This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC Discovery Grant, RGPIN-2019-04384), the Canada Foundation for Innovation (Project # 38710), and Major Innovation Fund, Alberta. We thank Dr. John Kastelic for editing the manuscript.

1. Preparation of buffer and reagents
<ol>
	<li>Make 500 mL of tryptic soy broth (TSB) (15 g of TSB powder and 500 mL of ultrapure water) and autoclave. 
	NOTE: This can be stored at room temperature for up to 3 months or at 4 &#176;C for up to 6 months.</li>
	<li>Make 500 mL of tryptic soy agar (TSA) (20 g of TSA powder and 500 mL of ultrapure water) and autoclave. 
	NOTE: This can be stored at 4 &#176;C for up to 3 months.</li>
	<li>Make 500 mL of Phosphate Buffered Saline (PBS; 4 g of NaCl, 0.1 g of KCl, 0.77 g of Na2HPO4, and 0.12 g of KH2PO4, 500 mL of ultrapure water), measure and adjust the pH to 7.2 &#177; 0.2 at 25 &#176;C and autoclave. 
	NOTE: This can be store at 4 &#176;C for up to 6 months.</li>
	<li>Make 200 mL of 1 M MgSO4 (49.294 g and 200 mL of ultrapure water) and sterilize via 0.22 &#181;m polyethersulfone filter.</li>
	<li>Make 500 mL of TSB with 10 mM MgSO4 (mTSB; 15 g of TSB powder, 500 mL of ultrapure water, and 5 mL of 1 M MgSO4) and autoclave; let the autoclaved media cool to room temperature. Apply the aseptic technique. Using a serological pipette, carefully transfer 5.0 mL of 1 M MgSO4; swirl gently to mix.</li>
</ol>
2. Preparation of bacterial culture
<ol>
	<li>To prepare Bacterial Research Laboratory Culture (BRLC), remove glycerol stock vial(s) from the freezer inventory and transfer to the laboratory.</li>
	<li>Use a disposable inoculation loop or equivalent, dip into the vial, remove a scraping of inoculum (frozen slush), and streak onto a TSA plate or equivalent within a level II biological safety cabinet. Incubate the plate at 37 &#177; 2 &#176;C for 15&#8722;18 h.</li>
	<li>Bag the prepared BRLC plates and store at 4 &#176;C. 
	NOTE: General expiry period for BRLC: 14 days at 4 &#176;C after generation.</li>
	<li>Inoculate a single colony of each E. coli O157 strain from the BRLC plates to be tested in 10 mL of TSB and statically incubate at 37 &#176;C for 18 h to reach 9 log10 CFU/mL.</li>
	<li>Prepare a serial dilution of the overnight cultures (the following morning), each E. coli O157 strain using mTSB containing 10 mM of MgSO4 to achieve 4-5 log10 CFU/ml (or other desired inoculum level).</li>
	<li>Mix an equal volume of an overnight culture of each strain to achieve 4-5 log10 CFU/mL in total (or as desired) to prepare the bacterial mixture.</li>
	<li>Immediately place the diluted bacterial culture at 4 &#176;C for pending use.</li>
	<li>Dilute the inoculum culture 10 or 100-fold and plate 0.1 mL aliquots of these dilutions on the TSA plates to obtain the isolated colonies.</li>
</ol>
3. Preparation of phage working solutions
<ol>
	<li>Propagate high-titer working stocks for each phage to be screened (&#8805;108 PFU/mL) by following the standard methods4. 
	NOTE: The general expiry period for the phage stocks is 3 months in a plastic bottle at 4 &#176;C after generation.</li>
	<li>To achieve the desired titer of, e.g., ~108 PFU/mL for lysis kinetics, dilute individual phage preparations with mTSB containing 10 mM of MgSO4. Prepare phage cocktails by mixing equal volumes of each working stock with the same titer in all the possible combinations.</li>
</ol>
4. Preparation of in-vitro lysis kinetics for individual phage and phage cocktails
<ol>
	<li>Prepare serial 10-fold dilutions of each phages in Columns 1-8 in sterile 96-well microplates to set up the microplate assay. 
	NOTE: Four phages against one bacterial strain can be tested in duplicate, in adjacent columns, on each plate. The remaining four columns are for controls, which are without phage, as well as mTSB blank (Figure 1).</li>
	<li>Place 180 &#181;L of mTSB to wells from Columns 1 to 12&#160;of the 96-well microplate.</li>
	<li>Add 20 &#181;L of diluted individual phages or phage cocktails (~108 PFU/mL) to wells 1-8 of the top row of the microplate (Row A).</li>
	<li>For the phage-free and Blank control, add 20 &#181;L of mTSB to the top well of Columns 9, 10, and 11.</li>
	<li>With a 12-channel pipette, dilute down the plate. Transfer 20 &#181;L from row to row. Mix the well contents by gentle, repeated aspiration, ejection (at least five times), and changing tips between dilutions. Remove 20 &#181;L from the last row (Row H). 
	NOTE: Using tips with filters is recommended to prevent cross-contamination.</li>
	<li>Set up a reservoir for each strain, use 1 mL pipette to transfer 2-3 mL of the diluted culture into the reservoir. For columns 1-10, use a multichannel pipette to add 20 &#181;L of the diluted bacterial culture to each well. Change tips between each addition.</li>
	<li>Cover and incubate the microplate at desired conditions (e.g., 37 &#176;C for 10 h or 22 &#176;C for 22 h).</li>
	<li>At 2 h or other desired intervals, remove the microplates from the incubator.</li>
</ol>
5. Determination of optical density
<ol>
	<li>Examine for optical density at 600 nm (OD600nm) using a Microplate Reader. 
	NOTE: It is advised to set up and save the assay protocol in the program before test plate preparation.</li>
	<li>Turn on the microplate reader and open program.</li>
	<li>Select the Simple mode in the Startup Options window and select Create a New Protocol in Task Manager (Supplementary Figure 1a,b).</li>
	<li>From the Select Plate Type window, choose 96 Well Plate from the drop-down list (Supplementary Figure 2).</li>
	<li>Select Absorbance for detection method, the Endpoint/Kinetic option for read type, and Monochromators for optics type. Click on OK (Supplementary Figure 3).</li>
	<li>For Read Step, enter 600 nm for wavelengths and select Normal for read speed. Click on OK (Supplementary Figure 4).</li>
	<li>To set the temperature for the assay, click on the Incubate checkbox and select Incubator On. Enter the desired temperature in the Temperature box. Click on OK. To prevent condensation on the plate lid during incubation, set a temperature gradient by entering a value (1-3) in the Gradient box. Click on OK (Supplementary Figure 5a). 
	NOTE: Click on the Preheat Before Continuing With the Next Step checkbox for the 37 &#176;C incubation temperature. This step is not required for the test condition at 22 &#176;C (RT).</li>
	<li>Next, click on the Kinetic checkbox to open the Kinetic Setup. Set the run time for 10 h for 37 &#176;C incubation or 22 h for RT. Enter 2 h for reading interval. Click on OK (Supplementary Figure 5b).</li>
	<li>Click on the Shake checkbox in the Procedure window to set up the shake condition, if needed, for each kinetic read (Supplementary Figure 6a).</li>
	<li>Select Linear for Shake Modes and change Duration to 30 s. Set Linear Frequency value to 731 cpm (2 mm), and then click on OK (Supplementary Figure 6b).</li>
	<li>In the Protocol Summary Dialogue window, click on Plate Layout and select Blanks, Assay Controls, and Samples. Click on Next (Supplementary Figure 7). 
	NOTE: Enter 1 in Number of Different Control Types for negative control.</li>
	<li>After defining the settings for each well type, click on Finish.</li>
	<li>Select a Well ID in the left-hand side interface, and then assign it to the matrix shown in the plate layout widow. Click on OK (Supplementary Figure 8).</li>
	<li>Click on the Read Plate button and save the protocol as a .prt file and click on Save (Supplementary Figure 9).</li>
	<li>Insert the plate and click on OK.</li>
	<li>Once the experiment is completed, save the experiment as a .xpt file and click on Save (Supplementary Figure 10).</li>
	<li>Export data to Excel by clicking on the Yes button in the message window box for further analysis (Supplementary Figure 11).</li>
	<li>Click on the Save Yes/No selection and the Don&#39;t Ask Again checkbox to save preference.</li>
</ol>
6. Data analyses
<ol>
	<li>Repeat at least two independent experiments as described above. Compile results from all the independent trials. Calculate the average and standard deviation of the OD600 from each phage-treated and -free culture.</li>
	<li>Perform square root of the OD values at 600 nm and analyze them using an appropriate statistical model for each bacterial strain and temperature. 
	NOTE: For SAS software, the MIXED model and the least-squares to differentiate means (P &#60; 0.05) are selected. For each strain, assign panels A&#8722;G to each phage treatment of which overall anti-O157 efficacy across time and MOIs differed (P &#60; 0.05).</li>
	<li>Define superior phage efficacy based on OD600nm value &#8804; 0.01 corresponding to no detectable bacterial growth (limit of detection:300 CFU/mL).
	<ol>
		<li>Analyze effects of time, incubation temperature, E. coli O157 strains, MOIs, and phage types on phage efficacy using an appropriate statistical model. 
		NOTE: For SAS software, use GLIMMIX with random measures.</li>
		<li>Calculate Odds Ratios to compare superior efficacy for different environmental and biological factors of interest.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Carvalho, C., Costa, A. R., Silva, F., Oliveira, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophages+and+their+derivatives+for+the+treatment+and+control+of+food-producing+animal+infections.">Bacteriophages and their derivatives for the treatment and control of food-producing animal infections.</a> Critical Reviews in Microbiology. 43 (5), 583-601 (2017).</li><li>Czaplewski, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatives+to+antibiotics-a+pipeline+portfolio+review.">Alternatives to antibiotics-a pipeline portfolio review.</a> The Lancet. Infectious Diseases. 16 (2), 239-251 (2016).</li><li>Cisek, A. A., Dabrowska, I., Gregorczyk, K. P., Wyzewski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+therapy+in+bacterial+infections+treatment:+one+hundred+years+after+the+discovery+of+bacteriophages.">Phage therapy in bacterial infections treatment: one hundred years after the discovery of bacteriophages.</a> Current Microbiology. 74 (2), 277-283 (2017).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Escherichia+coli+O157+on+beef+at+37,+22+and+4&#176;C+by+T5-,+T1-,+T4-and+O1-like+bacteriophages.">Control of Escherichia coli O157 on beef at 37, 22 and 4&#176;C by T5-, T1-, T4-and O1-like bacteriophages.</a> Food Microbiology. 51, 69-73 (2015).</li><li>Moye, Z. D., Woolston, J., Sulakvelidze, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+applications+for+food+production+and+processing.">Bacteriophage applications for food production and processing.</a> Viruses. 10 (4), (2018).</li><li>Ly-Chatain, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+factors+affecting+effectiveness+of+treatment+in+phages+therapy.">The factors affecting effectiveness of treatment in phages therapy.</a> Frontiers in Microbiololgy. 5, 51 (2014).</li><li>Dy, R. L., Rigano, L. A., Fineran, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage-based+biocontrol+strategies+and+their+application+in+agriculture+and+aquaculture.">Phage-based biocontrol strategies and their application in agriculture and aquaculture.</a> Biochemical Society Transactions. 46 (6), 1605-1613 (2018).</li><li>Bourdin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coverage+of+diarrhoea-associated+Escherichia+coli+isolates+from+different+origins+with+two+types+of+phage+cocktails.">Coverage of diarrhoea-associated Escherichia coli isolates from different origins with two types of phage cocktails.</a> Microbial Biotechnology. 7 (2), 165-176 (2014).</li><li>Niu, Y. D., Liu, H., Johnson, R. P., McAllister, T. A., Stanford, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+individual+bacteriophages+does+not+predict+efficacy+of+bacteriophage+cocktails+for+control+of+Escherichia+coli+O157.">Efficacy of individual bacteriophages does not predict efficacy of bacteriophage cocktails for control of Escherichia coli O157.</a> Frontiers in Microbiololgy. 12, 616712 (2021).</li><li>Niu, Y. D., Liu, H., Johnson, R. P., McAllister, T. A., Stanford, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+bacteriophage+T5virus+on+growth+of+Shiga+toxigenic+Escherichia+coli+and+Salmonella+strains+in+individual+and+mixed+cultures.">Effect of a bacteriophage T5virus on growth of Shiga toxigenic Escherichia coli and Salmonella strains in individual and mixed cultures.</a> Virology Journal. 17, 3 (2020).</li><li>Br&#252;ssow, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+needed+for+phage+therapy+to+become+a+reality+in+Western+medicine.">What is needed for phage therapy to become a reality in Western medicine.</a> Virology. 434 (2), 138-142 (2012).</li><li>Chan, B. K., Abedon, S. T., Loc-Carrillo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+cocktails+and+the+future+of+phage+therapy.">Phage cocktails and the future of phage therapy.</a> Future Microbiology. 8 (6), 769-783 (2013).</li><li>Nikolich, M. P., Filippov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+therapy:+developments+and+directions.">Bacteriophage therapy: developments and directions.</a> Antibiotics. 9 (3), 135 (2020).</li><li>Niu, Y. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+range+and+lytic+capability+of+four+bacteriophages+against+bovine+and+clinical+human+isolates+of+Shiga+toxin-producing+Escherichia+coli+O157:H7.">Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7.</a> Journal of Applied Microbiology. 107 (2), 646-656 (2009).</li></ol>

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This protocol described a robust approach for systemically evaluating phage efficacy against foodborne pathogens, including STEC9 and Salmonella10. One critical step is when diluting overnight culture of bacteria, using pre-chilled medium and manipulating the dilution with an ice bucket are recommend to minimize potential bacterial growth. In addition, phage dilution was prepared before diluting bacterial culture. The enumeration step 2.8 provided actual numbers of bacterial inoculum for calculation of the final MOI applied. For phage preparation, crude phage lysates prepared by filter phage infected in 4-6 h of bacterial culture is generally used. The critical step associated with phage infectivity is always to use phage work stocks prepared within 3 months. Extremely accurate pipetting (particularly when using a multichannel pipette) and uniformity of approach are also essential to obtain comparable and interpretable results. Modified TSB supplemented with 10 mM of Mg2+ was used to dilute phages, bacterial culture, and base medium to optimize adsorption and infection of phages.
As bacteria proliferate during the log phase, even below incubator temperature, it is recommended to use diluted overnight culture instead of log-phase culture, to minimize potential bacterial growth.
The proposed protocol has limitations. First, because microplate can only hold 200 &#181;L, prolonged incubation may cause substantial evaporation and is not recommended. In this case, the assay may not be suitable for slow-growing bacteria. Second, the proposed protocol was not able to monitor the amplification of phages. Third, this protocol could not monitor the development of phage resistance over time, a critical factor that determines the outcome of phage treatment11,12. Follow-up experiments are required to assess the further performance of the most influential cocktail in the screening in preventing the emergence of anti-phage mutants in an extensive broth culture system and other biological matrices.
In contrast to conventional antimicrobials, the biological nature of phages affects the complexity of biocontrol and therapeutic use in practical settings. Conventionally, rational selection of phage cocktails is primarily based on lytic activity and the host range of phages. Phage candidates with the strongest lytic activity and broadest host range are often recommended13,14. However, based on the current study, phages such as rV5 and T1, although alone not as virulent as T4 and T5, greatly facilitated overall biocontrol outcome when combined with T4 and/or T5. Consequently, to achieve superior efficacy of phage cocktails, systemic screening of antibacterial activity of potential phage combinations against targeted host strains under desired environmental conditions is recommended. In addition, the determination of receptors for phage candidates and the inclusion of phages with various receptors may prevent competition for host attachment, thwart rapid development of anti-phage mutants, and improve biocontrol outcomes13.
This method enabled accurate quantification of phage lysis kinetics in a high-throughput format. Furthermore, it allowed systematic evaluation of various biological and environmental factors on the antibacterial efficacy of an assortment of phages, thereby facilitating the formulation of phage cocktails with optimized outcomes. The method&#39;s future applications and development are assumed to involve in situ monitoring the efficacy of each phages within phage cocktails by fluorescence labeling of phages. In addition to the proposed protocol, understanding genetic determinants that promote synergistic and facilitated effects between phages when co-infecting one host would facilitate the formulation of appropriate phage cocktails with superior efficacy.

Bacteriophages (phages) are viruses that naturally invade bacterial cells, disrupting bacterial metabolism and causing lysis of the bacterium. In contrast to conventional antimicrobials (e.g., antibiotics), phage host spectrums are relatively narrow, only capable of infecting a targeted set of bacterial species or strains and thus should minimize collateral effects on microbiota that benefit animal and human health. With the rise of antimicrobial resistance (AMR), phages and their derivatives lead to alternative antimicrobials to control bacterial infectious diseases, including AMR bacterial infections in humans and animals1,2. Phages have confirmed therapeutic potential against &#62;20 bacterial pathogens that cause superficial infections and infections of the upper respiratory system and gastrointestinal tract of humans3.
In agricultural settings, a phage-based biocontrol strategy is the therapeutic use of phages to control bacterial pathogens. Phage biocontrol is well accepted as a green technology, effective at decontaminating foodborne pathogens (e.g., Shiga-toxin producing Escherichia coli (STEC), Salmonella, and Listeria) in various foods4,5. In addition, phages can be used as sanitizers to disinfect food processing surfaces and animal hides, which can be integrated into conventional antimicrobial systems (e.g., chemicals, steam, and hot water pasteurization) to enhance desired outcomes and reduce environmental impacts. The use of phages to reduce zoonotic bacteria in animals is also promising1. However, there is a need to address the technical challenges to improve outcomes from the phage biocontrol approach to be popularly applied in diverse food production systems. The main challenge is the impaired effectiveness of phages due to the development of bacteria-resistant mutants5 and changes in bacterial physiology due to exposure to environmental stressors6.
For minimizing the risk of phage resistance, phage cocktails (i.e., a combination of multiple phages) are proposed and have improved biocontrol potency in agriculture and aquaculture settings7. However, from several studies, it has been proved that phage cocktails did not always offer better effectiveness than the administration of a single phage. For instance, a cocktail of 3 T4-like phages had a narrower host range against E. coli strains8. Furthermore, AKFV33, a member of Tequintavirus, had greater efficacy than a cocktail of four phages in removing E. coli O157 from beef, despite the incubation temperatures applied4. Recently, it has been reported that the effectiveness of individual phages does not predict the efficacy of phage cocktails for control of O1579, as interactions among multiple phages can alter efficacy. Most importantly, numerous factors, such as phage genera and combinations, targeted strains and MOIs, and incubation temperatures and times, may impact interactions between phages. Therefore, carefully screening combinations of phages against specific bacteria to assess phage synergy or facilitation, or at least to ensure minimal phage antagonism under specific environmental conditions, is vitally important for optimal outcomes. Here, a method is described to systematically assess the efficacy of various phage combinations against foodborne pathogens under a range of environmental conditions. The benefit of this approach is to enable screening of all possible biotic and abiotic factors predicted to affect the antibacterial efficacy of phages in natural settings. In the protocol, STEC&#160;O157 and their infecting phages are employed as an example.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Essential supplies, reagents, and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculating loops</td>
			<td>VWR</td>
			<td>12000-806</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>1374361</td>
			<td>MgSO4.7H2O</td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lid</td>
			<td>Fisher</td>
			<td>FB0875713</td>
			<td>Diameter: 100 mm, sterile</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Fisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014382</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-300XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014405</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite Multi Pipette L12-20XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17013808</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Lite Pipette, Unv. SL-20XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014412</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips RT LTS 1000&#181;L FL 768A/8-low retention</td>
			<td>METTLER TOLEDO</td>
			<td>30389213</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips SR LTS 20&#181;L F 960A/5</td>
			<td>METTLER TOLEDO</td>
			<td>17005860</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips SR LTS 300&#181;L 768A/4</td>
			<td>METTLER TOLEDO</td>
			<td>17005867</td>
			<td>no filter</td>
		</tr>
		<tr>
			<td>Reservoir</td>
			<td>METTLER TOLEDO</td>
			<td>89094-662</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile, clear, 96-well flat-bottom polystyrene microplates with lids</td>
			<td>Fisher</td>
			<td>168055</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic soy agar (TSA)</td>
			<td>Sigma</td>
			<td>105458-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic soy broth (TSB)</td>
			<td>Sigma</td>
			<td>105459-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>T-Shaped Cell Spreaders</td>
			<td>VWR</td>
			<td>76299-566</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analog Vortex Mixer</td>
			<td>Fisher</td>
			<td>02-215-414</td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Microbiological Incubators</td>
			<td>Fisher</td>
			<td>50125590H</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stirrer Hotplates</td>
			<td>FIsher</td>
			<td>13-889-335</td>
			<td></td>
		</tr>
		<tr>
			<td>Polygon Stir Bars</td>
			<td>FIsher</td>
			<td>14-512-125</td>
			<td>length: 20 mm</td>
		</tr>
		<tr>
			<td>Synergy Neo2 Hybrid Multi-Mode Reader</td>
			<td>Fisher</td>
			<td>BTNEO2M</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SAS</td>
			<td>SAS Institute</td>
			<td>9.4</td>
			<td></td>
		</tr>
	</tbody>
</table>

bacteriophage effectiveness for biocontrol of foodborne pathogens evaluated via high-throughput settings

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant bacteria, novel antibacterial agents, are urgently needed. A bacteriophage-based biocontrol strategy is the therapeutic use of phages to control bacterial pathogens in agricultural settings. Phage biocontrol is increasingly accepted as a sustainable technology, effective at decontaminating foodborne pathogens. To ensure effective biocontrol outcomes, systematic screening of phage combinations against targeted bacteria under required environmental conditions is crucial. Antibacterial efficacy of phage cocktails may be affected by phage genera and combination, targeted bacterial strains, the multiplicity of infection, temperature, and time. To formulate a phage cocktail with superior efficacy, the proposed method was to systematically evaluate the effectiveness of individual phages and phage cocktails in killing foodborne bacterial pathogens under targeted conditions. Bacterial killing efficacy was monitored by measuring optical density at desired temperatures and durations. Superior phage efficacy was determined by complete inhibition of bacterial growth. The proposed method is a robust, evidence-based approach to facilitate formulating phage cocktails with superior antibacterial efficacy.

Following the protocol, a comparison of the anti-O157 phage efficacy with various phage combinations, temperatures, times, and MOIs was performed. Impacts of incubation temperature and times, MOIs, phages, and bacterial strains used on enhancing anti-E. coli O157 efficacy is presented in Table 1 (odds ratio table)9. The percentage (%) of optical turbidity measurement &#8804; 0.01 yielded was analyzed by each phage preparation under each experimental condition. Based on this analysis, anti-O157 phage efficacy was maximized after 14, 16, or 18 h of incubation at 22 &#176;C (P &#60; 0.001) and MOI = 1000 (P &#60; 0.001), with 75% and 89% of the growth of phage-treated culture being completely inhibited, respectively. In general, among 11 phage preparations, a cocktail of T1, T4, and rV5 was most effective (P &#60; 0.05) against O157. In addition, across incubation temperatures, times, MOIs, and phages tested, sensitivity to phages varied, with O157 strains tested with strain CO281-31N being most sensitive (P &#60; 0.001) and 3081 (P &#60; 0.001) being least sensitive to phages.
For understanding the phage-killing kinetics of each bacterial strain tested, OD600 value was plotted against each sampling time, MOIs, and phage treatment at each incubation temperature. Representative results from the efficacy of phages against E. coli O157 3081 at 37 &#176;C are shown in Figure 2. It is ensured that the growth curve of phage-free control culture is normal before assessing inhibition of growth curve of phage-treated culture. According to Figure 2, the inclusion of T4 completely inhibited bacterial growth at 37 &#176;C, at each sampling time at an MOI as low as 1. To overview how phage inhibited bacterial growth over time at various temperatures, irrespective of MOIs, the mean OD600nm value averaged from all the MOIs (MOI &#62; 0) was plotted against each sampling time and phage treatment (Figure 3). Compared to 37 &#176;C, particular phage killing efficacy, e.g., phages T4 and T1 + T4, was enhanced over 22 &#176;C. Another approach to summarize and compare overall anti-O157 effectiveness among phages is shown in Table 2 and Table 39. OD600 value was averaged from each sampling time and MOI and ranked phage efficacy from highest (A) to lowest (F: 37 &#176;C and D: 22 &#176;C). This table facilitates the identification of the best phage treatment. For example, for strain 3081, phages T4 and T5 + T4 (Panel A) were the most effective treatment at 37 &#176;C, whereas in addition to phage T4, phages T1 + T4, T1 + T4 + rV5, and 4 phage cocktails (Panel A) were the most effective treatment at 22 &#176;C.
This protocol also enabled us to compare phage efficacy against various bacterial strains targeted and customize phage preparation. For example, when selecting phages, at 37 &#176;C, excluding strain 3081, phage T1 + T4 + rV5 had the most significant effectiveness against all other strains, regardless of their phage types. At 22 &#176;C, the 4-phage cocktail was the most effective against all strains tested. The best MOIs resulted in complete lysis (OD600&#160;&#8804; 0.01), which warranted potential superior efficacy under experimental conditions (Table 2 and Table 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62812/62812fig01.jpg" /> 
Figure 1: Suggested layout of 96-well microplate assay. Serial 10-fold dilutions of each phage are prepared in Columns 1-8 of sterile 96-well microplates. Four phages against one bacterial strain can be tested in duplicate, in adjacent columns, on each plate. The remaining columns are for controls. <a href="https://www.jove.com/files/ftp_upload/62812/62812fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62812/62812fig02.jpg" /> 
Figure 2: Growth curves of E. coli O157 strain 3081 at 37 &#176;C treated and not treated with phages at each MOIs. The data were compiled from two independent trials. Bars present standard deviation. <a href="https://www.jove.com/files/ftp_upload/62812/62812fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62812/62812fig03.jpg" /> 
Figure 3: Growth curves of E. coli O157 strain 3081 strains. (A) Growth curves at 37 &#176;C. (B) Growth curves at 22 &#176;C. Each curve represents individual and mixed cultures, treated and not treated with phages across MOIs. The data were compiled from two independent trials. Bars present the standard error of the mean (adapted from Reference9 with permission). <a href="https://www.jove.com/files/ftp_upload/62812/62812fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Odds ratios comparing the likelihood of superior phage efficacy against E. coli O157 at incubation temperatures, incubation times, MOIs, phages, and strains of E. coli O157 (adapted from Reference9 with permission). <a href="https://www.jove.com/files/ftp_upload/62812/62812_Table 1_Final_.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Overall phage efficacy against E. coli O157 at 37 &#176;C (adapted from Reference9 with permission). <a href="https://www.jove.com/files/ftp_upload/62812/62812_Table 2_Final_.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Overall phage efficacy against E. coli O157 at 22 &#176;C (adapted from Reference9 with permission). <a href="https://www.jove.com/files/ftp_upload/62812/62812_Table 3_Final.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Figures 1-11: Snapshots of the microplate reader operation and procedures. <a href="https://www.jove.com/files/ftp_upload/62812/Niu_Supplementary Information.pdf" target="_blank">Please click here to download this File.</a>

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Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings

This protocol describes a robust method for using high-throughput settings to screen the antibacterial efficacy of bacteriophage cocktails.

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant ...

bacteriophage-effectiveness-for-biocontrol-foodborne-pathogens

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JoVE Journal

Immunology and Infection

2.7K Views.  University of Calgary. This protocol describes a robust method for using high-throughput settings to screen the antibacterial efficacy of bacteriophage cocktails.

Video: Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

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.

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.