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Bead Beating: A Primer (continued)

PLANTS

Pollen Homogenization

Pollen has a rigid cell wall that is most efficiently disrupted with zirconium beads. As the size range of pollen is very broad, bead size must be matched to the type of pollen. Undersized beads will have insufficient mass to crack open pollen, while using large beads will result in too few collisions. Pollen less than 10 µm in size should be disrupted with 200 µm zirconium beads, while pollen 10-50 µm in size should be processed with 400 µm zirconium beads. Large pollen (=50 µm) is most effectively homogenized with 800 µm zirconium beads. Depending upon the bead beater, samples can be processed in disruption tubes, deep well plates, 4 ml polycarbonate or polyethylene vials, and 15 ml polycarbonate vials.

Procedures

1. Pollen can be effectively disrupted by bead beating, though the choice of beads is dependent on the size of the pollen granules.  Pollen can be collected as a dry powder and weighed for processing.  The beads used to bead beat pollen are 100 µm zirconium (for pollen <10 µm), 400 µm zirconium (for pollen 10-50 µm), and 800 µm zirconium (for pollen larger than 50 µm).
2. Prepare samples according to the following table:
Disruption Tubes (2 ml) Deep Well Plates (square wells) 4 ml Vials 15 ml Vials
Sample Mass 10 mg 10 mg Up to 25 mg Up to 100 mg
Buffer Volume 500 µl 500 µl 1 ml 5 ml
Bead Volume 400 µl 300 µl 1 ml 3 ml
Processing Time 2-5 minutes 2-5 minutes 5 minutes 5-10 minutes
Bead Beater Speed High High High High
3. Process the samples as stated in the table. For heat sensitive samples, the processing can be divided into short bursts followed by a cooling period.
4. Ruptured pollen grains appear with a crack or split. Visual inspection can be used to estimate homogenization efficiency using a microscope (number of cracked grains/total grains in the field of view). If the efficiency is less than 60%, continue to homogenize the sample.
5. Lysate can then be processed as needed.

Suggested Products

Beads: 100 µm Zirconium (BAWZ 100-250-15), 400 µm Zirconium (BAWZ 400-250-35), 800 micron Zirconium (BAWZ 800-250-30)
Pre-filled Tubes: 100 µm Zirconium (PFAW 100-100-02), 400 micron Zirconium (PFAW 400-100-30), 800 µm Zirconium (PFAW 800-100-29)
Homogenization Vessel: See Appendix A
Homogenizers: See Appendix A