Permeabilization of Whole Cells to Lysozyme

Categorized: Outer Membranes

  1. Grow cells to mid log phase in Luria Broth.
  2. Centrifuge and resuspend in 5mM Na-azide, 5 mM Na-Hepes pH 7.0, to about 0.5 OD600. Note: KCN can be substituted for Na-azide
  3. Measure OD600 of cells only 10 seconds.
  4. Add lysozyme to give a concentration of 50 µg/ml. (measure OD 10 seconds)
  5. Add permeabilising compound. (EDTA, gentamicin, or polymyxin B) (measure OD for at least 1 minute).
  6. Lysis is measured as the decrease in OD600.
  7. Measure OD of controls:
  1. cells + antibiotic (no lysozyme)
  2. buffer + lysozyme + antibiotic (no cells)
  3. cells + lysozyme (no antibiotic)
  • If compound does show permeabilising activity, measure the effect of adding Mg2+.
  • Measure OD600 of cells.
  • Add lysozyme and measure OD600.
  • Add Mg2+ to final concentrationd of 3mM, 1mM, and 0.1mM; measure OD600.
  • Add antibiotic and measure OD600.