Bead Beating: A Primer (Part 3)
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
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: |
Protocol 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 |
Protocol Number
| 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. |
