2.3.1. Protocol for preparing sterilized tissues

1. Submerge sample (eggs, larvae, pupae, adult) in 0.2 % bleach for 3 min.

2. Rinse 3 times, with filter sterilized water, 1 minute each.

3. Rinse 3 times with 70 % ethanol, 3-4 min each.

4. In sterile hood, remove ethanol

5. Rinse twice with sterile water, 1 min each.

6. Remove water.

7. Place sample on sterile surface, i.e. top of tissue plate lid.

=> If eggs are used: place in depression well of sterilized slide or plate, i.e. black porcelain makes easier to see.

8. Add one drop of medium AmWH5 (Table 1).

9. Use sterile glass rod to gently crush eggs 2-3 taps per egg.

10. Add more medium AmWH5.

11. Using Pasteur glass pipette suck up medium AmWH5 with tissues.

12. Dispense into one well of a 24 multi-well tissue culture plate (3-6 eggs per well).

=> If larvae, pupae, or adults are used: then after surface sterilized, rinsed, and dry, sitting on sterile lid of plate in the hood:

8. Put medium AmWH5 into all the wells of four multi-well tissue culture plate, enough to cover bottom of each well.

9. Using sterilized fine tip metal forceps, grab dorsal surface of one bee abdomen, and tear small opening with second forceps. A haemolymph droplet will form.

10. Gently touch this droplet to the surface of medium to wick it from the bee’s body, into the medium.

11. Readjust your forceps to gently squeeze the abdomen or thorax and a second and third clear droplet can be collected in similar fashion.

12. The first three droplets can all be placed into one well.

13. Squeeze the bee’s body and cloudy droplets are now formed

14. Put these one droplet per well, until you run out of haemolymph.

15. Tear the abdomen from the thorax and head, or if this is a larvae, tear in half.

16. Working with the abdomen first (as the head material often results in contamination): Dip the abdomen in a well, gently shake, move to next well, repeat until no more cells are observed to come off the material (this can fill 1-3 plates).

17. Next process the dorsal half, if an undifferentiated larvae, or work with the thorax if sample is pupae/adult: Tear sample in the medium in well, move to next well, tear and shake, repeat until material is used up (about ½ to 1 full plate).

18. Now the head is last, put into medium and tear apart, makes one to three wells.

19. Plates are labelled genus species abbreviated, Am, Date, body part [haemolymph, head, abdomen, thorax, head (He., ab, Tx, hd)], passage number (P0).