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Bead Beating: A Primer (continued)

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MICROORGANISMS

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 extraction buffer.

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.

Procedure

1. A 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:
Matrix Sample Bead Mixture
Membrane Filter ¼ section of 47 mm membrane filter in 2 ml disruption tube 100 mg of each 100 µm and 400 µm silica beads
Soil/Sediment 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
Biofilm Stationary phase with biofilm which fits in a tube or vial (objective is to remove the film from the support). 1/6 volume of 1.0 mm zirconium beads.  Subsequent processing may require smaller beads.
2. 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.
3. Process 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.
4. The slower high throughput machines may need longer processing times than the HT Mini™ and HT 24™.

Suggested Products

Beads: 100 µm Zirconium (BAWZ 100-250-15), 100 µm Silica (BAWG 100-200-10), 400 µm Silica (BAWG 400-200-04), 1.0 mm Zirconium Beads (BAWZ-1000-250-33), 4 mm Glass Beads (BAWG 4000-200-18)
Pre-filled Tubes: Mixed 100 µm, 1.4 mm Zirconium & 4 mm Silica (PFMM 4000-100-28)
Homogenization Vessel: See Appendix A
Homogenizers: See Appendix A