From snowflake formation to growth of bacterial colonies II: Cooperative formation of complex colonial patterns ESHEL BEN-JACOB In nature, bacterial colonies must often cope with hostile environmental conditions. To do so they have developed sophisticated cooperative behaviour and intricate communication capabilities, such as direct cell ± cell physical interactions via extra-membrane polymers, collective production of extracellular `wetting’ fluid for movement on hard surfaces, long- range chemical signalling such as quorum sensing and chemotactic (bias of movement according to gradient of chemical agent) signalling, collective activation and deactivation of genes and even exchange of genetic material. Utilizing these capabilities, the bacterial colonies develop complex spatio-temporal patterns in response to adverse growth conditions. We present a wealth of beautiful patterns formed during colonial development of various bacterial strains and for different environmental conditions. Invoking ideas from pattern formation in non-living systems and using generic modelling we are able to reveal novel bacterial strategies which account for the salient features of the evolved patterns. Using the models, we demonstrate how bacterial communication leads to colonial self-organization that can only be achieved via cooperative behaviour of the cells. It can be viewed as the action of a singular feedback between the microscopic level (the individual cells) and the macroscopic level (the colony) in the determination of the emerging patterns. 1. Introduction Among evolving (non-equilibrium) systems, living organ- isms are the most challenging that scientists can study. A biological system constantly exchanges material and energy with the environment as it regulates its growth and survival. The energy and chemical balances at the cellular level involve an intricate interplay between the microscopic dynamics and the macroscopic environment, through which life at the intermediate mesoscopic scale is main- tained [1]. The development of a multicellular structure requires non-equilibrium dynamics, as microscopic imbal- ances are translated into the macroscopic gradients that control collective action and growth [2]. Much effort has been devoted to the search for basic principles of growth (communication, regulation and control) on the cellular and multicellular levels [3 ±10]. Armed with the new developments in the study of patterning in non-living systems, I set out to meet the challenge posed by living organisms. Of extreme impor- tance was the choice of starting point, that is which phenomena to study; it had to be simple enough to allow progress but also well motivated by the significance of the results. In addition, I wanted to be able to use my previous knowledge and expertise. Cooperative microbial behaviour was well suited to my requirements. 1.1. Complex bacterial patterns Traditionally, bacterial colonies are grown on substrates with a high nutrient level and intermediate agar concentra- tion. Such `friendly’ conditions yield colonies of simple compact patterns, which fit well the contemporary view of bacterial colonies as a collection of independent unicellular organisms (non-interacting `particles’). However, bacterial colonies in nature must regularly cope with hostile environmental conditions [5, 11]. Expecting complex patterns to be developed by stressed colonies, we created hostile conditions in a Petri dish by using a very low level of nutrients, a hard surface (high concentration of agar), or both. Indeed, we observed some very complex patterns (figure 1). Drawing on the analogy with diffusive patterning in non-living systems [12 ± 15], we can say that complex patterns are expected. The cellular reproduction rate that Author’s address: School of Physics and Astronomy, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel. Contemporary Physics, 1997, volume 38, number 3, pages 205 ± 241 0010-7514 /97 $12.00 Ó 1997 Taylor & Francis Ltd