Multi-scale Analysis of Bacterial Growth Under Stress Treatments

1. Cell culture, stress-induction, and sampling procedure

NOTE: Use sterile culture glassware, pipette tips, and growth medium filtered at 0.22 µm to avoid background particles. Here, cell cultures are grown in low autofluorescence rich defined medium (see Table of Materials)^7,8.

1. Streak the bacterial strain of interest from a frozen glycerol stock on a Luria-Broth (LB) agarose plate (with selective antibiotic if required) and incubate at 37 °C overnight (17 h).
   NOTE: The example experiment presented here uses Escherichia coli MG1655 hupA-mCherry. This strain produces the fluorescently tagged subunit α of the HU nucleoid associated protein, thus allowing light microscopy visualization of the chromosome in live cells⁹.
2. Inoculate 5 mL of medium with a single colony and grow at 37 °C with shaking at 140 rotation per minute (rpm) overnight (17 h). Flasks (≥50 mL) or large diameter (≥2 cm) test tubes must be used to ensure satisfactory aeration of the agitated culture.
3. The following morning measure the optical density at 600 nm (OD_600nm) and dilute the culture into a test tube containing fresh medium to an OD_600nm of 0.01. The total volume of the culture needs to be adjusted depending on the number of time points to be analyzed during the experiment.
4. Load a 200 µL sample of the culture into a microplate (0.2 mL per well of working volume with a clear transparent bottom) and place it in an automated plate reader (see Table of Materials) for OD_600nm monitoring during incubation at 37 °C.
5. Incubate the inoculated test tube at 37 °C with shaking (140 rpm) to OD_600nm = 0.2, corresponding to full exponential phase in rich medium.
   NOTE: It is critical to grow the cells for at least 4−5 generations before achieving proper exponential growth. The initial inoculum used in step 1.3 (OD_600nm = 0.01) needs to be adapted in the case of growth in poorer medium (i.e., where the exponential phase is reached below OD 0.2), or if more generations are wanted (e.g., for specific physiological studies or to extended stress treatments).
6. At OD_600nm = 0.2, take the following culture samples corresponding to the t₀ time point (exponentially growing cells before stress induction): (1) A 150 µL sample to be immediately loaded in the microfluidic apparatus for time-lapse microscopy imaging (see section 2); (2) A 200 µL sample for the dilution and plating assay (see section 3); (3) A 250 µL sample to be put on ice for flow cytometry analysis (section 4); (4) A 10 µL sample to be immediately deposited on an agarose-mounted slide for microscopy snapshot imaging (see section 5).
7. Expose the cell culture remaining in the test tube to the specific stress treatment you want to investigate and incubate at 37 °C with shaking (140 rpm).
   NOTE: The culture growing in the automated plate reader for OD_600nm monitoring should also be subjected to the stress treatment.
8. At relevant time points after the stress treatment (t₁, t₂, t₃, etc.), take the following cell samples from the stressed culture: (1) A 200 µL sample for the dilution and plating assay (see section 2); (2) A 250 µL sample to put on ice for flow cytometry analysis (section 3); (4) A 10 µL sample to be immediately deposited on an agarose-mounted slide for microscopy snapshot imaging (see section 4).
   NOTE: Each stress-inducing treatment has an efficiency that is dose- and time-dependent. Thus, it might be necessary to run preliminary tests to determine the dose and duration of treatment to be used for optimal results. This can be done by performing OD monitoring using an automated plate reader (potentially associated with plating assays) of a cell culture treated with a range of doses and exposure times. In the experiment presented here, the cell culture was treated with the cell division-inhibiting antibiotic cephalexin (Ceph.) at 5 µg/mL final concentration for 60 min. Cephalexin was then washed away by pelleting the cells in a 15 mL tube using centrifugation (475 g, 5 min), removing the supernatant, resuspending the cell pellet in an equal volume of fresh medium by gentle pipetting, and transferring into a clean tube. The washed cells were incubated at 37 °C with shaking (140 rpm) to allow recovery. The sample was taken at t₆₀ (60 min after cephalexin addition corresponding to the 'cephalexin-60min-treated' sample), t₁₂₀ (60 min after washing), and t₁₈₀ (120 min after washing).

2. Plating assay

NOTE: The plating assay allows for measuring the concentration of cells able to generate a CFU in the culture samples. This procedure reveals the rate at which one cell divides into two viable cells and allows to detect cell division arrests (e.g., increase of the bacterial generation time of cell lysis).

1. Prepare 10-fold serial dilutions up to 10^-7 of the 200 µL of culture sample in fresh medium. Plate 100 µL of the appropriate dilution on non-selective LB agarose plates in order to obtain between 3−300 colonies after overnight incubation at 37 °C.
   NOTE: Serial dilution in fresh medium must be performed rapidly to limit bacterial divisions. Alternatively, researchers might consider using a saline solution without a carbon source to prevent cell divisions during the dilution process.
2. The next day, count the number of colonies to determine the concentration of viable cells (CFU/mL) in each culture sample. Plot the CFU/mL as a function of time for untreated and treated cell cultures.

3. Flow cytometry

NOTE: The following section describes the preparation of cell samples for flow cytometry analysis. This analysis technique reveals the distribution of cell size and DNA content for a large number of cells. When possible, it is recommended to process the flow cytometry samples immediately. Alternatively, samples can be kept on ice (for up to 6 h) and analyzed simultaneously at the end of the day, once plating and microscopy imaging have been performed.

1. Dilute the 250 µL of culture sample to obtain 250 µL at a concentration of ~15,000 cells/µL (corresponding to an OD_600nm ~0.06) in fresh medium at 4 °C.
   NOTE: Incubation on ice will limit the growth and morphological modification of the cells. Alternatively, the user might consider performing fixation of the cells in 75% ethanol, as usually recommended for flow cytometry¹⁰.
2. For DNA staining, mix the bacterial sample with a 10 µg/mL solution of DNA fluorescent dye (ratio 1:1) and incubate in the dark for 15 min before analyzing the sample.
3. Pass the sample into the flow cytometer with a ~120,000 cells/min flow rate. Acquire forward-scattered (FSC) and side-scattered (SSC) light as well as DNA fluorescent dye fluorescence signal (FL-1) with the appropriate settings.
4. Plot the FSC and FL-1 cell density histograms to represent the distribution of cell size and DNA content in the cell population.

4. Snapshot microscopy imaging

NOTE: The following part describes the preparation of microscopy slides and image acquisition for population snapshot analysis. This procedure will provide information regarding the morphology of the cells (cell length, width, shape) and the intracellular organization of the nucleoid DNA.

1. Preheat the thermostated microscope chamber at 37 °C to stabilize the temperature before starting the observations. This chamber allows for temperature modulation of the microscope optics and sample stage during time-lapse experiments.
2. Prepare the agarose-mounted slides as described in Lesterlin and Dubarry⁷.
   1. Remove the plastic film from the bottom of the blue frame (see Table of Materials), leaving the hollowed plastic film on the other side. Stick the blue frame on a microscope glass slide.
   2. Pipette ~150 µL of melted 1% agarose medium solution and pour within the blue frame compartment. Rapidly cover with a clean coverslip to remove the excess liquid and wait a few min for the agarose pad to solidify at room temperature.
   3. When the cell sample is ready, remove the coverslip and the plastic film from the blue frame. Pour 10 µL of cell sample on the agarose pad and tilt the glass slide gently to spread the droplet. When all the liquid has been adsorbed, stick a clean coverslip on the blue frame to seal the sample. The microscopy slide is now ready for microscopy.
3. Place the slide on the microscope stage and perform image acquisition using transmitted light (with a phase contrast objective) and with light source excitation at the appropriate wavelengths (560 nm for mCherry).
4. Select fields of view that contain isolated cells in order to facilitate automated cell detection during image analysis. Make sure that at least 300 cells are imaged to allow robust statistical analysis of the cell population.

5. Microfluidics time-lapse microscopy imaging

NOTE: The following part explains the preparation of the microfluidic plates (see Table of Materials), cell loading, microfluidics program, and time-lapse image acquisition. This imaging procedure reveals the behavior of individual cells in real-time.

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 (see Table of Materials) 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 carefully 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.

6. Image analysis

NOTE: This section briefly describes the key steps of processing and analyzing snapshot and time-lapse microscopy images. Opening and visualization of microscopy images is done with the open source ImageJ/Fiji (https://fiji.sc/)¹¹. Quantitative image analysis is performed using the open source ImageJ/Fiji software together with the free MicrobeJ plugin¹² (https://microbej.com). This protocol uses the MicrobeJ 5.13I version.

1. Open the Fiji software and the MicrobeJ plugin.
2. For snapshot analysis, drop all images corresponding to one microscope slide (one sample) into the MicrobeJ loading bar to concatenate images and save the obtained image stacks file. For time-lapse data, just drop the image stack into the loading bar of MicrobeJ.
3. Run the automated detection of the cells' outlines based on the segmentation of phase contrast image and, if relevant, of the nucleoids based on the segmentation of the stained DNA fluorescence signal. Check the accuracy of the cell detection visually and use the MicrobeJ editing tool for correction if needed. Save the result file obtained.
   NOTE: The settings used for detection of E. coli cells are indicated in the Table of Materials (see Comments/Description column of MicrobeJ). For other bacterial species (especially for non-rod-shape bacteria), the user must refine the settings before detection (see MicrobeJ tutorial). For time-lapse images, running a semi-automated detection of the cells using the MicrobeJ editing tool might be preferred to allow focusing on the fate of individual cells (see MicrobeJ tutorial).
4. Click on the icon ResultJ to complete the analysis and obtain the ResultJ window. Many different types of output graphs can be generated from that point. Plot the normalized histograms of cell shape/length and mean nucleoid number per cell.