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Bead Beating: A Primer (continued)

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MICROORGANISMS

Fungal Homogenization

Filamentous fungi, pseudomycelia and fungal bodies can be homogenized with a variety of beads. Hyphae are relatively easy to shear, while dense mycelia and fruiting bodies (e.g., mushrooms, bracket fungi) can be much more difficult. Loosely packed mycelia are effectively disrupted with 800 µm zirconium or silica beads. However, as the density of the mycelium increases, as with fruiting bodies, larger beads and small grinding balls are needed to disrupt the thallus and shear the cells. Loosely packed mycelia can be homogenized with 1.7 mm zirconium beads, while solid fruiting bodies can be disrupted with 2.8 mm stainless steel balls. It is possible that fruiting bodies and other complex structures like lichens might require a mixture of beads and balls, much like environmental samples.Low binding beads can be used with dilute cultures to minimize loss of analytes. Depending upon the beat 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. Filamentous fungi can be difficult to sample as they tend to grow best as pellicles and on solid media. Some fungi will grow in liquid culture but, generally, not as dispersed cells. Consequently, mycelium must be collected as a mass of cells for homogenization. Tightly packed mycelia can be cut with scissors or a scalpel, while fungi cultured in broth can be collected by centrifugation or filtration. Pellicles can often be collected by plucking these from liquid cultures or scraping off membrane filters.
2. Prepare samples according to the following table:
Sample Type Disruption Tubes (2 ml) Deep Well Plates (square wells) 4 ml Vials 15 ml Vials
Mycelium
Pellet Size 50 µl 50 µl Up to 10 ml Up to 50 ml
Bead Size 800 µm Zr 800 µm Zr 800 µm Zr 800 µm Zr
Bead Mass 600 mg 600 mg 1200 mg 3 gm
Pellicle
Wet Weight 200 mg 200 mg 500 mg Up to 2 gm
Bead Size 1.7 mm Zr 1.7 mm Zr 1.7 mm Zr 1.7 mm Zr
Bead Mass 570 mg 570 mg 1200 mg 3 gm
Thallus
Wet Weight 200 mg 200 mg 500 mg Up to 2 gm
Ball Size 2.8 mm SS 2.8 mm SS 2.8 mm SS 2.8 mm SS
Number of Balls 8 8 15 30
Processing Time 2-5 minutes 2-5 minutes 5 minutes 5-10 minutes
Buffer Volume 600 µl 600 µl 1.5 ml 6 ml
Bead Beater Speed High High High High
3. Process the samples as stated in the table above. If pellicles are very dense, process as if they were a thallus, using 2.8 mm stainless steel balls. For heat sensitive samples, the processing can be divided into short bursts followed by a cooling period.
4. Sample homogenization can be monitored visually using a microscope. Hyphae should appear highly fragmented. Thallus should appear as a homogeneous slurry. If the sample is inadequately homogenized, repeat the processing and reassess the effectiveness.
5. Lysate can then be processed as needed.

Suggested Products

Beads: 800 µm Zirconium (BAWZ 800-250-30) for mycelia; 1.7 mm Zirconium (BAWZ 1700-300-22) for fungal balls, and 2.8 mm stainless steel balls (GBSS 089-1000-07) for thallus (e.g., mushroom)
Pre-filled Tubes: 800 µm Zirconium (PFAW 400-100-30) for mycelia; 1.7 mm Zirconium (PFAW 200-100-03) for pellicle, and 2.8 mm stainless steel (PFSS 2800-50-20) for thallus
Homogenization Vessel: See Appendix A
Homogenizers: See Appendix A