This article describes a method for tracking bacterial cell growth and division using a microfluidic device integrated with an inverted fluorescence microscope. The device allows for the confinement of bacterial cells in linear chains, facilitating long-term observation of individual cells.
Begin with a microfluidic device mounted on the stage of an inverted fluorescence microscope.
The device consists of a polymer chip featuring horizontal feeding channels. Each channel is lined with a series of narrow trenches sealed at one end and designed to confine bacterial cells in linear chains.
Within the trenches are non-motile bacterial cells that express a fluorescent protein, enabling their visualization.
Each feeding channel is connected to syringe pumps that deliver nutrient-rich medium at a controlled flow rate.
Initiate the flow to allow the medium to diffuse into the trenches and support bacterial growth.
Confined bacterial cells divide within the trench, forming a chain of progeny.
The trench design ensures newer cells at the open end are washed away, enabling long-term tracking of the oldest cells.
Capture images of trenches over time to track the growth and division of individual bacterial cells.
Carefully mount the loaded device onto a stage insert by taping the cover glass on either side of the PDMS to the bottom of the stage insert. Invert the stage insert device assembly so that the PDMS is facing up and place it on a soft surface such as a dust-free wipe.
Adjust the flow rate of the phase-one syringe pump to 35 microliters per minute. Then working with one lane at a time, insert the inlet needle and then the outlet needle that runs to the waste beaker. After visually inspecting the device for leaks, examine the outlet tubing for the appearance of excess cells that appear as a turbid stripe in the medium meniscus, which will slowly move towards the waste beaker.
Once the device has run for approximately 15 minutes, set the phase-one syringe pump to 1.5 microliters per minute and pause the flow. Bring the entire pump and device apparatus to an inverted fluorescent microscope and restart the phase-one medium flow at 1.5 microliters per minute. Carefully mount the stage insert with the device on to the microscope, using tape as necessary to route the inlet and outlet tubing.
Locate the appropriate positions on the device for imaging and begin imaging as desired. Note that the cells typically require a few hours to read steady-state, exponential phase growth, and the onset of image acquisition may be delayed as desired so that imaging begins after the equilibration period.
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