Creating Rapid Oxygen Oscillations in Microbial Single-cell Growth Analysis using a Microfluidic Double-layer Device

Microbial single-cell analysis using microfluidics coupled with time-lapse microscopy holds promise for investigating microbial growth behavior under confined environments with spatiotemporal resolution, offering insights that cannot be gained from conventional cultivation and analytical platforms. Oxygen plays a crucial role in determining microbial growth. However, temporal control in the range of seconds has not been implemented in microbial analysis using microfluidics. We have recently developed a double-layer PDMS microfluidic chip enabling temporal oxygen control in the tens-of-seconds range. The chip comprises an upper layer for gassing and a bottom layer for cultivation and fluid perfusion. The two layers are separated by a thin intermediate PDMS membrane, allowing fast gas exchange between the two layers. In this methodological paper, we explain the procedure for chip fabrication, characterization, and microbial cultivation using the chip. As representative results, we demonstrate the fast oxygen switching capability in tens of seconds, microbial cultivation, and time-resolved growth analysis under constant and oscillating oxygen conditions. This protocol aims to assist researchers interested in implementing temporal oxygen control in their microfluidic setup.