Preparation of spheroplasts from Streptococcus lactis

Abstract

Stationary phase cells of Streptococcus lactis resisted lysis by lysozyme under a variety of test conditions, although they lost 20% of their hexosamine. Lysozyme was bound by the cells and caused the optical density of the suspensions to rise. Incubation of the cells (0.5 mg dry weight/ml) in buffer with lysozyme (1 mg/ml), NaCl (0.5 M), or sodium dodecylsulfate (0.1%) caused prompt lysis. The cells were stabilized with 10% polyethylene glycol but not by 10–30% sucrose. Lysozyme and NaCl-treated cells in polyethylene glycol were spheroplasts because they were osmotically fragile, lost their shape but retained a rhamnose wall polymer. The spheroplasts retained all the DNA of the original cells, and 10% of the original numbers could be cultured on agar. However, they lost all their muramic acid and 60% of their hexosamine. In contrast to NaCl, sodium dodecylsulfate-treated cells were killed.