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Boll Weevil Processing

Boll weevil adults are processed in acid to free the pollen in the digestive system. The residue left after the procedure is mounted onto a glass slide and then scanned for pollen. To process boll weevils, use the following protocol:

Processing a boll weevil crop in an eppendorph tube. Processing whole boll weevils in 12mL test tubes.
TURN ON HOT BLOCK TO 100 degrees Celsius
1. Clear area to work.
2. Check safety equipment- gloves, face mask, lab coat.
3. Check for chemicals - ETOH, Sulfuric Acid, Acetic Anhydride, Glacial Acetic Acid, Sodium Bicarbonate, distilled water, Glycerin, Safranin stain.
4. Put centrifuge and vortex under hood.
5. Take water out from under hood.
6. Spin down 1.5 min
7. Decant carefully, do not pour out crop (white or brown)
8. Add water to top of ridges
9. Decant-vortex
10. Add Glacial Acetic Acid to top of ridges
11. Spin 1.5 min
12. decant, vortex
13. Add acetolysis mixture (9:1 acetic anhydride: sulfuric acid) pipette in ca. 3/4 mm to bottom of ridges
14. put in hot block for 12 min stir with stick every 4 min -REMOVE STICK EACH TIME-
15. add glacial acetic acid and stir very well
16. spin 1.5 min
17. decant, vortex
18. add water
19. spin 1.5 min
20. decant, vortex REPEAT STEPS 13-15
21. add ETOH, spin 1.5 min, decant, vortex (be careful when pouring out ETOH the pollen has a tendency to come out)
22. add 2 drops of saffrin stain and 2 drops of  glycerin
23. stir with stick (leave in tube)
24. place on hot block overnight at about 30°C, warm to touch.
TURN ON HOT BLOCK TO 100 degrees Celsius
1. Thaw boll weevils 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 wooden sticks
8. Place B.W.(s) in test tube with a wooden stick on top.
9. Crush B.W.'s with stick.
10. Make sure water is removed from the hood and water is turned off! Prepare acetolysis (USE FACEMASK).
  • Mark three 150 mL beakers, 1 250 mL beaker for waste acid, 1 600 mL beaker for sodium bicarbonate and 1 graduated cylinder.
  • Add 90% Acetic Anhydride
  • Add 10% H2SO4
  • Pour into graduated cylinder.
  • Caution water reacts violently with Acetolysis mixture!
11. Take sticks out and lay on clean surface in order.
12. Add acetolysis mixture to test tubes . put in hot block for 15 min stir with stick (use sticks on hood lip first ) every 3 min. -REMOVE STICK EACH TIME and place in Sodium Bicarbonate-
13. Add glacial acetic acid and stir very well
14. spin 3 min
15. decant,
16. vortex
17. add water
18. spin 3 min
19. decant,
20. vortex
21. REPEAT STEPS 17-20
25. Strain contents on 450 micrometer screen into beaker
26. discard material on screen
27. pour beaker back into test tube and spin for 3 min.
28. decant
29. repeat step 18 until beaker is empty
30. add ETOH, spin 3 min
31. decant
32. vortex (be careful when pouring out ETOH)
33. Pour into 1 dram vials with alcohol, label vials
34. add 2 drops of safranin stain and 2 drops glycerin to vials
35. stir with stick (leave in vial)
36. 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.