Organization of developing Escherichia coli colonies viewed by scanning electron microscopy

Abstract

Colony growth was initiated by inoculating minimal glucose agar with 1-,ul. spots of a plasmid-free Escherichia coli culture and incubating at 32°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. (20,24). Observations such as these indicated that regulatory systems could control the behavior of groups of procaryotic cells and that it should be possible to monitor specific multicellular morphogenetic events during bacterial colony development. This idea is not unprecedented. Other authors have proposed similar ideas based on studies of colony structure and variation (1,5,12).There are a number of studies of the early stages of bacterial colony development in the literature (7, 10, 13). These studies generally involved cultures growing on special slides or chambers and were limited to the first few hours of growth when most of the individual bacteria could be distinguished. To provide information about cellular changes at later stages of colony development, the scanning electron microscope (SEM) was used to examine the surface structures of E. coli colonies growing on agar medium in normal petri dishes. Previous work had shown that the SEM could be used to provide detailed information at the cellular level about the structure of mature Pseudomonas putida colonies without gross distortion of colony organization (18). The present study extended the earlier results by examining the colonies produced by a single culture at different stages of development, thereby documenting how patterns of multicellular organization could change over time.The background for these SEM studies was an investigation of bacterial morphogenesis on agar substrates, using time-lapse photography and video recording. The time-lapse studies concerned the growth of E. coli colonies and the formation of swarm colonies by Proteus mirabilis. The results significantly influenced my descriptions and interpretations of the SEM pictures in this report and thus require a brief summary. Kinetic analysis of living E. coli colonies revealed that expansion was not a uniform process but rather that different phases of activity could be discerned. To obtain the colonies for SEM examination, pla...

Bacteria are small but not stupid: cognition, natural genetic engineering and socio-bacteriology

“…One could see with the scanning electron microscope that E. coli cells change considerably with respect to size, shape, and how they arrange themselves in local populations during the course of colony morphogenesis (Shapiro, 1987). By light microscopy, it was possible to visualize cell-cell interactions showing that E. coli grow on agar surfaces by maximizing contact between cells rather than individual access to substrate (Shapiro & Hsu, 1989).…”

The Selective Value of Bacterial Shape

“…The tendency to align parallel with one another occurs even between the cells of different microcolonies as they collide (296). Furthermore, cell morphology changes in predictable ways in older colonies (295,296), forming demarcated zones filled with "cells of distinct sizes, shapes, and patterns of multicellular arrangement," including cells shaped as cocci, ovoids, bacilli, and filaments (294). Thus, the overall impression is one of directed morphological organization.…”

Thinking about Bacillus subtilis as a multicellular organism

“…Twenty years ago Shapiro had all the right ideas and tools to tackle multicellularity but his choice of strain was, in retrospect, misguided. He opted to carry out his genetic studies in M7124, "a strain that has been used as a standard bacteriophage and plasmid host for 18 years" [8].…”