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The transition from monolayers to multilayered structures in bacterialcolonies is a fundamental step in biofilm development. Observed acrossdifferent morphotypes and species, this transition is triggered within freelygrowing bacterial microcolonies comprising a few hundred cells. Using acombination of numerical simulations and analytical modeling, here wedemonstrate that this transition originates from the competition betweengrowth-induced in-plane active stresses and vertical restoring forces, due tothe cell-substrate interactions. Using a simple chainlike colony of laterallyconfined cells, we show that the transition sets when individual cells becomeunstable to rotations, thus it is localized and mechanically deterministic.Asynchronous cell division renders the process stochastic, so that all thecritical parameters that control the onset of the transition are continuouslydistributed random variables. Here we demonstrate that the occurrence of thefirst division in the colony can be approximated as a Poisson process in thelimit of large cells numbers. This allows us to approximately calculate theprobability distribution function of the position and time associated with thefirst extrusion. The rate of such a Poisson process can be identified as theorder parameter of the transition, thus highlighting its mixeddeterministic-stochastic nature.

Mono-to-multilayer transition in growing bacterial colonies