Corn Rootworm Processing

Corn rootworm beetles are processed in acid to free pollen from the digestive system. The residue left after the procedure is mounted onto a glass slide and then scanned for pollen. To process corn rootworms, use the following protocol:

TURN ON HOT BLOCK ALL THE WAY (~100 degrees Celsius)

1. Thaw corn rootworms if frozen.

2. Clear area to work.

3. Check safety equipment- gloves, face mask, lab coat.

4. Check for chemicals - ETOH, Sulfuric Acid, Acetic Anhydride, Glacial Acetic Acid, Sodium Bicarbonate, distilled water, Glycerin, Safranin stain.

5. Put centrifuge and vortex under hood.

6. Take water out from under hood.

7. Take out dissecting tray, forceps, and sticks

8. Place CRW(s) in eppendorph  tubes.

9. Crush CRW's with wooden stick.

10. Make sure water is removed from the hood and water is turned off! Prepare enough acetolysis to fill the tubes 3/4ths.(40 mL for 56 samples)

(USE FACEMASK).

• Add 90% Acetic Anhydride
• Add 10% Sulfuric Acid

11. Take sticks out and lay on clean surface in order.

12. Add acetolysis mixture to samples. Put in hot block for 12 min and stir with stick (use sticks on hood lip first ) every 3 min. -REMOVE STICK EACH TIME-

13. Remove from hot block and add glacial acetic acid and stir very well

14. spin 1.5 min

15. decant,

16. vortex

17. add water

18. spin 1.5 min

19. decant,

20. vortex

21. Repeat Steps 17 - 20.

26. add ETOH, spin 1.5 min

27. decant (be careful when pouring out ETOH)

28. vortex

29. add 2 drops of safranin stain and 2 drops glycerin to vials

28. stir with stick (leave in vial)

29. place on hot block overnight at about 30°C, warm to touch.

Do not leave pollen in ETOH. If you must leave them, try to leave them in water.