Permeabilization of Whole Cells to Lysozyme
Categorized: Outer Membranes
- Grow cells to mid log phase in Luria Broth.
- 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
- Measure OD600 of cells only 10 seconds.
- Add lysozyme to give a concentration of 50 µg/ml. (measure OD 10 seconds)
- Add permeabilising compound. (EDTA, gentamicin, or polymyxin B) (measure OD for at least 1 minute).
- Lysis is measured as the decrease in OD600.
- Measure OD of controls:
- cells + antibiotic (no lysozyme)
- buffer + lysozyme + antibiotic (no cells)
- 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.