Microscopic Analysis of Bacterial Growth Under Stress Using Microfluidic Chambers

Abstract

Source: Cayron, J., et al. Multi-scale Analysis of Bacterial Growth Under Stress Treatments. J. Vis. Exp. (2019).

This video demonstrates how to prepare and use a microfluidic plate for imaging bacterial cells under stress conditions. It outlines the process of introducing a bacterial culture and a stress-inducing antibiotic medium, followed by cell loading and microscopic setup. Time-lapse imaging in phase-contrast mode is then used to observe the development of elongated, filamentous cells with increased DNA content due to inhibited cell division.

Protocol

1. Microfluidics time-lapse microscopy imaging
   1. Remove the conservation solution from the microfluidic plate and replace it with fresh medium preheated to 37 °C, as described in the microfluidic software user guide.
   2. Seal the microfluidic plate to the manifold system and click on the Priming button.
   3. Place the microfluidic plate with the manifold system on the microscope stage and preheat at 37 °C for ~2 h before starting the microscopy acquisition.
      NOTE: This preheating step is critical to avoid the dilation of the microfluidic chamber, which would alter the focusing ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CellASIC ONIX Microfluidic System	Merck Millipore	CAX2-S0000	Microfluidic system
CellASIC ONIX2 FG	Merck Millipore	ONIX2 1.0.1	Microfluidic software
CellASIC ONIX2 Manifold Basic	Merck Millipore	CAX2-MBC20	Manifold system
E. coli strain carrying a chromosomal insertion for a hupA-mCherry fusion			Created by P1 transduction of hupA-mCherry in E. coli MG1655
Microfluidic Plates CellASIC ONIX	Merck Millipore	B04A-03-5PK	Plate for Microfluidic system
Microscope Nikon eclipse Ti	Nikon		Fluorescence microscope
MOPS EZ Rich Defined Medium (RDM)	Teknova	M2105	Growth rich medium, 10x MOPS Mixture, 0,132 M K₂HPO₄, 10x AGCU, 5x Supplement EZ, 20% Glucose. Filtered at 0.22 μm