Organization of developing Escherichia coli colonies viewed by scanning electron microscopy

Colony growth was initiated by inoculating minimal glucose agar with 1-microliter. spots of a plasmid-free Escherichia coli culture and incubating at 32 degrees C. Inoculations took place over a 3-day period, at the end of which the plates were fixed and dried for scanning electron microscopy. In this way, it was possible to examine the surfaces of colonies ranging in age from 0 to 68 h. Macroscopically, the colonies were organized into different concentric zones, and several morphological features could be seen to develop over this period. These included a shallow depression ring marking the site of inoculation, a deeper indentation ring whose position moved outward as the colony grew, an expanding plateau region between the two rings, a mound outside the indentation ring, and a flat brim extending onto the substrate which was either present or absent at different times. Microscopically, a variety of cell morphologies and cell arrangements were detected. Upon inoculation, the bacteria accumulated at the periphery of the inoculation spot but showed no other kind of order. For the first 7.5 h, all bacteria were rod shaped; at the end of this initial phase, a high degree of alignment was seen in the cells at the colony edge. By 24.5 h, both shorter more ovoid cells and longer filaments had begun to appear, and large multicellular arrays had formed. At later stages of colony development, morphologically distinguishable zones involving cells of different shapes and sizes had formed, and these zones often marked the boundaries of macroscopic features. The edges were particularly interesting and at 68 h displayed very sharp saw-toothed boundaries between concentrically organized groups of bacteria. There were some transient irregularities in the concentric organizations of growing colonies, and one colony had entered upon a distinct developmental pathway.