Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

Abstract

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g. cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

Inheritance of Cell-Cycle Duration in the Presence of Periodic Forcing

“…A detailed setup protocol can be found in Ref. [51]. Prior to microfluidic cultivation, the microfluidic chip was purged with fresh and sterile-filtered CGXII medium at 200 nL=min for 10 min.…”

Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations

“…An in-house developed microfluidic platform was used for C. glutamicum single-cell analysis (Grünberger et al, 2012;Gruenberger et al, 2013). The microfluidic device incorpo-rates a few hundred cultivation chambers with dimensions to ensure monolayer growth of isogenic microcolonies, with up to a few hundred cells maximum each.…”

“…The fabrication and improvement of user-friendly microfluidic chip devices for microbial cultivation provide an important platform for state-of-the-art live cell imaging approaches. In previous reports, we have described the design of a disposable microfluidic chip device that enables the analysis of isogenic bacterial populations under defined environmental conditions and allows single-cell analysis by time-lapse fluorescence microscopy at spatiotemporal resolution (Gruenberger et al, 2013).…”

Real‐time monitoring of fungal growth and morphogenesis at single‐cell resolution

“…Inlet and outlet were connected to a tubing and syringe pump system (NeMESYS, Cetoni GmbH, Germany) for continuous flow. For further information regarding setup and operation of the microfluidic system, the reader is referred to Grünberger et al [17]. After flushing the system (60 min) at a flow rate of 200 nL min −1 , the desired positions for high-throughput investigations were selected (on average 50 positions, depending on the morphological state).…”