Capsules of Diplococcus pneumoniae type I11 and Klebsiella pneumoniae type I were examined in the electron microscope using ruthenium red in combination with osmium tetroxide. The ability of this combination stain and fixative to stain polysaccharides has been well established. A slightly acid pH was necessary to obtain satisfactory capsular polysaccharide staining.The capsules of both Diplococcus pneumoniae and Klebsiella pneurnoniae were stained by the ruthenium red before alcohol dehydration and embedding in Epon 812. The capsule of D. pneumoniae had the appearance of a tightly woven mat. The capsule of K. pneumoniae had a fibrous appearance. Ruthenium red was found inside the plasma membrane of D. pneumoniae, but remained outside the cell membrane of K. pneumoniae.

Slime layers are being studied in our laboratories in an attempt to understand their functions in the control of pollution in natural streams. A method for fixing, staining, and embedding microorganisms in the intact slime has been developed. In this method, epoxy resin discs are placed in a holder and are introduced into a simulated stream. After various periods of time the discs are punched out of the holder into the fixative. The disc with the attached slime is fixed, stained (4% osmium tetroxide plus ruthenium red), dehydrated, and embedded in epoxy resin so that thin sections can be cut through the vertical plane of the slime mass. Such thin sections permit detailed examination of the attached layer, the surface-slime interface, the spatial relationships between cells in the vertical slime structure, and the strands of extracellular material between and around cells. No special attachment structures were noted as the cells appeared to be attached to the surface by extracellular material alone. This material was observed in strands and netlike forms between cells which are positioned 1 to 4,m apart in the slime.

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg2+, the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system. P. aeruginosa on exposure to EDTA. The investigations described in this paper were undertaken, therefore, to elucidate the effect of EDTA on the ultrastructure of the cell envelope of P. aeruginosa.