Microfluidic Time-Lapse Microscopy to Study Antibiotic Persistence in Bacteria

Abstract

Source: Oms, T., et al. Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli. J. Vis. Exp. (2023)

This video presents a protocol for monitoring individual bacterial cells expressing a fluorescently labeled DNA-binding protein within a microfluidic system. It illustrates how antibiotics inhibit most cells, while a subpopulation of persister cells survives the treatment and regrows following antibiotic removal.

Protocol

Microfluidic time-lapse microscopy imaging

NOTE: The following section describes the preparation of the microfluidic plate as well as the time-lapse image acquisition and image analysis procedure. The aim of this experiment is to observe and analyze the persistence phenotype upon antibiotic treatment at the single-cell level. The data collected during this experiment can be used to generate a wide range of results depending on the question addressed and/or the fluorescent reporters used during the experiment. In the experiment described here, quantitative analysis of the cell length and HU-mCherry fluorescence, reflecting the nucleoid organization in persister and non-persister cells, was carried out.

1. Bacterial cell culture for microfluidic time-lapse microscopy
   1. Inoculate 5 mL of medium (3-[N-morpholino] propanesulfonic acid (MOPS) glycerol 0.4%, supplemented with a selective antibiotic if required) with an isolated colony in a glass tube (≥25 mL), and place the tube in a shaking incubator set to 37 °C and 180 rpm overnight (between 15 h and 19 h).
   2. The next day, measure the OD₆₀₀ nm, and dilute the culture in fresh temperature-adjusted medium (37 °C, MOPS glycerol 0.4%) in a glass tube to a final OD₆₀₀ nm of ~0.001. Let the culture grow overnight (between 15 h and 19 h) in a shaking incubator at 37 °C and 180 rpm to obtain an early exponential-phase culture on the next day.
2. Preparation of the microfluidic plate and time-lapse microscopy imaging
   NOTE: Microfluidic experiments can be performed in commercially available microfluidic devices (as described here) or in in-house produced microfluidic systems.
   1. Remove the conservation solution (if present) from every well of the microfluidic plate, and replace it with a fresh culture medium.
      NOTE: If the microfluidic plate contains a waste outlet well, the conservation solution of the outlet well should be removed but not replaced by the medium.
   2. Seal the microfluidic plate with the manifold system by clicking on the Seal button or through the microfluidic software (first select Tool, followed by Seal Plate).
      NOTE: To seal the plate, uniform pressure should be applied to the plate and the manifold by manually squeezing the plate against the manifold. If performed correctly, the note "sealed" should appear on the ONIX2 interface. It is important not to apply any pressure to the glass slide to avoid any potential risk of breaking the manifold.
   3. Once sealed, perform a first priming sequence (click on Run Liquid Priming Sequence on the microfluidic software interface).
      NOTE: Run Liquid Priming Sequence corresponds to 5 min of perfusion at 6.9 kPa for wells 1-5, followed by 5 min of perfusion at 6.9 kPa for well 8, and a final perfusion round for well 6 for 5 min at 6.9 kPa. The Run Liquid Priming Sequence allows the removal of the conservation solution that may still be present in the channels connecting the different wells.
   4. Incubate the plate in a thermostatically controlled cabinet of the microscope at the desired temperature (here, 37 °C) for a minimum of 2 h before the start of the microscopy imaging.
   5. Start a second Run Liquid Priming Sequence before beginning the experiment.
   6. Seal off the microfluidic plate by clicking on Seal off on the microfluidic software interface. Replace the medium in well 1 and well 2 with 200 μL of fresh medium, in well 3 with 200 μL of fresh medium containing the antibiotic (here, ofloxacin (OFX) at 5 μg·mL⁻¹), in well 4 and well 5 with 200 μL of fresh medium, in well 6 with 200 µL of fresh medium, and in well 8 with 200 μL of the culture sample (from step 1.2) diluted to an OD₆₀₀ nm of 0.01 in the fresh medium.
   7. Seal the microfluidic plate as described in step 2.2 and place it on the microscope objective inside the microscope cabinet.
      NOTE: Make sure to place a drop of immersion oil onto the microscope objective before placing the microfluidic plate.
   8. In the microfluidic software, click on Cell Loading to allow the cell loading into the microfluidic plate.
      NOTE: The Cell Loading step comprises 15 s of perfusion at 13.8 kPa for well 8, followed by 15 s of perfusion at 27.6 kPa for well 6 and well 8, and a final perfusion round for well 6 for 30 s at 6.9 kPa. The density of the cells in the microfluidic plate is critical for the experiment. The first part of this microfluidic protocol consists of growing bacteria for 6 h in a fresh medium before the antibiotic treatment. After 6 h of growth, the cell density has to be sufficient to detect the rare persister cells (in the conditions used in this study, the persister cells generate at a frequency of 10⁻⁴). If the cell density is too high, it is difficult to distinguish the individual cells, which prevents precise single-cell analysis. As the growth rate is directly dependent on the medium, the density of cells in the microscopy fields should be assessed before launching the experiment.
   9. Set an optimal focus using transmitted light mode, and select several regions of interest (ROIs) where an appropriate cell number is observed (up to 300 cells per field).
      NOTE: Select at least 40 ROIs to make sure that the rare persister cells are imaged.
   10. On the microfluidic software, click on Create a Protocol. Program the injection of fresh medium at 6.9 kPa for 6 h (well 1-2), followed by the injection of the medium containing the antibiotic at 6.9 kPa for 6 h (well 3), and, finally, the injection of fresh medium at 6.9 kPa for 20 h (wells 4-5).
       NOTE: A bacterial culture diluted to an OD₆₀₀ nm of 0.01 allows bacteria to grow in the microfluidic chamber for 6 h, ensuring that the cells are in the exponential growth phase. Depending on the number of cells introduced into the microfluidic device during the loading step (see step 2.8), the duration of the growth phase can be adapted to obtain up to 300 cells per ROI. As persistence is a rare phenomenon, increasing the number of cells per ROI improves the possibility of observing persister cells. The cell number should, however, not exceed 300 cells per ROI, as this makes single-cell analysis tedious.
   11. Perform microscopy imaging in time-lapse mode with one frame every 15 min using transmitted light and the excitation light source for the fluorescent reporter. Here, a 560 nm excitation light source for the mCherry signal was used (580 nm LED at 10% power with filter 00 [530-585 ex, 615LP em, Zeiss] and 100 ms exposure for mCherry). The Zeiss-compatible Zen3.2 software was used for cell imaging.
3. Image analysis
   NOTE: The opening and visualization of the microscopy images are performed with the open-source ImageJ/Fiji software (https://fiji.sc/). The quantitative image analysis is performed using the open-source ImageJ/Fiji software and the free MicrobeJ plugin (https://microbej.com). In this protocol, the MicrobeJ 5.13I(14) version was used.
   1. Open the ImageJ/Fiji software on the computer, and drag the hyperstack time-lapse microscopy images into the Fiji loading bar. Use Image > Color > Make Composite to fuse the different channels of the hyperstack. If the channels of the time-lapse experiment do not correspond to the desired color (e.g., the phase contrast is shown in red instead of gray), use Image > Color > Arrange Channels to apply the appropriate color to the channels.
   2. Open the MicrobeJ plugin and detect the bacterial cells using the manual editing interface. Delete the automatically detected cells and manually outline the persister cells of interest frame by frame.
      NOTE: Different settings can be used to automatically detect individual cells. Manual detection was used here as the analyzed persister cells form long filaments, which are rarely detected correctly using automatic detection.
   3. After detection, use the Result icon in the MicrobeJ manual editing interface to generate a ResultJ table. Save the ResultJ file and use it to gain insights into different parameters of interest in the single-cell analysis. In this protocol, the mean fluorescence of the HU-mCherry intensity, the cell length, and the cell area of individual cells were exported.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Axio Observer	Zeiss		Inverted fluorescence microscope
Fiji	ImageJ	https://fiji.sc/	Image software; Schindelin et al. if used in publication
E. coli K-12 MG1655 CF1648 (fnr+)		BE10	Wt reference strain, lab strain
E. coli K-12 MG1655 CF1648 (fnr+) hupA-mCherry::FRT-kan-FRT		BE16	HU-mCherry fusion integrated via P1 transduction at the native locus, lab strain
CellASIC ONIX Microfluidic System	Merck	CAX2-S0000	Microfluidic system
Microfluidic Plates CellASIC ONIX	Merck	B04A-03-5PK	Plate for microfluidic system
CellASIC ONIX2 FG	Merck	ONIX2 1.0.1	Microfluidic software
CellASIC ONIX2 Manifold Basic	Merck	CAX2-MBC20	Manifold system
MicrobeJ	Imagej/Fiji plugin	https://www.microbej.com/	Microscopy image analysis plugin. Ducret et al.for in publication; Detection settings: For bacteria : Area (μm2): 0,1-max; Length (μm): 0,5-max; Width (μm): 0,6-max; Range (μm): 0,5-max; Angularity (rad): 0-0,3; 0-max for all other parameters.
Zeiss® immersion Oil 518F	Zeiss		Immersion oil to increase resolution of microscope
Zen3.2 Pro	Zeiss		Microscopic image acquisition and processing software
Ofloxacin	Merck	82419-36-1	Fluoroquinolone antibiotic used to treat the bacterial cells
CaCl₂·2H₂O	Merck	1.02382.0250	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
CoCl₂·6H₂O	Merck	1.02539.0100	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
CuSO₄·5H₂O	Merck	1.02790.0250	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
D-(+)-glucose	Sigma Aldrich	G7021-1KG	Carbon source for MOPS glucose 0.4% growth medium
FeSO₄·7H₂O	VWR Chemicals	24244.232	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
Glycerol	Merck	56815	Carbon source for MOPS glycerol 0.4% growth medium
H₃BO₃	Sigma-Aldrich	B6768-1KG	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
K₂HPO₄	Merck	1.05099.1000	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
KOH	Merck	1.05029.1000	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
K₂SO₄	Merck	1.05153.0500	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
MgCl₂·6H₂O	Merck	1.05832.1000	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
MnCl₂·4H₂O	Merck	1.05927.0100	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
MOPS, Free Acid ULTROL® Grade	Merck	475898-500GM	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
NaCl	Sigma-Aldrich	S5886-1KG	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
(NH₄)₆Mo₇O₂₄·4H₂O	Merck	1.01180.0250	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
NH₄Cl	Merck	1.01145.0500	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
N-[Tris(hydroxyméthyl)-méthyl]-glycine	Merck	1.08602.0250	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium
ZnSO₄·7H₂O	Merck	1.08883.0500	For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium