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Protoplasts were prepared from Streptococcus sanguis and some S. mutans serotypes by use of lysozyme (EC 3.2.1.17) under particular conditions: cells had to be grown in dl-threonine (20 mm) and harvested in early exponential phase. The efficiency of protoplast formation was enhanced by two additional steps: plasmolysis (in 12% PEG), prior to addition of lysozyme, and a swirling phase, after the enzymic action. This procedure allowed us to obtain clean protoplasts, with only 0·5% contamination by bacterial cell walls. Up to 90% protoplast lysis was obtained in 0·5 m-NaCl. Cytoplasmic membrane purification was achieved by centrifugation on a glycerol cushion.
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