Microtiter plate tests for segregation of bioluminescent bacteria

Abstract

It 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.

Phoretic interactions and oscillations in active suspensions of growing Escherichia coli

“…At first glance this result appears to be unexpected, because the rate of bacterial growth is of the order of h −1 [ 3 , 6 , 7 ]. In the previous studies, we interpreted similar results by assuming temporal changes of cell behaviour due to their metabolic flexibility [ 21 , 23 , 24 ]. It is known that intracellular oscillations may emerge in a living cell [ 30 ].…”

“…Illustrative snapshots are shown in figure 1 a (see also electronic supplementary material, movie S1). The bioluminescence along the circular contact line and across the diameter of the well was measured using the image processing software [ 24 ]. Typical examples of the space–time plots of bioluminescence are shown in figure 1 b .…”

“…The observed fluctuations in the number of aggregates during 24 h recordings are illustrated by figure 1 c . The self-organization of bacteria in a microtitre plate well was characterized by the average number of bioluminescent aggregates ( m ) formed along the three-phase contact line [ 24 ].

Figure 1.

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“…The cultures of E. coli and their mutants were prepared as described in [ 20 , 21 , 23 , 24 ]. Cells were grown in LB (Luria–Bertani) broth medium.…”

“…Optical density measurements at 600 nm (OD600) were used to monitor the concentration of cells. The freshly harvested mid-log phase cultures (OD600 = 0.4–0.6) were chosen for examination [ 20 , 21 , 23 , 24 ]. The induction of the culture was provided by adding a stock solution of isopropyl-β- d -thiogalactoside into LB medium up to a final concentration of 1 mM.…”