Bead Beating: A Primer (continued)
Environmental Sample Homogenization
Air, water, soil, sediments, and surfaces supporting
biofilm can loosely be grouped as environmental samples.
A common characteristic of environmental samples is the presence of both
biotic and abiotic components, with the former representing a diverse
collection or community of microbes, animals and plants (to various
degrees). Depending upon the density of the biotic
component, environmental samples are often concentrated by centrifugation or
filtration prior to homogenization. The biotic component
of air and water can be concentrated by membrane filtration. The filter and retained materials are then subsequently homogenized
by bead beating. Water can also be centrifuged to pellet
microorganisms, though this can be impractical with larger volumes. Biofilms
grown on the inside surface of glass tubes can be disrupted by placing the
tube in a vial with a bead mixture. The homogenization of
soil and sediments must not only disrupt the biological component, but also
disaggregate soil particles that may house microorganisms. Soil and sediments can be processed directly with mixed beads and an
In all cases, environmental samples usually require a
mixture of different beads and balls to effectively disrupt the range of
organisms and particles present. Samples with significant
amounts of abiotic matrices can be homogenized with a combination of one or
more large beads to facilitate general breakage. A
mixture may include a couple of large balls (e.g., 4 mm) to break up soil
particles, beads ranging in size from 800 µm – 1.4 mm to facilitate fungi
breakage and 100 µm beads to disrupt bacteria. Depending upon the beat
beater, samples can be processed in disruption tubes, deep well plates, 4 ml
polycarbonate vials, and 15 ml polycarbonate vials.
mixture containing small (100 µm), medium (400-800 µm), and large (1.4 mm to
4 mm) beads is recommended for environmental samples.
Typically, the mixture contains many small beads and only one or two large
beads. Following are recommended bead combinations for
homogenization in 2 ml disruption tubes:
¼ section of 47 mm membrane filter in 2
ml disruption tube
100 mg of each 100 µm and 400 µm silica
250 mg in 2 ml disruption tube
100 mg each of 100 µm silica and 500 mg
of 400 µm beads and two 4 mm glass beads
Stationary phase with biofilm which fits
in a tube or vial (objective is to remove the film from the
1/6 volume of 1.0 mm zirconium beads.
Subsequent processing may require smaller beads.
||Prepare samples as described above and add extraction
buffer. The recipe and volume of the buffer will vary
based on subsequent processing steps, but, in general, the volume should be
kept between 0.5 and 1.0 ml. For larger vials, the amount
of sample and beads can be scaled up accordingly.
samples on high speed for 2 minutes. For heat labile
samples, shorter processing bursts can be alternated with cooling periods,
e.g., process for 15 seconds then cool for 2 minutes.
slower high throughput machines may need longer processing times than the HT
Mini™ and HT 24™.