We thank Daniel May, Marc Chevrette, and Don Hoang for critical reading of the manuscript. This work, including the efforts of Cameron R. Currie, was supported by the University of Wisconsin-Madison, Office of the Vice Chancellor for Research and Graduate Education with funding from the Wisconsin Alumni Research Foundation, funding also provided by the National Institutes of Health Centers for Excellence for Translational Research (U19-AI109673-01). Reed M. Stubbendieck was supported by a National Library of Medicine training grant to the Computation and Informatics in Biology and Medicine Training Program (NLM 5T15LM007359). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Informed consent was obtained from the donor&#39;s parents, and the Human Subjects Committee at the University of Wisconsin-Madison approved the study (Institutional Review Board [IRB] approval number H-2013-1044).
1. Sample Culture
NOTE: This procedure is used here for the study of interactions among bacteria isolates from the human nasal cavity. In principle, the following methods are applicable to any culture condition. Brain-heart-infusion broth (BHI) is used for general propagation of nasal bacteria. All plates are solidified using 1.5% agar. For this study, samples are taken from saline solution flushed into a donor&#39;s nose (nasal lavage), transferred into microcentrifuge tubes, and frozen at -80 &#176;C.
<ol>
	<li>Use standard culture techniques to plate 100 &#181;L of each of the thawed lavage samples onto BHI plates.</li>
	<li>Incubate the plates aerobically at 37 &#176;C for 1 week.</li>
	<li>After incubation, select &#8805;2 colonies of each distinct morphotype per plate and passage the isolates aerobically on BHI plates by streaking a colony from the initial plate onto a new BHI plate and incubating the plate at 37 &#176;C. Repeat until the bacterial cultures are pure. 
	NOTE: Bacterial isolates can be identified through colony morphology, gram-staining, 16S rRNA gene sequencing, or another method. However, knowing the isolate&#39;s identity is not necessary for continuing the protocol.</li>
	<li>Cryopreserve all bacterial isolates at -80 &#176;C after combining 1 mL of 50% glycerol with 1 mL of bacterial overnight culture (see section 3) in cryotubes.</li>
</ol>
2. 3D Printing Stamps
NOTE: Polycarbonate was selected as the stamping material due to its high glass transition temperature (147 &#176;C) that exceeds standard autoclave temperatures (121 &#176;C), which minimizes the potential for deformation after repeated uses.
<ol>
	<li>Load the 3D printer with polycarbonate filament.</li>
	<li>Apply white school glue (polyvinyl acetate) to the print bed to aid in the adhesion and minimize warping of the inoculation stamp during the print.</li>
	<li>Load the .STL model file (Supplementary Data File) for the inoculation stamp (Figure 1) into the 3D printer software.</li>
	<li>Print the inoculation stamp at a 290 &#176;C nozzle temperature, 60 &#176;C bed temperature, and layer height of 0.38 mm.</li>
	<li>Wrap the stamp in the aluminum foil and sterilize by autoclaving for 1.5 h on a gravity cycle with 15 min of drying. 
	NOTE: Though polycarbonate is hygroscopic, the stamps only retain approximately 0.5% water weight after autoclaving.</li>
</ol>
3. Preparation of Overnight Cultures
<ol>
	<li>Using a serological pipette, pipette 3 mL of sterile BHI broth into 14 mL culture tubes.</li>
	<li>Using a sterile 1 &#181;L inoculating loop, inoculate a bacterial colony into the broth. Swirl the loop to ensure the clump disperses into the broth. Vortex the culture tubes briefly before incubation.</li>
	<li>Incubate the culture tubes at 37 &#176;C overnight (~16 h) on a shaker at 250 rpm.</li>
	<li>Vortex to break up the clumps of cells once the bacterial cultures reach sufficient&#160;turbidity (OD600&#160;&#8805; 1).</li>
</ol>
4. Preparation of Bioassay Plates
NOTE: Bioassay plates are prepared in a laminar flow hood to maintain sterility.
<ol>
	<li>Prepare BHI media with 1.5% agar and sterilize by autoclaving according to manufacturer instructions.</li>
	<li>After autoclaving, cool the BHI media to 55 &#176;C in a temperature-controlled water bath.</li>
	<li>Using a serological pipette, pipette 3 mL of molten BHI media into each of the 12 wells on a 12 well plate. Ensure that the wells are as exact and even as possible. One liter of media will yield ~27 bioassay plates.</li>
	<li>Allow the agar to set overnight.</li>
</ol>
5. Inoculating Bioassay Plates with the Test Organism
NOTE: A test organism refers to the organism for which the production of inhibitory activity (e.g., antibiotic production) is determined using the co-culture interaction assay. For this experiment, the test organisms are Actinobacteria isolated from nasal lavages samples.
<ol>
	<li>Inoculate the test organism on a bioassay plate by inserting a sterile 10 &#181;L inoculating loop into the overnight culture and streaking a culture droplet over the left third of a plate well. 
	NOTE: It is recommended to only streak one-third of the well, as overgrowth of the well prohibits later inoculation of target organisms.</li>
	<li>Repeat until all 12 wells on the plate have been inoculated with the test organism.</li>
	<li>Incubate plates upside down at the appropriate temperature for 7 days. At higher temperatures (&#8805;37 &#176;C) or in drier climates, store the plates in a humid container to prevent the plates from drying out.</li>
</ol>
6. Preparation of Target Organisms
NOTE: A target organism refers to the organism whose inhibition status is determined using the co-culture interaction assay. For this experiment, the target organisms are Staphylococcus spp. isolated from nasal lavages samples.
<ol>
	<li>After incubating the bioassay plates for 6 days, prepare overnight cultures of the specified target organisms, as done above (see section 3).</li>
</ol>
7. Target Organism Inoculation
NOTE: After overnight incubation, ensure that the cultures are turbid (OD600&#160;&#8805; 1). Some bacterial cultures may flocculate at the bottom of the culture tube. Vortex the culture tubes to disperse clumps and assess the culture turbidity.
<ol>
	<li>Prepare the target plate by filling each well of an empty 12 well plate with 1.8 mL of BHI and 200 &#181;L of the target overnight culture.</li>
	<li>Unwrap and place a sterile inoculation stamp into the target plate. Gently swirl the cultures around in the wells, taking care to ensure that the cultures do not cross contaminate neighboring wells.</li>
	<li>Lift the inoculation stamp and ensure that there is a droplet of diluted target culture on each stamp tip.</li>
	<li>Prepare a monoculture control plate by placing the inoculation stamp on an uninoculated bioassay plate and gently rock the stamp so that a culture drop inoculates each well. Once the inoculation stamp is removed, a droplet of culture should be visible in the wells of the bioassay plate.
	<ol>
		<li>If any wells are not inoculated with the inoculation stamp due to uneven levels of media, spot 3 &#181;L of diluted overnight culture onto the right side of the wells using a pipette.</li>
	</ol>
	</li>
	<li>Inoculate the bioassay plates as done above (see step 7.4.1), but align the stamp so that the tips align with the right side of the 12 well plate. Ensure that the stamp does not contact the existing bacterial colony when inoculating the wells.</li>
	<li>Carefully remove the stamp and place back into the target plate.</li>
	<li>Repeat the inoculation for each bioassay plate until all the plates are inoculated.</li>
	<li>Incubate bioassay plates upside down at the appropriate temperature for 7 days.</li>
</ol>
8. Scoring
<ol>
	<li>After co-culturing the test and target organisms for 1 week, score the interactions based on the following visual assessment:
	<ol>
		<li>Score the wells with target organism growth that exhibits growth that is indistinguishable from the monoculture control as &#34;0&#34; (no inhibition) (Figure 2A,B).</li>
		<li>Score the wells with target organism that exhibits diminished growth compared to the control as &#34;1&#34; (weak inhibition) (Figure 2C).</li>
		<li>Score the wells where the target organism did not grow as &#34;2&#34; (strong inhibition) (Figure 2D).</li>
	</ol>
	</li>
</ol>

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The authors declare no competing interests.

Antibiotics and other secondary metabolites that mediate microbial interactions are useful for a multitude of applications, including drug discovery. Herein, a protocol for co-culture assays to assess large numbers of microbial interactions is presented. These co-culture interaction assays are a simple, affordable, scalable, and high throughput means to investigate many pairwise combinations of microorganisms in tandem. Target organisms are spotted next to test organisms in a well of a 12 well plate using an inoculation ...

In nature, microorganisms rarely exist in isolation; consequently, they are constantly interacting with other organisms. Therefore, studying how microorganisms interact with each other is essential to understanding a multitude of microbial behaviors1. Microbial interactions can be mutualistic, commensal, or antagonistic. These in teractions can affect not only the microorganisms themselves but also the environments and hosts that the microorganisms colonize1,2.
Many scientists study microbial interactions to identify new antimicrobial molecules. One of the first clinically important antimicrobial molecules was found through the study of microbial interactions. Sir Alexander Fleming observed a contaminating Penicillium spp. isolate that inhibited the growth of a Staphylococcus strain, which led to the discovery of the commonly used antibiotic penicillin3. Characterization of the mechanisms that microorganisms use to antagonize their competitors remains a fruitful resource for the discovery of antimicrobial molecules. For example, it was recently shown that Streptomyces sp. strain Mg1 produces antibiotic linearmycins, which have a lytic and degradative activity against Bacillus subtilis4.
Further, a non-ribosomally synthesized peptide named lugdunin was recently discovered after the observation that nasal commensal Staphylococcus lugdunensis inhibits Staphylococcus aureus5. Studies have also shown that mutualistic interactions between microorganisms are equally as powerful as antagonistic interactions for the discovery of antimicrobial molecules. For example, many fungus-farming ants in the tribe Attini harbor symbiotic bacteria called Pseudonocardia on their exoskeleton that produces antifungal molecules to inhibit an obligate pathogen of their fungal crop6. As the study of microbial interactions has been beneficial for discovering antimicrobial molecules, the use of high throughput screens may result in the discovery of new antimicrobial molecules.
With respect to the cost and ease of performance, the methodologies used to study microbial interactions range from simple to complex. For instance, an agar plug assay is an inexpensive and simple method that can be used to investigate antagonism between multiple microorganisms7. However, an agar plug assay is not an efficient procedure and can be labor-intensive for many pairwise combinations.To assess the effects of microbially produced products on target isolates of interest in a high throughput manner, many laboratories use disk diffusion assays8. These assays are easy and inexpensive and can be scalable to higher numbers of samples7. However, this assay requires the generation of microbial extracts and may produce misleading results for certain combinations of target organisms and antibiotics, such as Salmonella and cephalosporins9.
The preceding approaches rely on isolated components to elicit a response in a target organism, instead of allowing microorganisms to interact with each other. This is of note because interactions between microbes may elicit the production of &#34;cryptic&#34; antimicrobial molecules that are not produced in monoculture. For instance, it was recently shown that the antimicrobial keyicin is only produced by a Micromonospora sp. when co-cultured with a Rhodococcus sp. that is isolated from the same sponge microbiome10. More complex interaction methodologies circumvent this potential monoculture hindrance. For instance, the iChip is useful for isolating rare and difficult to cultivate bacteria from environmental samples and allows for the observation of microbial interactions through growth in situ11. To investigate interactions in detail, matrix assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF-IMS) can be used. This approach provides detailed information on the composition and distribution of small molecules and peptides produced by interacting microbial colonies with high spatial resolution. MALDI-TOF-IMS&#160;has also been used in multiple studies of bacterial interactions to characterize the mechanisms of competition12,13,14,15. However, MALDI-TOF-IMS often requires laborious sample preparation, specialized expertise to operate the equipment, and expensive and specialized mass spectrometers. For these reasons, it is a difficult technique to use for high throughput studies. Thus, a simple, scalable, and high throughput co-culture assay for microbial interactions that overcomes many limitations of the above approaches would be beneficial.
Here, a protocol for high throughput microbial co-culture is presented. This assay is simple and easily incorporated into preexisting studies of microbial interactions. In contrast to many commonly used methods for the study of microbial interactions, our method is simple, inexpensive, and is amenable to investigating large numbers of interactions. These assays are not only easy to perform, but the materials are widely available from most laboratory suppliers or public resources (e.g., libraries and makerspaces). Consequently, this assay is advantageous as a first line of investigation to identify and parse interesting patterns among many pairwise combinations of microorganisms, which may be especially useful for the investigation of microbial ecology.

<table><tbody><tr><td>1 &amp;mu;L disposable polystyrene inoculating loops, blue</td><td>VWR</td><td>12000-806</td><td></td></tr><tr><td>10 &amp;mu;L disposable polystyrene inoculating loops, yellow</td><td>VWR</td><td>12000-810</td><td></td></tr><tr><td>12-well cell culture plate, sterile with lid</td><td>Greiner bio-one</td><td>665 180</td><td></td></tr><tr><td>14 mL polystyrene round bottom tube, 17 x 100 mm &lt;sup&gt;2&lt;/sup&gt;style, nonpyrogenic, sterile</td><td>Falcon</td><td>352057</td><td></td></tr><tr><td>2.0 self standing screw cap tubes with caps, sterile</td><td>USA scientific</td><td>1420-9710</td><td></td></tr><tr><td>25 mL serological pipet</td><td>Cell Treat</td><td>229225B</td><td></td></tr><tr><td>Agar, bacteriological</td><td>VWR</td><td>J637</td><td></td></tr><tr><td>Brain Heart Infusion Broth</td><td>Dot Scientific</td><td>DSB11000-5000</td><td></td></tr><tr><td>Polycarbonate filament, white, 3 mm diameter</td><td>Keene Village Plastics</td><td>12.1-3MM-WH-581.2-1KG-R</td><td></td></tr><tr><td>School Glue</td><td>Elmer&#039;s</td><td>EPIE304</td><td></td></tr><tr><td>Taz 6 3D printer</td><td>Lulzbot</td><td></td><td></td></tr></tbody></table>

high throughput co-culture assays for the investigation of microbial interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

Co-culture interaction assays can be used to understand microbial interactions, identify patterns of interest, and uncover microbial isolates with intriguing activities. In these assays, a test organism is monocultured on one side of a 12 well agar plate and incubated for 7 days. Subsequently, a target organism is spotted next to the test organism and the two microbes were co-cultured for 7 days before scoring for the growth phenotype of the target organism. The assays are scored based on...

Watch this Scientific Journal Video about High Throughput Co-culture Assays for the Investigation of Microbial Interactions at JoVE.com

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

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Research

JoVE Journal

Immunology and Infection

9.7K Views.  University of Wisconsin-Madison. Ons protocol biedt een hoge doorvoermethode voor het screenen van een groot aantal paarswise microbiële interacties in tandem om interacties van belang te identificeren die microbioomonderzoek en antimicrobiële ontdekking kunnen helpen. Deze methode is gemakkelijk toepasbaar op het bestuderen van microbiële interacties in verschillende systemen, zolang de micro-organismen beminnelijk zijn voor cultuur onder laboratoriumomstandigheden. Het belangrijkste voordeel van deze techniek is dat het...

Video: High Throughput Co-culture Assays for the Investigation of Microbial Interactions

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Biology

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Bioengineering

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

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.

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 ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Protocol for Co-Culturing Sucrose-Secreting Cyanobacteria with Fungal Partners

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being explored as a method for solar-driven bioproduction. One approach involves the use of CO2-fixing cyanobacteria that secrete organic carbon to support the metabolism of a co-cultivated heterotroph, which in turn transforms the carbon into higher-value goods or services. In this protocol, a technical description to assist the experimentalist in the establishment of a co-culture combining a sucrose-secreting cyanobacterial strain with a fungal partner(s), as represented by model yeast species, is provided. The protocol describes the key prerequisites for co-culture establishment: Defining the media composition, monitoring the growth characteristics of individual partners, and the analysis of mixed cultures with multiple species combined in the same growth vessel. Basic laboratory techniques for co-culture monitoring, including microscopy, cell counter, and single-cell flow cytometry, are summarized, and examples of nonproprietary software to use for data analysis of raw flow cytometry standard (FCS) files in line with FAIR (Findable, Accessible, Interoperable, Reusable) principles are provided. Finally, commentary on the bottlenecks and pitfalls frequently encountered when attempting to establish a co-culture with sugar-secreting cyanobacteria and a novel heterotrophic partner is included. This protocol provides a resource for researchers attempting to establish a new pair of co-cultured microbes that includes a cyanobacterium and a heterotrophic microbe.

This protocol provides a comprehensive guideline for the setup and quantitative monitoring of co-cultures, including photoautotrophic sugar-secreting cyanobacteria and heterotrophic yeasts.

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

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