Spheroplast production by lysozyme and ethylenediaminetetraacetate (EDTA) was examined as a means of obtaining osmotically sensitive cells for studies of enzyme localization. Physiologically young cells plasmolyzed with 0.5 M sucrose in 0.01 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7, 8, or 9) were quantitatively converted to plasmolyzed osmotically sensitive rods after lysozyme treatment. Although such cells were osmotically sensitive, a 1:1 dilution in Tris buffer was necessary for conversion of rods into spheroplasts. Addition of EDTA resulted in a rapid conversion of the plasmolyzed spheroplasts into spherical structures devoid of a plasmolysis vacuole. These structures, which we call EDTA-lysozyme spheroplasts, contained a number of attached membranes. We believe that this conversion results from a weakening of the outer trilaminar component of the cell wall by EDTA, resulting in the collapse of the plasmolysis vacuole. Dilution of sucrose below 0.15 M also resulted in the collapse of the plasmolysis vacuole. Both the lysozyme spheroplasts and the EDTA-lysozyme spheroplasts were osmotically sensitive. Thin sections of the EDTA-lysozyme spheroplasts demonstrated that the outer trilaminar component of the cell wall was broken, exposing large areas of the cytoplasmic membrane to the environment.

Wilson, University of Wisconsin. centrifugation packs the cells but permits the slime to be decanted. The washed cells were used immediately or stored at-10 C. Viable cells were counted by spreading 0.10 ml aliquots of appropriate dilutions on plates of Burk's medium containing agar. Direct counts were made in a Spencer bright-line hemocytometer for phase microscopy with a Zeiss Ph 2 objective and a 10 X compensating ocular. Turbidity was determined with a Klett-Summerson colorimeter using a red filter.

Several procedures were evaluated for separating and concentrating indigenous microorganisms from soil without the occurrence of growth. Electron microscopy of nontangential, thin sections through these cells revealed that all of the cells examined were less than 0.9 gm in diameter, and up to 72% were "dwarf' cells less than 0.3 gim in diameter. Some were small enough that they should not be resolved with the light microscope. Approximately 27% had a fine structure bearing some resemblance to that of a bacterial cyst or microcyst, but this value may be low because cells having their outer layers partially stripped off were not included in the count. Approximately 25% showed a distinct periplasmic space, which often contained stainable material. Other fine structure features are presented together with frequencies of occurrence for the populations examined.

COTA-ROBLES, EUGENE H. (University of California, Riverside). Electron microscopy of plasmolysis in Esche7ichia coli. J. Bacteriol. 85:499-503. 1963.-Escherichia coli cells plasmolyzed in 0.35 M sucrose reveal plasmolysis at one tip of a cell or in the center of dividing cells in which protoplast partition has been complete. Central plasmolysis reveals that protol)last

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