PERMEABILIZATION OF WHOLE CELLS TO LYSOZYME

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)

1. If compound does show permeabilising activity, measure the effect of adding Mg²⁺.
2. Measure OD₆₀₀ of cells.
3. Add lysozyme and measure OD₆₀₀.
4. Add Mg²⁺ to final concentrationd of 3mM, 1mM, and 0.1mM; measure OD₆₀₀.
5. Add antibiotic and measure OD₆₀₀.

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Updated 01 December 2000.