Bead Beating: A Primer (continued)

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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:
    3. 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.

Suggested Products

Beads: 400 µm Zirconium (BAWZ 400-250-30) for Saccharomyces; 200 µm Zirconium (BAWZ 200-250-07) for Pichia
Pre-filled Tubes: 400 µm Zirconium (PFAW 400-100-30) for Saccharomyces; 200 µm Zirconium (PFAW 200-100-03) for Pichia
Homogenization Vessel: See Appendix A
Homogenizers: See Appendix A

Products

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