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Bead Beating: A Primer (continued)

MICROORGANISMS

Bacterial Homogenization

Small size and cell concentration/density are important parameters to consider when bead beating bacteria. Whether disrupting bacterial cultures directly or following concentration by centrifugation, bead beating of bacteria is most effective with 100 µm zirconium or silica beads. With dilute samples, low binding beads will reduce the loss of analytes during processing. Depending upon the beat beater, samples can be processed in 0.5 ml or 2 ml disruption tubes, deep well plates, 4 ml polycarbonate or polyethylene vials, and 15 ml polycarbonate vials. Larger volumes or samples with high concentrations of cells typically require longer processing times. Likewise, linear homogenizers require longer processing times than the HT Mini™ or HT 24™.

Procedure

1. Both dilute and concentrated bacteria can be homogenized effectively. Pellet bacterial cells by centrifugation and resuspend in a suitable homogenization buffer. It is important to limit the size of a pellet that is resuspended for bead beating. Samples that are too thick are difficult to disrupt since the small beads must not only crack cells, but also fight against the viscosity of the suspension. Generally, the pellet from 2 ml of cultured broth can be disrupted in 2 ml tubes or plates, while the pellet from up to 15 ml of culture broth can be disrupted in 4 ml vials; a pellet from up to 100 ml culture broth can be disrupted in 15 ml vials.
2. Prepare samples according to the following table:
Disruption Tubes (2 ml) Deep Well Plates (square wells) 4 ml Vials 15 ml Vials
Buffer Volume 600 µl 600 µl 1.5 ml 6 ml
Bead Volume (100 micron beads) 400 µl 300 µl 0.8 ml 3 ml
Processing Time 2-5 minutes 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 (known as pulsing).
4. Sample homogenization cannot be monitored visually because the cells are too small.  Disruption can be checked using a marker enzyme, such as lactate dehydrogenase, which is liberated from cells during processing.
5. Proceed to purification and/or analysis when cells are sufficiently disrupted.

Suggested Products

Beads: 100 µm Zirconium (BAWZ 100-250-15)
Pre-filled Tubes: 100 µm Zirconium (PFAW 100-100-02)
Homogenization Vessel: See Appendix A
Homogenizers: See Appendix A