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The cell walls of a selected isolate ofStaphylococcus aureus FDA 209P were observed undergoing progressive disintegration when exposed to lysostaphin (1 unit/ml) in 24% NaCl solution. Electron micrographs of ultrathin sections of test cells after exposure to lysostaphin for 2 min showed only superficial evidence of lytic damage. However, an average of 89% of these cells were osmotically fragile, and 21% were damaged beyond their capacity to regenerate cell walls and to grow as normal staphylococci. The 68% (average) of the osmotically fragile cells which retained the capacity to revert to normal staphylococci were designated spheroplasts. Neither perforations of the cell walls nor separation of the cell walls from the plasma membranes were observed in the micrographs of these 2-min spheroplasts. Thus, it appears that the osmotic fragility of these and possibly all lysostaphin-induced staphylococcal spheroplasts results from the hydrolysis of a critical number of the pentapeptide cross-linkages of the murein of the cell wall. Electron micrographs of cells exposed to lysostaphin for 5 to 10 min showed perforations and more extensive damage, including the separation of walls from the plasma membranes and the disintegration of large sections of the walls. Smaller numbers of spheroplasts (21 and 8%) were recovered from these 5- and 10-min preparations; those recovered probably represent cells which were attacked more slowly than the majority by the lytic enzyme. The nonrevertible, osmotically fragile cells that retained segments of cell wall were designated protoplast-like bodies. After 20-min exposure to lysostaphin, all of the cell wall was digested away from most of the cells, and true staphylococcal protoplasts were produced. These lysostaphin-induced, osmotically fragile forms appear to have different osmotic properties from the staphylococcal “protoplasts” reported by other investigators and should serve as the basis for a variety of fundamental investigations.

Electron Microscopy and Viability of Lysostaphin-induced Staphylococcal Spheroplasts, Protoplast-like Bodies, and Protoplasts