temperature variation and to transfer the larvae into individual rearing cells. We recommend crystal polystyrene grafting cells (ref CNE/3, NICOPLAST Society), having an internal diameter of 9 mm.

8) Before use, the cells are submerged for 30 min in 0.4% MBC (methyl benzethonium chloride) in water, and then dried in a laminar-flow hood. MBC can be replaced by chloride tablets generally used for nursing bottle sterilisation.

9) Each cell is placed into a well of a 48-well tissue culture plate, which was previously half filled with a piece of dental roll wetted with 15.5% glycerol in 0.4% MBC.

10) The young larvae are transferred with a grafting tool (a thin paint brush for example) from the frame into individual plastic cells previously filled with 20 μl of diet A (Table 8).

11) The larvae are fed once a day (except day 2) with a micropipette. Diet composition varies according to larval age (Fig. 9, Table 8). The diet is warmed at 34°C prior to each use.

12) The plates are placed into a hermetic Plexiglas desiccator (NALGENE 5314-0120 or 5317-0180 or similar, according to the required volume), provided with a dish filled with K2SO4 saturated solution in order to maintain a water-saturated atmosphere.

13) The desiccator is placed into an incubator at 34 ± 0.5°C. This parameter is crucial considering that susceptibility to a compound may vary significantly according to temperature (Medrzycki et al., 2010).

14) At D7 (pre pupa stage), the plates are transferred into a hermetic container containing a dish filled with a saturated NaCl solution in order to maintain 80% relative humidity. The container is then placed into an incubator at 34°C.

15) At D15, each plate is transferred into a crystal polypropylene box (11 x 15 x 12cm) with a cover aerated with a wire mesh, and containing a piece of comb with a small plastic royal pheromone diffuser in its centre (Bee Boost®), fixed with a wire.

16) Emerging bees are fed with syrup and pollen powder delivered using bird feeders or similar structures. The boxes are kept in the hermetic container.

Fig. 9. Steps of a brood in vitro test.

1298JDE revised Fig9

Table 8. Composition of the diets provided to larvae (Aupinel et al., 2005, summarised in Crailsheim et al. 2013). (Example: to prepare 20g of diet A (Crailsheim et al., 2013). - Mix 1.2 g glucose, 1.2g fructose and 0.2g yeast extract into 7 ml water, and then adjust until 10 ml with water. Mix 10g of this solution with 10g of royal jelly.





Royal jelly (%)




Yeast extract (%)




D glucose (%)




D fructose (%)




Dry matter (%)