The overall purpose of this method is to analyze the effect of stress treatment on bacterial cells, both at the single cell and on the population level. It provides a global vision of the effect of stress on several aspect of bacterial growth, including cell viability, morphology, and DNA content. It also allows characterizing the population recovery.
This method could be used in pharmaceutical industry to screen for the activity of new antimicrobial molecule and appreciate their impact on bacteria viability and growth. Stress-inducing treatment has an inefficiency, such as dose and time-dependent. It may be necessary to run preliminary tests to determine the optimal dose and the duration of treatment before starting this protocol.
The visual demonstration of this method allows picturing important details of each critical steps and how to perform them properly. To begin, inoculate five milliliters of MOPS EZ Rich Defined Medium with a single colony of E.Coli MG1655 HU-PAmCherry and grow at 37 degrees Celsius with shaking at 140 rotations per minute overnight. The following morning, extract 100 microliters of the sample and dilute it with 900 microliters of medium to measure the optical density at 600 nanometers.
Then, dilute the culture in a test tube containing fresh medium to an OD600 nanometers of 0.01. Incubate the inoculated test tube at 37 degrees Celsius with shaking at 140 RPM until the optical density reaches 0.2, corresponding to full exponential phase in rich medium. Then, aliquot the culture samples for time-lapse microscopy imaging, the dilution and plating assay, the flow cytometry analysis, and the microscopy snapshot imaging.
Expose the remaining 4.4 milliliters of cell culture in the test tube to specific stress treatment. For example, 4.4 microliters of cephalexin at five micrograms per milliliter and incubate at 37 degrees Celsius with shaking at 140 RPM. Prepare ten-fold serial dilutions, up to 10 to the minus seventh, of the 200 microliters of culture sample in fresh medium.
Mix between each dilution by very gentle vortexing or by inverting the tube. Plate 100 microliters of the appropriate dilution on non-selected LB agarose plates and incubate overnight at 37 degrees Celsius. The next day, count the number of colonies to determine the concentration of viable cells in each culture sample.
Plot the CFU per milliliter as a function of time for the untreated and treated cell cultures. Measure the OD600 of the culture on a spectrophotometer. Dilute the culture sample with fresh medium at four degrees Celsius to obtain a sample with OD600 at 0.06, corresponding to approximately 15, 000 cells per microliter.
For DNA staining, mix the diluted bacterial sample with a 10 microgram per milliliter solution of DNA fluorescent dye at the volume ration of one to one and incubate in the dark for 15 minutes. Before injection of the sample, mix the sample by very gentle vortexing or by inverting the tube. Pass the sample into the flow cytometer at a flow rate of approximately 120, 000 cells per minute.
Acquire forward-scattered and side-scattered light as well as DNA fluorescent dye/fluorescent signal with the appropriate settings. Plot the forward-scattered cell density histogram to represent the distribution of cell size and plot the DNA fluorescent dye/fluorescent signal cell density histogram to represent the DNA content in the cell population. First, preheat the thermostated microscope chamber at 37 degrees Celsius to stabilize the temperature.
Prepare the agarose-mounted slides as described in the manuscript. Place the slide on the microscope stage and perform image acquisition using transmitted light with a face contrast objective and with light source excitation at 560 nanometers for mCherry. 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. First, remove the conservation solution from the microfluidic plate and replace it with fresh medium preheated to 37 degrees Celsius as described in the Microfluidic Software User Guide. Attach the microfluidic plate to the manifold system.
And in the microfluidic software, click on the Seal button to seal on the plate to the manifold system. After that, click on the Priming button. Position the microfluidic plate with the manifold system on the microscope stage and preheat at 37 degrees Celsius for two hours to avoid the dilation of the microfluidic chamber.
Seal off the microfluidic plate on the microfluidic system by clicking on the Seal Off button. Replace the medium from well eight with 150 microliters of culture sample and replace the medium from well one to five with the desired medium, with or without the stress-inducing reagent. Seal the microfluidic plate and place it on the microscope stage.
In 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. Carefully focus in transmitted-light mode and select several fields of view that show isolated bacteria and are not overcrowded, around 10 to 20 cells per 100 square microliters.
In the microfluidic software, click on the Run a Custom Sequence button, then program the injection of the stress-inducing medium during 10 minutes at two PSI, followed by injection at one PSI for the wanted duration of the stress treatment. Perform microscopy imaging in the time-lapse mode with one frame every 10 minutes, using phase contrast in transmitted light and a 560 nanometer excitation light source for the mCherry signal if required. Start the microscopic image acquisition and the microfluidic injection protocol at the same time.
Open the Fiji software and the MicrobeJ plugin. For snapshot analysis, drop all images corresponding to one microscope slide 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.
Run the automated detection of the cell's outlines based on the segmentation of phase contrast image and run the detection of the nucleoids based on the segmentation of the stained DNA fluorescent signal. Check the accuracy of the cell detection visually and use the MicrobeJ editing tool for correction if needed. Save the result file obtained.
Click on the icon ResultJ to complete the analysis and obtain the ResultJ window. Many different types of output graphs are generated. Plot the normalized histograms of cell shape or length and mean nucleoid number per cell.
This study analyzed the behavior of Escherichia coli K-12 cells during transient exposure to cephalexin, an antibiotic that specifically inhibits cell division. Cell cultures growing in the presence of cephalexin exhibited similar OD600 increases as the unstressed cultures. The concentration of viable cells did not increase when cephalexin was present.
Cells started dividing again when cephalexin was removed and eventually reached a concentration equivalent to the unstressed culture after two hours. Different stresses resulted in different uncoupling of the OD and CFU per milliliter curves, depending on the effect induced. Exposure to cephalexin provoked a parallel increase of cell size and DNA content.
When cephalexin was removed, the population cell size and DNA content gradually decreased and became similar to the unstressed population after two hours. Cephalexin provoked the formation of long cells with normal cell width then no division septa. Quantitative image analysis confirmed the cell size and DNA content increase.
Time-lapse images confirm that cell elongation and chromosome replication and segregation were not inhibited by exposure to cephalexin. Analysis of the filamented cell lineage showed that cell division restarted roughly 20 minutes after washing away the drug. It is crucial to make sure that the bacterial population is in full exponential growth before inducing stress treatments.
Stress-inducing treatment used for these approaches could be hazardous for experimental, like genotoxic compound. So handle compound carefully and wear glove, mask, goggle, and lab coat.
