Protoplast and Cytoplasmic Membrane Preparations from Streptococcus sanguis and Streptococcus mutans

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Abstract
Protoplasts were prepared from S. 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 2 additional steps: plasmolysis (in 12% polyethylene glycol) prior to addition of lysozyme and a swirling phase after the enzymic action. This procedure yielded 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.