Spatial anisotropy in Escherichia coli.

There has been little indication of spatial orientation of structural elements within cells of Escherichia coli. Typical electron micrographs of E. coli indicate a randomly oriented nuclear material embedded in a substantially isotropic cytoplasm (3), although exceptions to this have been reported (2). Interpretation of recent microscopic observations of plasmolyzed cells of E. coli now lead us to suggest a collapse of the cytoplasm into planes which, in connected daughter cells, are at right angles to each other. E. coli B/r from local laboratory stock was grown in air-bubbled nutrient broth at 37 C. At concentrations of 107 to 108 cells/ml, samples were centrifuged and the pellets were suspended in 1 M sucrose. Cells were fixed and observed under dark-phase contrast. Glutaraldehyde, at a final concentration of 2%, served for the initial fixation in preparing the cells for electron microscopy. These cells were collected on a membrane filter (Millipore Corp., Bedford, Mass.; 0.22,m pore size) by aspiration at 10 to 25 cm of Hg, postfixed on the filter for 1 hr in 1% OS04, and then prepared for sectioning as described elsewhere (1). Thin sections giving gold-to-silver interference colors were double-stained in uranyl acetate and lead citrate. A phase photomicrograph of normal cells is shown in Fig. la. The two pairs shown in Fig. lb represent common forms following plasmolysis, each with one daughter that appears normal and one which shows bright rather than dark contrast. Similar pairs could be observed moving in the medium; however, in these instances the areas of bright and dark contrast repeatedly reversed their relative positions. Maximal contrast occurred simultaneously in daughter cells, one being brighest when the other was darkest. We suggest that plasmolysis resulted in the cytoplasm collapsing into planes and that, in daughter cells, these planes were at right angles to each other. Observations normal to and along such planes involved different optical paths (concentration of cytoplasm X refractive increment X distance). The optical path across the plane was changed little, since the increased concentration was compensated for by a decreased distance, whereas this compensation was absent for the optical path along the plane. Normal darkcontrast phase microscopy assumes optical paths through the cells to be only slightly different from that through the medium. When these conditions are not met, the destructive interference required to form a dark image is lacking, and constructive interference may lead to the bright images of Fig. lb. As cells rotated about their long axis, the direction of observation with respect to these planes changed and resulted in the observed contrast reversal. Electron microscopy lends additional weight to this interpretation (Fig. lc, d, e, and f). When stained for the same period of time, the plasmolyzed cells revealed a more electron-dense cytoplasm than the corresponding controls. Crosssections verify that plasmolysis did involve a cytoplasmic collapse into planes and that the collapse could involve the cell wall as well. Longitudinal sections confirmed expectations based on the proposed hypothesis. It is not clear whether the source of this orientation lies within the cytoplasm, the nuclear material, or at the inner surface of the cell envelope. Even if it is not present in normal cells, the factors providing orientation under plasmolysis must exist. Perhaps the planes are related to some aspect of deoxyribonucleic acid orientation, such as the points of contact with the plasma membrane, and that the replication process leaves this feature of daughter genomes oriented at right angles to each other.