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 of the microscope during the time-lapse experiment and compromise image acquisition.
   4. Seal off the microfluidic plate. Replace the medium from well 8 with 150 µL of culture sample and replace the medium from well 1 to 5 by the desired medium with or without the stress-inducing reagent.
   5. Seal the microfluidic plate and place it on the microscope stage.
   6. On the microfluidic software run the cell loading procedure. Check that the loading of the cells is satisfactory by looking under the microscope in transmitted light. Run the cell loading procedure a second time if the cell density in the chamber is insufficient.
   7. Perform careful focus in transmitted light mode and select several fields of view that show isolated bacteria. It is important to select fields that are not overcrowded to be able to monitor the growth of isolated cells over time (~10–20 cells per 100 µm² is recommended). This will also facilitate cell detection during image analysis.
   8. On the microfluidic software, click on the Create a Protocol button. Program the injection of growth medium for 1–2 generation time equivalents to allow for the cells to adapt (optional). Then program the injection of the stress-inducing medium during 10 min at 2 psi, followed by injection at 1 psi for the wanted duration of the stress treatment. If you intend to analyze the recovery of the cells after stress, program the injection of fresh growth medium for the wanted duration.
      NOTE: In the experiment presented here, cephalexin was injected for 10 min at 2 psi, followed by 50 min at 1 psi. Then, fresh growth medium was injected at 2 psi for 10 min, followed by 3 h at 1 psi.
   9. Perform microscopy imaging in time-lapse mode with 1 frame every 10 min using phase contrast in transmitted light and a 560 nm excitation light source for the mCherry signal if required.
      NOTE: It is important to start microscopic image acquisition at the same time as the start of the microfluidic injection protocol.

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