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Mycobacterium Cell Disruption Methods

Mycobacterium sp. are distinct microorganisms from typical gram positive and gram negative bacteria as they have a heavy outer layer composed of lipids, mycolic acids (long chain fatty acids), polysaccharides (arabinoglycan), and peptidoglycan.  This layered cell wall is resilient to chemical and enzymatic lysis, thus mechanical breakage is typically used for the extraction of nucleic acids and protein. Typical methods for disrupting cells include bead beating and sonication.  With bead beating, the most efficient lysis occurs when using 100 µm beads, though larger beads will work as well.

References

Yanna Liang, Dale R. Gardner, Charles D. Miller, Dong Chen, Anne J. Anderson, Bart C. Weimer, and Ronald C. Sims. Study of Biochemical Pathways and Enzymes Involved in Pyrene Degradation by Mycobacterium sp. Strain KMS. AEM (2006) p. 7821-7828, Vol. 72, No. 12

    Protocol - Bead Beating

  1. Using either 100 µm zirconium or silica beads, fill a microfuge tube or microtiter plate well approximately 1/6 full with beads.  Beads are available in bulk in three grades (acid washed, molecular biology grade, and low binding) or in pre-filled disruption tubes.

  2. Add sample containing the bacteria so that the tube or well is no more than 1/2 full.  Depending upon the goal, cells may be homogenized in a DNA/RNA lysis buffer or homogenization buffer for protein isolation.  In either case, avoid buffers with detergents as foaming during bead beating will hinder disruption.  If detergents are needed for subsequent steps in the process, they can always be added after the bead beating step.

  3. Cap the tubes or seal the plate with a push on mat or strip caps.  For microwell plates, the cap must fit tightly to avoid leakage.

  4. Place the tubes in the bead beater (such as a Pulsing Vortexer or HT Mini) and plates in a high throughput homogenizer.  Lock in place and run for 2-5 minutes on high speed.

  5. Place the tubes/plates in a centrifuge and spin to pellet cell debris.  The supernatant can be removed to a different tube or plate for subsequent processing.