Bacterial Growth on a Microfluidic Chip to Study Antibiotic Drug Resistance

Abstract

Source: Liu, P. C., et al. Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection. J. Vis. Exp. (2016)

This video demonstrates the use of a microfluidic chip to visualize and compare the growth and morphology of wild-type and trimethoprim-resistant E. coli strains under trimethoprim antibiotic stress.

Protocol

1. On Chip Growth Experiment

1. Before loading into the microfluidic device, flush it with deionized water for 1 hr and M9 medium for 1 hr.
2. Thaw the daily frozen sample at room temperature for 5 min and inoculate a new test tube with fresh M9 medium. Place the sample in a shaker incubator at 37 °C overnight.
3. Dilute the microbial sample 10-fold with M9 medium and load it into the microfluidic device by pressuring the microbial sample container at 5 psi. Then load the trimethoprim (TMP) drug medium into the chip by pressuring the drug medium container at 5 psi. Execute the mixing between the microbe and drug by actuating the micromechanical valve between the two chambers on the microfluidic chip for 15 min.
4. Place the microfluidic chip in the microscope incubator mounted on the inverted microscope. Acquire the cell image in the growth chamber every hr for 8 hr with the CCD camera mounted on the inverted microscope.
5. Use the custom program script to calculate the cell numbers through a threshold algorithm on the cell image data. Note that the cell number data are averaged typically over three microfluidic chambers.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Inverted microscope	Leica Microsystems	Leica DMI-LED	used for microfluidic measurement Use X40 objective NA=0.55
Microscope Incubator	Live Cell Instrument	CU-109	used for microfluidic measurement
Solenoidal valves	Pneumadyne	S10MM-31-12-3	Normally open 1.3 Watt 12 Vdc
USB interface card	Hobby Engineering	USBIO24-R Digital I/O Module	for microfluidics measurement
Air compressor	Rocker Scientific	ROCKER 440	Pressure source for microfluidcs Max. Pressure 80 Psi
Male luer-lock fittings to 1/8" barb	ValuePlastics.com	MTLL230-1	used for microfluidic control
1/8" barb to 10-32 threaded port	ValuePlastics.com	B-1	used for microfluidic control
Female luer-lock fittings to 10-32 threaded port	ValuePlastics.com	KFTL-1	used for microfluidic control
NPN darlington transistor 500mA, 40V (2N6427)	DigiKey.com	2N6427GOS-ND	used for microfluidic control
10kOhm, carbon film resistor, 0.25W	DigiKey.com	P10KBACT-ND	used for microfluidic control
Tantalum capacitor, 10uF, 25V, 10%	DigiKey.com	478-1841-ND	used for microfluidic control
Andor CCD camera	Andor	Zyla 4.2 Plus SCMOS	used for microfluidic on chip imaging
Trimethoprim	Sigma
Difco M9 minimal salt 5x	BD		Medium