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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
Matrix | Sample | Bead Mixture |
Membrane Filter | 1/4 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. |
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 |