AbstractIt has been recently shown that bioluminescence imaging can be usefully applied to provide new insights into bacterial self‐organization. In this work we employ bioluminescence imaging to record images of nutrient rich liquid cultures of the lux‐gene reporter Escherichia coli in microtiter plate wells. The images show that patterns of inhomogenous bioluminescence form along the three‐phase contact lines. The paper analyzes the dependencies of the average number of luminous aggregates (clouds) on various environmental factors. In particular, our results show that optimal (neutral) pH and high aeration rates determine the highest mean number of clouds, and that spatiotemporal patterns do not form in the pH buffered suspensions. In addition, a sigmoidal (switch‐like) dependence of the number of aggregates on the rate of aeration was observed. The obtained bioluminescence imaging data was interpreted by employing the Keller–Segel–Fisher (KSF) model of chemotaxis and logistic growth, adapted to systems of metabolically flexible (two‐state) bacteria. The modified KSF model successfully simulated the observed switch‐like responses. The results of the microtiter plate tests and their simulations indicate that the segregation of bacteria with different activities proceeds in the three‐phase contact line region. Copyright © 2015 John Wiley & Sons, Ltd.