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Microfluidics has enabled various research projects in the field of microbial single-cell analysis. In particular, single-use microfluidic cultivation devices combined with automated time-lapse imaging provide powerful approaches for analyzing microbial phenomena at the single-cell level. High spatiotemporal resolution facilitates individual cell identification and tracking, delivering detailed insights into areas like phenotypic population heterogeneity, which can be highly relevant to the fate of a microbial population and may negatively impact the efficiency of biotechnological fermentations. New tools need to be developed to access the origin of population heterogeneity and understand its functional role. In this study, we present a microfluidic device for batch cultivations inside picoliter-sized cultivation chambers that can be reversibly isolated from continuous medium supply. Therefore, the cultivation broth is simply replaced by a continuous flow of humidified air, removing any medium residue along the supply channels but preserving five picoliters of cultivation medium inside the cultivation chambers in a highly parallel manner. Living cells can grow inside our microfabricated batch chambers, which can accommodate up to several hundred cells. The chamber height approximately matches the diameter of a single cell, facilitating cell growth in monolayers that are ideal for image-based cell analysis. We successfully demonstrated the growth of Escherichia coli during continuous medium perfusion and batch cultivation conditions. As expected, the cells grew exponentially under continuous medium influx until the maximum chamber capacity was reached, but when they were cultivated under batch conditions, cellular growth underwent an exponential phase, followed by a stationary phase with obvious morphological changes.