Three-dimensional characterization of rippling behavior in dense colonies of Myxococcus xanthus

One of the characteristic collective phenomena displayed by the bacterium Myxococcus xanthus is its ability to self-organize into wave-like structures termed ripples. Rippling has been previously explained using a simple model that 1) invokes coordinated cellular reversals that produce wave fronts that reflect off of each other and, 2) dictates a wavelength that is a function of the cell speed and reversal time. However, experimental support for this model comes from cell tracking in thin, sub-monolayer groups of cells whereas rippling is typically observed in much denser colonies. In this work, we characterize the properties of rippling in three dimensions within dense populations of M. xanthus. Using a surface profilometer, we demonstrate that rippling waves span 6 to 20 cell layers in height, with a characteristic wavelength that is dependent on the stiffness and composition of the underlying substrate. We use spinning-disk confocal microscopy and fluorescent-cell labeling to measure individual cell motility within rippling waves. Unlike the predictions from the aforementioned model, we do not find synchronization of cellular reversals, correlation between reversal events and wave crest collisions, or the correct dependance of rippling wavelength on cell speed and reversal frequency. We further our investigation into the structure of formed wave crests using dual view light sheet microscopy. We image volumes of dense cells with high isotropic resolution from we can observe overall ordering of cells within waves.