7.2.1. Primary extract
1. Mix 2 ml of extraction
buffer (Table 4) per 1 g of bee tissue.
2. Prepare a primary virus extract by either:
- Grinding the bee tissues in liquid nitrogen in a mortar-and-pestle.
- Liquidizing in an automatic blender.
Using a large-volume bead mill.
(see also section 8.2.; “Sample Homogenisation”)
3. Transfer to a solvent-resistant container.
4. Add 0.5 ml of chloroform or carbon tetra-chloride per 1 g of bee tissue.
5. Shake vigorously by hand.
6. Centrifuge at 8,000 g and 4oC for 15 minutes.
7. Carefully collect the supernatant.
8. Discard the organic phase.
9. Remove 10 µl for virus analysis by RT-qPCR or ELISA (see section 9.3. and the BEEBOOK article on molecular methods (Evans et al., 2013) to determine the viral purity of the extract.
10. The crude extract at this stage is appropriate for long-term storage. Add glycerol to a final concentration of 50%, aliquot and store at -80oC.