Name: Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes (Video) | JoVE Journal
Uploaded: 2025-02-14T14:30:00.000+00:00
Duration: 7 min 44 s
Description: Learn time-lapse epifluorescence microscopy for visualizing persister cells in Pseudomonas aeruginosa and Staphylococcus aureus. This protocol enables single-cell analysis of antibiotic survival mechanisms.

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5 minutes

We thank Ms. Susan Staurovsky at the UConn Health Center for Cell Analysis and Modeling Microscopy Facility for her assistance with microscopy experiments. We thank Dr. Mona Wu Orr and the Essentials of Staphylococcal Genetics and Metabolism Workshop for their protocols and advice on cloning in P. aeruginosa and S. aureus, respectively. This work was supported by funding from the National Institutes of Health (NIH; DP2GM146456-01 and 1R01AI167886-01A1 to W.W.K.M., 1F30DE032598-01A1 to P.J.H., and 1F31DK136259-01A1 to T.J.L.). The funders had no role in the design of our experiments or preparation of this manuscript.

Visualizing Antibiotic Persistence in Bacterial Pathogens

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The authors have no conflicts of interest to declare.

We have found that the success of a time-lapse microscopy experiment hinges on the quality of the agarose pads and their stability throughout the course of imaging. The stainless-steel chamber-enclosed agarose pads are relatively easy to prepare, resulting in consistently planar samples that can be stably imaged over dozens of hours. This enables the imaging of tens of thousands of cells in a single experiment and increases the likelihood of detecting rare phenotypic variants, like persister cells, in a population.
This agarose pad preparation method presents an easily implementable alternative to previously published methods. Our protocol does not require the technical precision of microfluidic device fabrication or the dexterous manipulations of agarose &#34;sandwich&#34; methods, making it easier to achieve consistent preparations from run to run14,16,36. Furthermore, the system is cost-effective. The stainless-steel chamber is sterilizable and reusable (unlike single-use plastic chambers) and the setup does not require specialized equipment16,37. The chamber is easily fit to different microscopy systems using commercially available stage inserts. Additionally, because bacteria are immobilized at the agarose-cover glass interface, we have had success with tracking highly motile bacteria such as P. aeruginosa while still allowing for morphological changes (Figure&#160;4, Supplementary Video 4). Other single-cell imaging techniques, such as the &#34;mother machine&#34;, confine cells to channels that preclude observation of morphological changes other than filamentation36.
For success with this protocol, there are some critical steps and parameters to keep in mind. For pad preparation, it is important to thoroughly heat and melt the agarose, as any remaining agarose crystals will cause light diffraction and affect image quality. Similarly, one must take care to pipette the agarose into the chamber without introducing air bubbles. To ensure that the thickness of the agarose pads remains consistent and to limit sample drift, it is important to allow the pad to equilibrate&#8212;typically for 15 min&#8212;in the humidified, temperature-controlled enclosure before imaging begins. Another factor that can cause poor image quality is humidity control: low humidity will result in the agarose pad dehydrating and shrinking, whereas high humidity (or improper sample warming in the chamber) could cause the warm air to condense on the sample and distort the imaging. An example of suboptimal time-lapse imaging due to condensation can be found&#160;Supplementary Video 8.
A limitation of the current set-up is that the culture media cannot be exchanged, which prevents continuous tracking of individual bacteria before, during, and after antibiotic treatment. We anticipate that coupling the agarose pad with flow cells or microfluidic devices that permit culture media exchange could enable populations to be tracked during nutritional or environmental change. Another parameter of the current design that could be improved is sample aeration. The O-ring seal, screw-top design of the interchangeable coverslip dish enables better sample aeration compared with set-ups that require the use of wax or grease-based sealant to seal the pads16. However, aeration in the sealed chamber may still be limited and may not support the growth of obligate aerobes, though this remains to be tested.
The time-lapse imaging sample preparation protocol that we present in this article enables thousands of bacteria to be tracked as they respond to or recover from antibiotic treatment. This method is also highly generalizable and has a variety of potential applications beyond persister biology. For example, the agarose pad and divider set-up allows for the seeding of spatially separated cell samples yet permits cell-cell communication via diffusion through the agarose pad. We are currently exploring this set-up&#39;s potential for testing how the exchange of secreted products affects cell growth in multispecies communities. We anticipate that this protocol will provide a low barrier of entry to time-lapse microscopy for the new investigator and limitless variations for the seasoned microbiologist to explore.

Bacterial pathogens can evade the effects of antibiotics through two primary mechanisms: antibiotic resistance, which involves genetic changes, and phenotypic tolerance, which involves non-genetic changes. Antibiotic resistance is a genetically encoded phenomenon that confers the ability of a given bacterial cell to not only survive but also replicate in the presence of an antibiotic1. Phenotypic tolerance, which can encompass antibiotic-tolerant or antibiotic-persistent bacteria, occurs when cells withstand bactericidal antibiotic treatment without gaining the ability to replicate in the presence of an inhibitory concentration of the antibiotic1,2. What differentiates tolerance from persistence is that tolerance refers to the ability of the whole population to survive treatment, whereas persistence refers to a subset of an isogenic but phenotypically heterogeneous population that survives antibiotic treatment. When a clonal culture is treated with bactericidal antibiotics and survivors that remain in the culture are plotted against time on a log-linear scale, a biphasic curve is usually detected when persisters are present. On these curves, the first phase shows that the majority of the population is killed relatively quickly, and the second phase indicates that an antibiotic persistent fraction is killed at a slower rate or not at all1,2.
Antibiotic persistence presents a major burden on global healthcare systems. For instance, Staphylococcus aureus and Pseudomonas aeruginosa persisters, which are the focus of this article, are thought to cause antibiotic-recalcitrant infections, including recurrent airway infections in patients with cystic fibrosis and chronic wound infections3,4. Therefore, further elucidating persister cell biology and phenotypic programs is critical. While progress has been made in understanding how persisters form and resuscitate, critical knowledge gaps pertaining to the coordination of metabolic reprogramming and molecular events in individual cells that underlie persistence remain5,6,7,8.
Effectively studying persistence has proven to be a technical challenge. Since persistence is only observable in a small subset of a bacterial population, techniques that sample bulk bacterial populations often fail to capture relevant biological information1,2,8,9,10. Furthermore, since phenotypic changes that underlie persistence are transient and not heritable, tracking the fate of persister cells can be complex1,8,9,10,11. Once bacterial persisters resume growth, they can divide and give rise to both persisters and non-persisters, which makes it impossible to enrich for pure persister populations by culturing. These challenges highlight the need for techniques that can fulfill the following criteria: 1) the ability to capture biological information of live, single cells and 2) the ability to be used in tandem with fluorescent dyes, probes, sensors, and reporters that allow phenotypes of individual cells in heterogeneous populations to be interrogated over time.
Recent advancements in single-cell technologies have provided an avenue to effectively investigate bacterial heterogeneity and overcome these hurdles in studying persistence12,13. Some of these techniques include fluorescence microscopy, flow cytometry/fluorescence-activated cell sorting, microfluidics, and single-cell RNA sequencing12,13. Here, we describe protocols for elucidating single-cell persister physiology using epifluorescence time-lapse microscopy of transcriptional or translational reporter strains. Fluorescence microscopy is a powerful technique that fulfills the criteria for studying persister phenotypes, namely, the ability to identify which individual cells in a large population propagate after antibiotic removal and can thus be defined as persisters. With the introduction of automated camera technologies and incubated chambers, capturing live bacterial cells is widely accessible across the field of microbiology. Crucially, time-lapse microscopy offers the ability to visualize single cells in real-time, over the course of hours and even days, which makes it possible to track bacteria before, during, and after antibiotic treatment14,15,16. Insights from these investigations harnessing time-lapse microscopy have immense potential to generate insight into the complex mechanisms of persister biology.

<table><tbody><tr><td>BaSiC</td><td>GitHub</td><td>https://github.com/marrlab/BaSiC</td><td></td></tr><tr><td>Certified Molecular Biology Agarose</td><td>Biorad</td><td>1613101</td><td></td></tr><tr><td>Fiji-ImageJ</td><td>NIH</td><td>https://imagej.net/software/fiji/downloads</td><td></td></tr><tr><td>Interchangeable Coverglass Dish</td><td>Bioptechs</td><td>190310-35</td><td>35 mm ICD for preparing agarose pads; comes with 30 mm (#1.5) coverslips</td></tr><tr><td>Lumencor Spectra 7 LED light engine</td><td>Lumencor</td><td>https://lumencor.com/products/spectra-light-engine</td><td>Spectra 7 LED light engine</td></tr><tr><td>MetaMorph</td><td>Molecular Devices</td><td>Premier version 7.10.5</td><td></td></tr><tr><td>pco.edge 4.2 bi sCMOS camera</td><td>Excelitas</td><td>https://www.excelitas.com/product/pcoedge-42-bi-usb-scmos-camera</td><td>sCMOS camera</td></tr><tr><td>PeCon live cell incubation chamber</td><td>peCon</td><td>https://www.pecon.biz/</td><td></td></tr><tr><td>Thomas Scientific Round cover glass, #1.5 thickness, 25 mm, 100 pack</td><td>Fisher Scientific</td><td>NC1272770</td><td>25 mm (#1.5) coverslips</td></tr><tr><td>Zeiss Axiovert 200M microscope</td><td>Zeiss</td><td>https://www.zeiss.com/microscopy/en/products/light-microscopes/widefield-microscopes/axiovert-for-materials.html</td><td>Inverted microscope with Plan-Apochromat 63x/1.40 Oil Ph3 M27 objective, Lumencor Spectra 7 LED light engine, and pco.edge 4.2 bi sCMOS camera (6.5 mm pixel size)</td></tr></tbody></table>

time-lapse epifluorescence microscopy imaging of pseudomonas aeruginosa and staphylococcus aureus heterogeneous phenotypes

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to&#160;underlie clinical treatment failure.&#160;Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited.
This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus)&#160;during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.

Successful introduction of reporter plasmids into P. aeruginosa and S. aureus is indicated by growth on the correct selective antibiotics and can be confirmed by colony PCR and/or sequencing. The modified strains should be verified as phenotypic reporters by subjecting them to conditions in which the gene of interest is known to be induced, and the resultant fluorescence can be measured by flow cytometry, spectrophotometry, or epifluorescence microscopy (Figure 1).
To facilitate the selection of an antibiotic dose(s) that will be used for subsequent experiments, perform concentration-dependent antibiotic persister assays for the P. aeruginosa or S. aureus strains of interest. Concentration-dependent assays typically result in a biphasic curve with a steep initial slope at lower antibiotic concentrations and a plateau or less steep slope at higher concentrations. However, for some antibiotic-species pairs, a distinct biphasic curve may not result. For example, the curve for S. aureus delafloxacin curve is clearly biphasic (Figure 3A), but the P. aeruginosa levofloxacin curve is not (Figure 3B)15. In this scenario, we would choose a concentration that is at least 10x the MIC (e.g., 5 µg/mL, which is about 15x the MIC for P. aeruginosa)15. However, because 15x levofloxacin MIC results in only ~0.001% P. aeruginosa survivors, we use 1 µg/mL levofloxacin treatment if we want to see persisters when imaging cells as they recover on antibiotic-free agarose pads (Supplementary Video 6); otherwise, the number of fields of view needed to image multiple persisters becomes prohibitive.
At the start of imaging, the ideal sample and agarose pad preparation should appear planar throughout the field of view, free of large debris, wrinkles, or air bubbles, and with evenly distributed single cells. Obtaining well-distributed single cells may require optimization of the sample dilution or resuspension. For S. aureus, cells tend to form small clusters and need to be vortexed thoroughly before seeding to the agarose pad (Figure 4 and Figure 5). For P. aeruginosa, cells may form aggregates encased in a sticky extracellular matrix in suspension; it is necessary to pipette these samples thoroughly and disrupt the aggregates for imaging single cells. 
After an imaging experiment's conclusion, a successful time-lapse of images will appear in focus, stably illuminated, and with minimal drift in the x-y plane throughout the experiment. Supplementary Video 7 represents an optimal image acquisition: t is the phase channel of Supplementary Video 4 before shade or drift correction. Loss of focus can occur if condensation (from over-humidification or insufficient sample warming) causes water droplets to form on the top 25 mm coverslip, distorting the light and pushing the focal plane outside of the autofocus algorithm's maximal search range (Supplementary Video 8). Variation in illumination usually indicates insufficient immersion oil at the time of imaging. If the stage moves too quickly, the oil on the objective may drag behind and still be catching up when the images are acquired. This can be mitigated by adjusting the acquisition controls to slow the speed of movement or adding a pause between the movement to the next position and the image acquisition. Major sample drift will look like many cells streaking across the field of view while some stay in place (Supplementary Video 9). This typically occurs later in experiments because the agarose pad has dehydrated due to insufficient humidity control. The agarose pad preparation presented in this paper was designed to facilitate sample stability, but properly warming/humidifying the sample and its surrounding environment is necessary for optimal image acquisition.
<img alt="Persister cell microscopy; gene expression levels, fluorescent focus over time; diagram." class="xfigimg" src="/files/ftp_upload/67617/67617fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Fluorescent reporter strains illuminate the expression of a gene of interest. (A) S. aureus was transduced with a GFP transcriptional reporter for a gene of interest per Protocol 1. The reporter strain was treated for 24 h with antibiotic, washed with PBS, and then seeded onto an agarose pad made from CA-MHB plus propidium iodide (1.6 µM) and chloramphenicol (10 µg/mL for reporter plasmid maintenance) for imaging during recovery (Supplementary Video 1). (B) P. aeruginosa was transformed with a plasmid bearing a mScarlet-linked translational reporter for a protein of interest26. The reporter strain was treated for 5 h with antibiotic, washed with PBS, and then seeded onto an agarose pad made from BSM plus Tet (75 µg/mL; for reporter plasmid maintenance) for imaging during recovery (Supplementary Video 2). <a href="https://www.jove.com/files/ftp_upload/67617/67617fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bacterial growth on agar plates, dilution series, colony observation, microbiology experiment setup." class="xfigimg" src="/files/ftp_upload/67617/67617fig2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Propagating and harvesting bacteriophage. (A) The six plates show six different amounts of diluted phage stock on the lawns of S. aureus RN4220. The red outlines indicate the three plates that would be harvested, from the plate with the most clearing (bold red outline; 1 x109 PFU/mL) to the next two dilutions (1 x108 and 1 x 107 PFU/mL). The black arrows point to individual plaques. (B) To harvest phage from the plates, scrape the soft agar layer (left), transfer the slurry to the next dilution plate (center), and, after pooling the soft agar from all three plates together, combine into a conical tube for centrifugation (right). <a href="https://www.jove.com/files/ftp_upload/67617/67617fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Survival fraction vs antibiotic concentration graph, S. aureus, P. aeruginosa, drug efficacy analysis." class="xfigimg" src="/files/ftp_upload/67617/67617fig3.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Representative concentration-dependent persister assays. Concentration-dependent fluoroquinolone persistence was assessed in stationary-phase (A) S. aureus (against delafloxacin) and (B) P. aeruginosa (against levofloxacin). Subsequent experiments utilize 5 µg/mL delafloxacin (red circle) because S. aureus killing had plateaued at this concentration. A dosage of at least 1 µg/mL levofloxacin (red circle) would be utilized for subsequent experiments with P. aeruginosa. Note that the bacterial killing does not plateau for P. aeruginosa, but there is still a less steep "second phase" of the biphasic curve that indicates a persistent subpopulation. Panel 3B has been adapted with permission from Hare et al.15. <a href="https://www.jove.com/files/ftp_upload/67617/67617fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="S. aureus and P. aeruginosa time-lapse microscopy showing PI+ cell changes over time. Diagrams included." class="xfigimg" src="/files/ftp_upload/67617/67617fig4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Imaging bacterial phenotypes during antibiotic treatment. Stationary-phase (A) S. aureus and (B) P. aeruginosa cells were seeded onto agarose pads containing fluoroquinolone antibiotics and monitored during treatment: 5 µg/mL delafloxacin for S. aureus (Supplementary Video 3) and 5 µg/mL levofloxacin for P. aeruginosa (Supplementary Video 4)15. Propidium iodide (PI; 16 µM for P. aeruginosa, 1.6 µM for S. aureus) was added to the pads to mark dead or dying cells. S. aureus cells remain largely intact and alive in the presence of the FQ, whereas most P. aeruginosa cells undergo drastic morphological changes, including forming round spheroplasts, before they lyse and die. <a href="https://www.jove.com/files/ftp_upload/67617/67617fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Staphylococcus aureus and Pseudomonas aeruginosa growth time-lapse microscopy, bacterial proliferation." class="xfigimg" src="/files/ftp_upload/67617/67617fig5.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Tracking persisters during recovery. (A) S. aureus and (B) P. aeruginosa populations were seeded onto agarose pads containing fresh media after they had been treated with fluoroquinolones (5 µg/mL delafloxacin for S. aureus and 1 µg/mL levofloxacin for P. aeruginosa) and monitored during their post-treatment recovery (Supplementary Video 5 and Supplementary Video 6). The perisisters seen are indicated with green arrows in the first two frames in each panel, and they remained intact and viable during antibiotic treatment. After an initial lag period, the persisters started to divide and gave rise to new progeny (indicated with green circles). <a href="https://www.jove.com/files/ftp_upload/67617/67617fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Coverslip preparation and cell seeding process, setup diagram and components for microscopy study." class="xfigimg" src="/files/ftp_upload/67617/67617fig6.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6: Microscope sample preparation. (A) Schematic of the sample preparation workflow using an interchangeable coverslip dish ("chamber"). (B) Picture of the disassembled chamber and its individual components. (C) Picture of the fully assembled chamber. <a href="https://www.jove.com/files/ftp_upload/67617/67617fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video 1: S. aureus persister. Video file containing the images in Figure 1A. In brief, S. aureus bearing a GFP transcriptional reporter for a gene of interest was treated for 24 h with antibiotic, washed with PBS, then seeded onto an agarose pad made with CA-MHB plus propidium iodide (1.6 µM) and chloramphenicol (10 µg/mL) for imaging during recovery. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS1_S-aureus_persister_video.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 2: P. aeruginosa persister. Video file containing images in Figure 1B. In brief, P. aeruginosa bearing an mScarlet-linked translational reporter for a protein of interest was treated for 5 h with antibiotic, washed with PBS, then seeded onto an agarose pad made with BSM plus Tet (75 µg/mL) for imaging during recovery26. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS2_Paeru-fluorescent-persister.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 3: S. aureus during antibiotic treatment. A stationary-phase culture of S. aureus grown in rich chemically defined media was seeded to agarose pads made from the culture's cell-free conditioned media with propidium iodide (1.6 μM) and delafloxacin (5 μg/mL). <a href="https://www.jove.com/files/ftp_upload/67617/VideoS3_S-aureus_treatment.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 4: P. aeruginosa during antibiotic treatment. A stationary-phase culture of P. aeruginosa grown in BSM was seeded to agarose pads made from cell-free conditioned media from a culture of P. aeruginosa grown in BSM in parallel; the agarose pad also contained propidium iodide (16 µM) and levofloxacin (5 µg/mL). This video has been adapted with permission from Hare et al.15. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS4_P-aeruginosa_treatment.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 5: S. aureus during post-antibiotic recovery. S. aureus was grown to stationary phase in rich chemically defined media. The stationary-phase cultures were treated with 5 µg/mL delafloxacin in test tubes for 24 h, washed with PBS, and then seeded to antibiotic-free CA-MHB agarose pads containing propidium iodide (1.6 µM) for imaging. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS5_S-aureus_recovery.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 6: P. aeruginosa during post-antibiotic recovery. A stationary-phase culture of P. aeruginosa grown in BSM was treated with 1 µg/mL levofloxacin in test tubes for 7 h, washed with PBS, then seeded to antibiotic-free BSM agarose pads containing propidium iodide (16 µM) for imaging. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS6_P-aeruginosa_recovery.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 7: Example of optimal image acquisition. This video is the phase channel of Supplementary Video 4 before image processing as an example of an optimal time-lapse acquisition. Note the minimal drift, stable illumination, and maintenance of focus throughout the experiment. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS7_uncorrected-Paeru.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 8: Example of suboptimal image acquisition due to condensation. This video shows part of an experiment when the image acquisition was affected by poor focus, likely due to condensation on the chamber due to improper heating of the sample and/or over-humidification of the imaging environment. The sample being imaged was levofloxacin-treated P. aeruginosa during post-antibiotic recovery on a BSM agarose pad. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS8_Suboptimal_Imaging_Condensation.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 9: Example of suboptimal image acquisition due to drift. This video shows part of an experiment when the image acquisition was affected by sample drift, likely due to dehydration and the shrinking/lifting of the agarose pad from the coverslip. The sample being imaged was P. aeruginosa on an agarose pad containing levofloxacin and propidium iodide. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS9_Suboptimal_Drift.avi" target="_blank">Please click here to download this video.</a>
Supplementary File 1: 25mm-3D-divider-for-35mmBioptechs.stl <a href="https://www.jove.com/files/ftp_upload/67617/25mm-3D-divider-for-35mmBioptechs.stl" target="_blank">Please click here to download this file.</a>

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize&#160;phenotypic variants, such as persisters, by time-lapse epifluorescence&#160;microscopy.

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment ...

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