This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.
Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.
Polymicrobial communities are common in nature, spanning a variety of environmental1,2,3 and human niches4,5. Some of the most notorious polymicrobial infections in human disease include dental biofilms6, chronic wounds7,8, and respiratory infections in individuals with chronic obstructive pulmonary disease9, ventilator-associated pneumonia10, and the genetic disease cystic fibrosis (CF)11,12. These infections are often composed of diverse microbial species; however, a recent focus on the interactions between the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Pseudomonas aeruginosa has emerged. Studies including patients coinfected with these organisms and in vitro analyses reveal both competitive and cooperative interactions that can have profound and unexpected influences on disease progression, microbial survival, virulence, metabolism, and antibiotic susceptibility (reviewed in detail by Hotterbeekx et al. 201713 and Limoli and Hoffman 20193).
The growing interest in interspecies interactions during infection has yielded a variety of methods for studying polymicrobial community behaviors. Typically, these studies have utilized plate or broth-based assays to investigate the phenotypic differences between monoculture and coculture. For example, coculturing P. aeruginosa and S. aureus on solid surfaces has allowed for visualization of growth inhibition or changes in colony phenotype, pigment, or polysaccharide production14,15,16. Mixed species biofilms, on biotic or abiotic surfaces, as well as coculturing bacterial species in liquid culture also has enabled measurement of changes in growth, metabolism, antibiotic tolerance, competition and viability, in addition to gene and protein expression17,18. While these bulk culture experiments have revealed insight into how P. aeruginosa and S. aureus might influence one another on the community-scale, they are unable to answer important questions about the interactions that take place at the single-cell level. The method presented here adds to the approaches for studying interspecies interactions by focusing on the changes in movement, cell viability, and gene expression of single cells within a cocultured community over time.
Single-cell interactions can widely differ from the interactions that take place in a bulk community of cells. Through single-cell analysis, heterogeneity within a community can be quantified to study how spatial placement of cells influence community dynamics or how gene and protein expression levels change within individual members of a group. Tracking cells can also provide insight into how single cells move and behave over time, through multiple generations. By tracking cell movement and changes in gene expression concurrently, correlations can be made between gene fluctuations and corresponding phenotypes. Previous protocols for studying individual bacterial species at the single-cell level have been described. These studies take advantage of live-imaging cells over time in a single plane, and have been useful for observing phenotypes like cell division and antibiotic susceptibility19,20. Additional live-imaging microscopy has been utilized to monitor growth, motility, surface colonization and biofilm formation of single bacterial species21,22,23. However, while these studies have been insightful for understanding the physiology of bacteria in monoculture, experiments for observing single-cell behavior of multiple bacterial species over time in coculture are limited.
Here, previous protocols used for single-species imaging have been adapted to study interactions between P. aeruginosa and S. aureus. These organisms can be differentiated under phase contrast based on their cell morphologies (P. aeruginosa are bacilli and S. aureus are cocci). Development of this method recently enabled the visualization of previously undescribed motility behaviors of P. aeruginosa in the presence of S. aureus24. P. aeruginosa was found to be capable of sensing S. aureus from a distance and responding with increased and directional single-cell movements towards clusters of S. aureus cells. P. aeruginosa movement towards S. aureus was found to require the type IV pili (TFP), hair-like projections whose coordinated extension and retraction generate a movement called twitching motility25.
These studies demonstrate the utility of this method for interrogating earlier interactions between species. However, imaging at high cell densities at later interaction time points is difficult given that single layers of cells can no longer be identified, which mostly poses issues during post-imaging analysis. Given this limitation, the method is best suited for earlier interactions that could then be followed up with traditional macroscopic assays at higher cell densities representative of later interactions. Additional limitations of this method include the low-throughput nature, since only one sample can be imaged at a time and the cost of the microscope, camera, and environmental chamber. Moreover, fluorescence microscopy poses risks to the bacterial cells like phototoxicity and photobleaching, therefore limiting the frequency that fluorescence images can be acquired. Lastly, the agarose pads used in this method are highly susceptible to changes in the environment, making it critical to control for conditions like temperature and humidity, given that the pads can start to shrink or expand if the conditions are not correct. Finally, while this method does not mimic the host environment, it provides clues for how different bacterial species respond on surfaces, which can be followed-up in assays designed to mimic environmental/host conditions.
This method differs from previous studies tracking single-cell movement, in that cells are inoculated between a coverslip and agarose pad, restricting cells to the surface. This enables cell tracking over time in a single plane; however, limits cycles of transient surface engagement observed when cells are submerged in liquid26. An additional benefit of imaging bacteria under an agarose pad is that it mimics macroscopic plate-based sub-surface inoculation assays classically used to examine P. aeruginosa twitching motility25. In this assay, bacterial cells are inoculated between the bottom of a Petri dish and the agar, keeping the cells in a single plane as they move across the bottom of the dish outward from the point of inoculation, much like this microscopy protocol.
The time-lapse microscopy protocol for visualizing interspecies interactions presented here consists of 1) preparing the bacterial sample and agarose pad, 2) selecting microscope settings for imaging acquisition and 3) post-imaging analysis. Detailed visualization of cell movement and tracking can be performed by acquisition of images at short time intervals by phase contrast. Fluorescence microscopy can also be utilized to determine cell viability or gene expression over time. Here, we show one example of adaptation for fluorescence microscopy through the addition of viability dyes to the agarose pads.
NOTE: A full description and catalog numbers for all supplies in this protocol can be found in the Table of Materials.
1. Preparation of M8T minimal media
Day 1:
2. Preparation of bacterial overnight cultures
Day 2:
3. Subculture of bacterial strains
4. Preparation of materials for pad molds
5. Preparation of agarose pads
NOTE: The pads are prepared with the M8T minimal media as the nutrient source for the bacteria used in this protocol. However, the nutrients used in the pads can be modified for different organisms.
6. Preparation of bacterial cells and inoculating pads
7. Setting up the microscope for live imaging
8. Optional: Modifications for live/dead imaging
9. Data analysis
Successful use of the described method will result in a series of frames that generate a video in which the interspecies interactions can be observed over time. The schematic in Figure 1 provides a visual to highlight the key steps involved in preparing materials for imaging. Use of this method has allowed the demonstration of P. aeruginosa cells exhibiting different behaviors in monoculture versus in coculture with S. aureus. Compared to the P. aeruginosa cells in monoculture that remain grouped in rafts, when in coculture with S. aureus, P. aeruginosa has increased single-cell motility towards S. aureus colonies (Figure 2A-2B). Fluorescently labeled bacterial strains also allow for visualizing mixed populations of bacterial species. Using GFP-labeled P. aeruginosa permitted the observation that after P. aeruginosa single cells increase in motility, they will surround and eventually invade S. aureus colonies (Figure 2C). Utilization of fluorescently-marked cells also allows for visualization of P. aeruginosa cell invasion into the S. aureus colonies for the first time (Figure 2C, bottom).
While the protocol yields high quality images for observing interspecies interactions, there are several common problems that can lead to poor quality frame sequences. Inconsistent humidity over the duration of the experiment and incorrectly dried pads are two common problems that lead to drift resulting from the pad shifting and dragging the cells with it out of the FOV (Figure 3A). Changes in humidity affect the dryness of the agarose pads. Increased humidity makes the pads too wet, allowing moisture to settle between the pad and the bottom of the glass-bottom dish. The moisture leaves a layer of liquid thick enough for motile cells to swarm or swim through and does not keep bacterial cells in a single plane. Meanwhile, decreases in humidity dry out the pad faster, which cause cells to drift early on. Another common mistake when using this method with fluorescence is acquiring fluorescent images too frequently or exposing bacterial cells to fluorescent light for too long. Short acquisition intervals for fluorescent images repeatedly excite fluorophores with high intensity light of a specific wavelength. Excited fluorophores then react with oxygen, causing degradation of the fluorophore. The resulting photobleaching depletes fluorescence until more of the fluorophore can be expressed and folded, but does not harm the bacterial cells themselves (Figure 3B). Loss of fluorescence, however, does interfere with measurements of gene/protein expression, leaving the cells markerless until the fluorophore can be fully synthesized and excited again. Moreover, the oxygen interacting with excited fluorophores can form reactive oxygen species (ROS). These ROS radicals then damage bacterial cells, ultimately becoming toxic and resulting in cell death within a few cell divisions (Figure 3C). The processes of photobleaching and phototoxicity can be easily seen as cells will first lose their fluorescence completely, and in subsequent frames, the cells will stop dividing and may eventually die (Figure 3B-3C). One final common issue when setting up the experiment is starting with too many cells per FOV or with cells too close to each other, which is generally less than 20 µm apart. Crowded cells in the first frame will yield clusters of dividing cells that simply merge into each other as they grow rather than interact. Additionally, cells that begin too close in proximity may not have sufficient time to establish a gradient of secreted signals to which the other species can respond (Figure 3D) whereas distant bacterial cells may not have the chance to encounter one another within the duration of the experiment.
Figure 4 shows an example of how bacterial cell viability can be visualized with this protocol by adding propidium iodide to the agarose pads. Propidium iodide is impermeable to live cells, but can enter cells with damaged membranes and bind to nucleic acids. Here mid-log GFP-labeled WT S. aureus were treated with media alone or with cell-free supernatant derived from P. aeruginosa and imaged immediately following treatment. Three different channels were imaged: Phase, TxRed and GFP. Bright green cells indicate live cells actively expressing GFP as seen for the S. aureus treated only with media (Figure 4A), and red cells indicate dead propidium iodide-stained S. aureus cells, after being treated with P. aeruginosa supernatant (Figure 4B). While only one time point is shown, this method can be adapted to determine cell viability during time-lapse live imaging.
Several post-imaging analyses can be performed to quantify aspects of interspecies interactions. For example, cell tracking can provide measurements for the directedness of P. aeruginosa single cell movements towards a cluster of S. aureus. The movements of individual P. aeruginosa cells are tracked from the frame a cell leaves the raft through the frame in which the cell reaches the S. aureus cluster (Figure 5A). The distance between the P. aeruginosa raft and the S. aureus cluster provide the Euclidian distance, D(E), while the total track lengths provide the accumulated distance, D(A) (Figure 5B). Directedness of each cell is calculated as a ratio of D(E)/D(A). In the coculture experiments, WT P. aeruginosa moved towards WT S. aureus with significantly higher directedness than towards S. aureus ΔagrBDCA, a mutant lacking Agr-regulated secreted factors, previously determined to be necessary for directional motility towards S. aureus24 (Figure 5C).
Figure 1: Schematic of the imaging setup protocol.
Overview of critical steps for preparation of bacterial cultures and agarose pads. Created with BioRender.com. Please click here to view a larger version of this figure.
Figure 2: Time-lapse, live-imaging microscopy shows differences in P. aeruginosa behavior when cocultured with S. aureus.
Representative snap shots of P. aeruginosa (bacilli, green) in monoculture (A) and in coculture with S. aureus (cocci, unmarked) (B). (C) Fluorescently-labeled bacteria allow for visualization of P. aeruginosa single cells invading S. aureus clusters. Phase contrast and GFP channel overlay (top) and GFP channel alone (bottom). Please click here to view a larger version of this figure.
Figure 3: Representative snap shots of common imaging issues that lead to poor image acquisition.
(A) Improperly dried pads and inconsistent humidity lead to drifting of cells across the FOV over the duration of imaging. The position of the founding cell is marked in each frame (green rod). (B) Photobleaching from exposure to light for too long will deplete detectable levels of fluorescence for a period of time, but does not kill the cells. (C) Phototoxicity as a result of frequent exposure to light leads to cell death. The first signs of phototoxicity are seen when cells stop fluorescing and fail to divide. (D) High initial inoculum crowds cells in the FOV and prevents observation of interspecies interactions. Please click here to view a larger version of this figure.
Figure 4: Comparison of live versus dead S. aureus cells.
Representative snap shots for GFP-labeled WT S. aureus treated with either medium alone (A) or cell-free P. aeruginosa supernatant (B). Cells were immediately imaged following treatment. Live cells (green) actively express GFP and exclude propidium iodide, while dead cells (red) lose GFP fluorescence and membrane permeabilization allows propidium iodide to enter the cells and bind nucleic acids. Please click here to view a larger version of this figure.
Figure 5: Cell tracking analysis of P. aeruginosa in coculture with S. aureus.
Previously, this method was used method to perform cell tracking of WT P. aeruginosa in coculture with either WT or ΔagrBDCA S. aureus. (A) Representation of P. aeruginosa single-cell tracks in a coculture with S. aureus ΔagrBDCA. (B) Schematic for the Euclidian distance (D(E)) and accumulated distance (D(A)) measurements used to determine directedness ((D(E)/D(A)). (C) Directedness measurements of single WT P. aeruginosa cells in coculture with WT and ΔagrBDCA S. aureus. This figure has been modified from Limoli et al. 201924. Please click here to view a larger version of this figure.
Supplemental File. Please click here to view a larger version of this figure.
The methods presented here describe a protocol for live-cell imaging of bacterial species interactions at the single-cell level with modifications for other applications including cell tracking and monitoring cell viability. This method opens new avenues for studying single-cell behaviors of microorganisms in coculture with other species over time. Specifically, the protocol demonstrates the usefulness of this coculture method in observing bacterial surface behaviors, particularly when studying organisms that have both surface and liquid-associated appendages for motility. For example, by limiting bacterial movement to a surface in a single plane of focus, the increased and directional pili-mediated motility of P. aeruginosa in response to S. aureus can be visualized.
As previously mentioned, achieving optimal results with this imaging method requires consideration of several conditions, including temperature and humidity of the sample and imaging instruments (i.e., objectives and stage). For more tips and troubleshooting, see the Supplemental File 1. A critical step in successful use of the protocol is preparation of the agarose pads, as inadequately drying pads is one of the most common problems. As shown in Figure 3A, if the pads are not dried long enough, drift appears at the beginning of the time-lapse imaging, whereas pads that are dried for too long start to shrink and the cells drift out of the FOV a few hours into imaging. Ensuring that all materials are pre-warmed and maintained at uniform temperature and humidity over the duration of the experiment, through use of a stage-top incubator and damp lint-free wipes, will help reduce drift. It is also advised that a backup pad is always made in case the first pad is not dried properly or tears during transfer from the mold to the sample dish. Additionally, it is important to use a low-autofluorescence medium, both for growing bacterial cultures and for making the pads in order to minimize the background fluorescence from the medium when imaging the cells. It is recommended to use minimal media for microscopy, since rich media often have high autofluorescence. Starting with a low-density inoculum and even spatial distribution of cells in the FOV are also key factors in this method. Specifically, in prior studies evaluating P. aeruginosa and S. aureus interactions, this allowed the bacteria to generate a sufficient gradient of secreted factors that can then be detected by the other species present (Figure 2B).
Despite the benefits of this method, there are also limitations including its price, low-throughput nature, fluorescence restrictions, and high dependence on controlling environmental conditions. Figure 3B-3C show the major limitations of using fluorescence microscopy. As discussed in the results section, if the fluorescent images are captured in short intervals, photobleaching and phototoxicity can occur. In order to avoid these two outcomes, fluorescent image intervals should be taken far enough apart and with as low fluorescent light exposure time as possible to still adequately visualize the fluorophore. Additionally, when determining the interval for fluorescence imaging, it is important to consider the maturation time of each fluorophore. The fluorophores used in the P. aeruginosa and S. aureus strains shown in Figure 2, Figure 3 and Figure 4, for example, have a maturation time of about 20 minutes and can therefore be excited every 20 minutes without concern of potential photobleaching effects. Meanwhile, another disadvantage of this method is that it does not allow for observations of late interspecies interactions once cells reach a high cell density. In order to visualize individual bacterial cells, they must remain in a single plane of focus. However, once the population reaches a high cell density, the cells begin to grow in more than one plane.
This method can be modified to study different phenotypes such as cell viability (Figure 4) and expression of genes of interest (data not shown). Figure 4 shows an example of how the method has been adapted to visualize bacterial viability by adding propidium iodide to the agarose pads. Another application of this method is measuring bacterial gene/protein expression in coculture with another organism through fluorescent reporters. For example, multiple fluorophores can be incorporated into a plasmid vector or the bacterial chromosome to simultaneously study the expression of different genes or proteins. Here it is important to select fluorophores that do not have overlapping excitation and emission spectra. Lastly, the use of bacterial cell tracking in post-imaging analysis enables the directionality (Figure 5), speed and acceleration, among other measurements, to be calculated as well24.
Overall, this coculture imaging method adapted from previously described monoculture protocols enhances the ability to visualize behaviors of multiple bacterial species in coculture. This method offers the opportunity to study microbes from a mixed-culture perspective, which will increase understanding of how each species alters its behaviors in a single-cell manner, ultimately providing new insight into how bacterial species interact in polymicrobial environments.
The authors have nothing to disclose.
This work was supported by funding from the Cystic Fibrosis Foundation Postdoc-to-Faculty Transition Award LIMOLI18F5 (DHL), Cystic Fibrosis Foundation Junior Faculty Recruitment Award LIMOLI19R3 (DHL), and NIH T32 Training Grant 5T32HL007638-34 (ASP). We thank Jeffrey Meisner, Minsu Kim, and Ethan Garner for sharing initial protocols and advice for imaging and making pads.
Agarose pads | |||
35 mm Glass Bottom Dish with 20 mm Micro-well #1.5 Cover Glass | Cellvis | D35-20-1.5-N | One for agarose pad molds, one for experiment |
KimWipes | Kimberly-Clark Professional | 06-666A | |
Low-Melt Agarose | Nu-Sieve GTG/Lonza | 50081 | For making agarose pads |
Round-Bottom Spatulas | VWR | 82027-492 | |
Round-Tapered Spatulas | VWR | 82027-530 | |
Silicon Isolators, Press-to-Seal, 1 well, D diameter 2.0 mm 20 mm, silicone/adhesive | Sigma-Aldrich | S6685-25EA | For agarose pad molds |
Sterile Petri Plates, 85 mm | Kord-Valmark /sold by RPI | 2900 | |
Tweezers | VWR | 89259-944 | |
M8T Minimal Media | |||
D (+) Glucose | RPI | G32045 | |
KH2PO4 | RPI | P250500 | |
MgSO4 | Sigma-Aldrich | 208094 | |
NaCl | RPI | S23025 | |
Na2HPO4.7H2O | Sigma-Aldrich | 230391 | |
Tryptone | BD Biosciences | DF0123173 | |
Microscope | |||
Andor Sona 4.2B-11 | Andor | 77026135 | Camera. 4.2 Megapixel Back-illuminated sCMOS, 11 μm pixel, 95% QE, 48 fps, USB 3.0, F-mount. |
Filter Cube GFP | Nikon | 96372 | Filter cube |
Filter Cube TxRed | Nikon | 96375 | Filter cube |
H201-NIKON-TI-S-ER | Okolab | 77057447 | Stagetop incubator |
Nikon NIS-Elements AR with GA3 and 2D and 3D tracking | Nikon | 77010609, MQS43110, 77010603, MQS42950 | Software for data analysis |
Nikon Ti2 Eclipse | Nikon | Model Ti2-E | Microscope |
CFI Plan Apo ƛ20x objective (0.75NA) | Nikon | MRD00205 | Objective |
CFI Plan Apo ƛ100x oil Ph3 DM objective (1.45NA) | Nikon | MRD31905 | Objective |
ThermoBox with built-in fan heaters | Tokai Hit | TI2TB-E-BK | Enclosure |
Bacterial Strains | |||
Pseudomonas aeruginosa PA14 (WT) | PMID: 7604262 | Non-mucoid prototroph | |
Pseudomonas aeruginosa PA14 (WT) pSMC21 (Ptac-GFP) | PMID: 9361441 | ||
Pseudomonas aeruginosa PAO1 (WT) pPrpoD-mKate2 | PMID: 26041805 | ||
Staphylococcus aureus USA300 LAC (WT) | PMID: 23404398 | USA300 CA-Methicillin resistant strain LAC without plasmids | |
Staphylococcus aureus USA300 LAC (WT) pCM29 (sarAP1-sGFP) | PMID: 20829608 | ||
Staphylococcus aureus USA300 LAC △agrBDCA | PMID: 31713513 | ||
Viability Stain | |||
Propidium Iodide | Invitrogen | L7012 | LIVE/DEAD™ BacLight™ Bacterial Viability Kit |