Bead Beating: A Primer (continued)
MICROORGANISMS
Yeast Homogenization
Yeast is a vegetative single cell fungus that can be
effectively cracked open via bead beating. Small yeast cells, such as
Pichia, are best homogenized with 200 µm zirconium beads. Larger yeast
cells, such as Saccharomyces, are best disrupted with 400 µm silica
or zirconium beads. Yeast samples with low cell numbers can be homogenized
with low binding beads, which lose fewer analytes to non-specific
adsorption. Depending upon the bead beater, samples can be processed in 2 ml
disruption tubes, deep well plates, 4 ml polycarbonate or polyethylene
vials, and 15 ml polycarbonate vials.
Procedures
| 1. |
Both
dilute and concentrated yeast can be homogenized effectively.
Pellet yeast 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.
Resuspended yeast pellets can be very viscous, which will work against
efficient disruption. Generally, the pellet from 2 ml of
cultured broth can be disrupted in 2 ml tubes or plates, cells from up to 15
ml culture broth in 4 ml vials, and a pellet from up to 100 ml culture broth
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 (400 µm beads)
|
400 µl
|
300 µl
|
0.8 ml
|
3 ml
|
|
Processing Time
|
5 minutes
|
5 minutes
|
5-10 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. |
Disruption of yeast can be monitored microscopically.
Using phase contrast, intact yeast cells appear refractile (bright) as the
cells act like a small lens. Disrupted yeast cells look black
or gray, which are often termed “ghosts”. Visually
inspect the yeast cells (dilution may be needed) and count or estimate the
percentage of ghost cells in the population. Typically,
bead beating should yield 90% or greater ghost cells. |
| 5. |
Continue
cell processing if sufficient lysis has not occurred. |
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