FIG. 1.

Flowchart for optimal fractionation of A. faecalis. All procedures were performed at 30°C. Cells were resuspended in 0.5 M glucose plus 1 mM EDTA in 0.2 M Tris-HCl, pH 7.4 (GET buffer), to a concentration of 1 g (wet weight) of cells per 8 ml of buffer. Lysozyme, which was first suspended in water, was then added to a concentration 1 mg/ml. Phenylmethylsulfonyl fluoride (a protease inhibitor) was added to 100 μg/ml. Under conditions when some release of cytoplasmic components was suspected, 20 mM MgCl₂ plus 10 μg of DNAse I per ml were added the supernatant to reduce viscosity due to release of DNA. Disruption of spheroplasts that were separated from the periplasm was accomplished by a 10-fold osmotic shock followed by mild ultrasonication (30 W of power for 10 min).