Microfluidic Patterning and Fluorescence-Based Tracking of Single-Cell Bacterial Growth

Abstract

Source: Pioli, R., et al., Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly. J. Vis. Exp. (2021)

This video demonstrates the method of capturing single fluorescently tagged Escherichia coli (E. coli) cells in microfluidic traps using a carbon-free suspension and controlled flow, followed by nutrient addition to trigger growth and fluorescence imaging of microcolonies.

Protocol

1. Bacterial patterning

1. One day prior to the experiment, grow a population of Escherichia coli (strain MG1655 prpsM-GFP). Inoculate the culture directly from the frozen stock and grow overnight for 20 h in lysogeny broth (LB) medium in a shaker incubator at 37 °C. Add 50 µg/mL of kanamycin for the cells to retain the prpsM-GFP (green fluorescent protein) plasmid.
2. On the day of the experiment, set the box incubator (Figure 1A) at 37 °C several hours before the experiment to ensure a uniform and stable temperature before starting. Then, set up the syringe pump and the heated glass plate on the microscope stage (

Results

Figure 1: Schematic of the platform for sequential capillarity-assisted assembly in a microfluidic channel. <.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD 10 mL Syringe (Luer-Lock)	BD	300912	Used to flush fresh Lysogeny broth into the microfluidic channel
Box Incubator	Life Imaging Services		Used to ensure a uniform and constant temperature in the channel
Centrifuge	Eppendorf	5424R	Used to replace the overnight media with fresh minimal media
Centrifuge vial	Eppendorf	30120086	1.5 mL
CETONI Base 120	CETONI GmbH		Syringe pump
H401-T-CONTROLLER	Okolab		Controller of the heated glass plate
H601-NIKON-TS2R-GLASS	Okolab		Heated glass plate
Insulin syringes, U 100, with luer	Codan Medical ApS	CODA621640	1 mL syringe used to withdraw the liquid suspension during the patterning process
LB Broth, Miller (Luria-Bertani)	Fisher Scientific	244610	Lysogeny broth flushed into the microfluidic channel
Masterflex transfer tubing	Masterflex	HV-06419-05	0.020'' ID, 0.06'' OD
MOPS (10x)	Teknova	M2101	Diluted tenfold with milliQ water and used to replace the overnight medium
Nikon Eclipse Ti2	Nikon Instruments		Microscope
Potassium phosphate dibasic	Sigma Aldrich	P3786	Added to MOPS 1x
Tween 20	Sigma Aldrich	P1379	Used to ensure an optimal receding contact angle during the patterning process