This research was supported by the Agriculture and Food Research Initiative competitive grant 2015-67012-22773 from the USDA National Institute of Food and Agriculture to K.L.H.

1. Preparation of Supernatant to Be Tested for Activity
<ol>
	<li>Prepare a 0.5 mg/ml stock solution of mitomycin C by dissolving the appropriate amount into sterile 0.1 M MgSO4 buffer (e.g. 1 mg mitomycin C/2 ml buffer). Store stock at 4 &#176;C in a light protected container (mitomycin C is light sensitive).</li>
	<li>Inoculate a single colony of P. syringae into 3 ml of King&#39;s medium B (KB) broth14. Incubate over night with shaking (200 rpm) at room temperature (21-25 &#176;C).</li>
	<li>The following morning, dilute the broth culture 1/100 into 3 ml of fresh KB broth. Incubate for 3-4 hr with shaking at room temperature. Add mitomycin C (0.5 &#181;g/ml, final concentration). Incubate the culture over night with shaking at room temperature. 
	NOTE: Mitomycin C causes double stranded DNA breaks, thus stimulating the cell&#39;s SOS response, leading to the production of both bacteriophage and bacteriocins.</li>
	<li>Pellet 1-2 ml mitomycin C induced cultures by centrifugation at 20,000 x g for 5 min in a bench top microcentrifuge.</li>
	<li>Remove and sterilize culture supernatants either by passing the supernatant through a 0.22 &#181;m pore size filter or by treating the supernatant with chloroform (100 &#181;l chloroform per 1 ml supernatant).
	<ol>
		<li>If using chloroform, vortex the mixture for 15 sec and let incubate at room temperature for 1 hr. Following incubation, centrifuge the mixture at 20,000 x g for 5 min. 
		NOTE: Chloroform efficiently kills cells by solubilizing their membranes. The benefit of using chloroform over filter sterilization is that it is cheaper and easier to scale-up if processing many (&#62;20) samples at a time. There may be a possibility that a given killing activity is lost following chloroform treatment as a result of partitioning into the organic phase.</li>
		<li>Remove the upper, aqueous phase using a 1 ml pipet to a fresh, sterile 1.5 or 2.0 ml microfuge tube. Be careful not to transfer any of the lower chloroform layer (it is better to remove less of the aqueous phase to avoid carry-over).</li>
		<li>Incubate the transferred supernatant uncapped in a fume hood to allow the residual chloroform to evaporate (several hours). Store supernatants at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Separation and Concentration of High Molecular Weight Bactericidal Compounds by Polyethylene Glycol (PEG) Precipitation
<ol>
	<li>To the sterile supernatant, add NaCl and PEG 8000 to 1 M and 10% final concentrations, respectively. Repeatedly invert the sample until both the NaCl and PEG are completely dissolved. Incubate samples in an ice bath for 1 hr or overnight at 4 &#176;C.</li>
	<li>Centrifuge samples at 16,000 x g for 30 min at 4 &#176;C. A pellet should form at the bottom of the centrifuge tube. Decant the supernatant and resuspend the pellet in desired volume (such as 1/10 or 1/100 of the original supernatant volume) of buffer (10 mM Tris, 10 mM MgSO4, pH 7.0) by repeated pipetting.</li>
	<li>Remove residual PEG by two sequential extractions with an equal volume of chloroform.
	<ol>
		<li>Combine chloroform with the resuspended pellet and vortex for 10 to 15 sec, then centrifuge the mixture at 20,000 x g for 5 min. Transfer the upper, aqueous phase to a fresh microfuge tube. Repeat this extraction until no white interface between the aqueous and organic phase is visible (usually 2 extractions total). Allow residual chloroform to evaporate from extracted supernatants in a fume hood.</li>
	</ol>
	</li>
</ol>
3. Preparation of Overlay and Testing Supernatants for Activity
<ol>
	<li>Inoculate a single colony of a P. syringae strain to be tested for sensitivity into 3 ml of KB, incubate over night with shaking at room temperature.</li>
	<li>The following morning, back dilute the culture 1/100 into fresh KB. Incubate 3-4 hr with shaking at room temperature.</li>
	<li>Prepare sterilized water agar by autoclaving a suspension 0.35-0.7% (w/v) agar in ultrapure water. 
	NOTE: A stock of soft water agar can be melted in a microwave and reused repeatedly, fresh overlay does not need to be prepared for each experiment. If water agar is reused, it is critical to ensure it is completely melted following microwaving. If not, the overlay will have a grainy texture upon solidifying that will make interpretation difficult. If this occurs, melt the agar for several minutes longer in the microwave than done previously.</li>
	<li>Maintain the molten soft agar in a 60 &#176;C water bath prior to use. Using a sterile serological pipet, transfer 3 ml of soft agar to a sterile culture tube. Return the culture tube to water bath to maintain in a molten state.</li>
	<li>To pour the overlay, first allow the molten agar to cool (it should feel warm but not hot to touch), but do not allow to solidify.</li>
	<li>In a sterile hood, inoculate 100 &#181;l of the tester strain culture into the soft agar and vortex to mix. Rotate culture by hand for 10-15 sec, then pour onto a bottom agar (KB agar). Tilt the plate in all directions to ensure the soft agar evenly covers the bottom agar. 
	NOTE: The bottom agar can be any solidified (1.5% agar) medium on which the test strain grows vigorously. For a standard 100 mm diameter Petri dish, use ~20 ml melted medium. The bottom agar can be prepared several weeks ahead of time (if maintained at 4 &#176;C without drying) or can be prepared the same day. If prepared the same day as performing the overlay, it is best to do so prior to step 3.4.</li>
	<li>Cover the plate and allow it 20-30 min to solidify. Be careful not to disturb the plate while solidifying.</li>
	<li>Once solidified, spot 2-5 &#181;l of supernatant (generated in sections 1 and 2, above) onto the overlay. Allow the plates to incubate over night at room temperature. Observe and record results the following morning. 
	NOTE: It may be useful to perform and spot from a serial dilution of the supernatant. This will allow researchers to distinguish between bacteriophage and bacteriocin clearing activity. In this case, it is recommended to perform 1:5 or 1:10 dilutions.</li>
</ol>

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	<li>Cavera, V. L., Arthur, T. D., Kashtanov, D., Chikindas, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriocins+and+their+position+in+the+next+wave+of+conventional+antibiotics.">Bacteriocins and their position in the next wave of conventional antibiotics.</a> Int J of Antimicrob Agents. 46 (5), 494-501 (2015).</li><li>Blaser, M. J., Cardon, Z. 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=Toward+a+Predictive+Understanding+of+Earth's+Microbiomes+to+Address+21st+Century+Challenges.">Toward a Predictive Understanding of Earth's Microbiomes to Address 21st Century Challenges.</a> MBio. 7 (3), e00714-e00716 (2016).</li><li>Hibbing, M. E., Fuqua, C., Parsek, M. R., Peterson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+competition:+surviving+and+thriving+in+the+microbial+jungle.">Bacterial competition: surviving and thriving in the microbial jungle.</a> Nat Rev Microbiol. 8 (1), 15-25 (2010).</li><li>Michel-Briand, Y., Baysse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pyocins+of+Pseudomonas+aeruginosa.">The pyocins of Pseudomonas aeruginosa.</a> Biochimie. 84 (5-6), 499-510 (2002).</li><li>Ghequire, M. G. K., De Mot, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomally+encoded+antibacterial+proteins+and+peptides+from+Pseudomonas.">Ribosomally encoded antibacterial proteins and peptides from Pseudomonas.</a> FEMS Microbiol Rev. 38 (4), 523-568 (2014).</li><li>Cascales, E., Buchanan, S. K., 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=Colicin+Biology.">Colicin Biology.</a> Microbiol and Mol Biol Rev. 71 (1), 158-229 (2007).</li><li>Brown, S. P., Le Chat, L., De Paepe, M., Taddei, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+of+Microbial+Invasions:+Amplification+Allows+Virus+Carriers+to+Invade+More+Rapidly+When+Rare.">Ecology of Microbial Invasions: Amplification Allows Virus Carriers to Invade More Rapidly When Rare.</a> Curr Biol. 16 (20), 2048-2052 (2006).</li><li>Grinter, R., Roszak, A. W., Cogdell, R. J., Milner, J. J., Walker, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Crystal+Structure+of+the+Lipid+II-degrading+Bacteriocin+Syringacin+M+Suggests+Unexpected+Evolutionary+Relationships+between+Colicin+M-like+Bacteriocins.">The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins.</a> J Biol Chem. 287 (46), 38876-38888 (2012).</li><li>Hockett, K. L., Renner, T., Baltrus, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+Co-Option+of+a+Tailed+Bacteriophage+into+a+Killing+Complex+in+Pseudomonas.">Independent Co-Option of a Tailed Bacteriophage into a Killing Complex in Pseudomonas.</a> MBio. 6 (4), (2015).</li><li>Iacobellis, N. S., Lavermicocca, P., Grgurina, I., Simmaco, M., Ballio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytotoxic+properties+of+Pseudomonas+syringae+pv.+syringae+toxins.">Phytotoxic properties of Pseudomonas syringae pv. syringae toxins.</a> Physiol Mol Plant Pathol. 40 (2), 107-116 (1992).</li><li>Baltrus, D. A., Hendry, T. A., Hockett, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+Genomics+of+Pseudomonas+syringae.">Ecological Genomics of Pseudomonas syringae.</a> Genomics of Plant-Associated Bacteria. , 59-77 (2014).</li><li>Gratia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+Relationships+between+Lysogenic+Bacteria+and+Particles+of+Bacteriophage.">Numerical Relationships between Lysogenic Bacteria and Particles of Bacteriophage.</a> Ann Inst Pasteur. 57, 652 (1936).</li><li>King, E. O., Ward, M. K., Raney, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+simple+media+for+the+demonstration+of+pyocyanin+and+fluorescin.">Two simple media for the demonstration of pyocyanin and fluorescin.</a> J Lab Clin Med. 44, 301-307 (1954).</li><li>Heng, N. C. K., Wescombe, P. A., Burton, J. P., Jack, R. W., Tagg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Diversity+of+Bacteriocins+in+Gram-Positive+Bacteria+.">The Diversity of Bacteriocins in Gram-Positive Bacteria .</a> Bacteriocins. (Chapter 4), 45-92 (2007).</li><li>Kawai, Y., Saito, T., Uemura, J., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Detection+Method+for+Bacteriocin+and+Distribution+of+Bacteriocin-producing+Strains+in+Lactobacillus+acidophilusGroup+Lactic+Acid+Bacteria+Isolated+from+Human+Feces.">Rapid Detection Method for Bacteriocin and Distribution of Bacteriocin-producing Strains in Lactobacillus acidophilusGroup Lactic Acid Bacteria Isolated from Human Feces.</a> Biosci Biotechnol Biochem. 61 (1), 179-182 (2014).</li></ol>

The authors have nothing to disclose.

The soft-agar overlay technique described here has been widely applied for many decades by researchers interested in bacteriophages or bacteriocins. The main benefits of this approach are that it is simple, cheap and relatively easy to interpret. By combining the soft-agar overlay with PEG precipitation and serial dilution, antimicrobial agents can be separated into high vs. low molecular weight agents, and replicative vs. non-replicative agents (i.e. bacteriophage vs. tailocin, respectively). Finally, incorporation of targeted genetic manipulation (deletion and complementation) of putative bacteriocin or prophage loci can firmly establish the identity of a given antagonistic compound. We have recently used this method to describe a new bacteriophage-derived bacteriocin prevalent among Pseudomonas syringae strains9.
The growth media and conditions indicated in this protocol work well for Pseudomonas syringae and would likely suffice for other Gram-negative bacteria that grow vigorously under these conditions. However, researchers using this method will want to optimize the timing, culture medium, induction method, and incubation temperature for their system. The critical parameters include inducing the producing culture while in the logarithmic growth phase, as well as seeding the soft-agar overlay with sufficient logarithmic phase culture to ensure a uniform bacterial distribution, but not excessive culture, which will obscure potential clearing zones. There are many bacteriocins that are not induced as part of the SOS response, but rather are induced through peptide-based quorum sensing systems (particularly Gram-positive bacteriocins15), thus, a researcher may want to screen several different induction methods (such as modifying nutrient content of inducing medium, or allowing for extended incubation of the producing strain).
When using this technique, the soft-agar overlay must be thoroughly melted and maintained at 55-60 &#176;C prior to the addition of the seeding strain. We have had several experiments where the soft-agar was not thoroughly melted (though visually it appeared to be) and resulted in an overlay with a grainy appearance. Results from a grainy overlay can still be generally interpretable, however, this is dependent on the strength of the inhibition (weaker inhibition will be more difficult to observe). A final, confounding variable to consider when using this technique is that the temperature of the molten agar is allowed sufficient time to cool prior to inoculation with the seeding strain, so as to avoid killing the seeding strain. To avoid this issue, we ensure the soft-agar is warm, but not hot, to touch. Along these lines, we have also observed that if we give inadequate time for a seeding culture to acclimate to the molten agar, prior to pouring the overlay, we recover generally poor bacterial growth, which makes interpretation of specific inhibition difficult or impossible to interpret. This is why we allow 10-15 additional seconds of incubation between vortexing and pouring the overlay. The trade-off between maintaining the agar in a completely molten state (hotter is better) but not lethal to the seeding strain (hotter is not better) is one that will likely need to be determined by a researcher through trial and error.
If a PEG precipitation step is used, it would be beneficial to include a negative control where a sterile broth medium is processed identically to the culture supernatant. This will ensure that killing activity is not the result of residual PEG or chloroform within the sample supernatant.
As both bacteriophages and bacteriocins tend to be highly specific, it is not uncommon to encounter no apparent killing activity with a given combination of strains. This can result from a variety of strain-specific resistance mechanisms, one being that the tester strain either lacks or has an unrecognized version of the receptor needed for targeting by a given bacteriocin or bacteriophage.
An alternative method, bacteriocin screening method has been described by Kawai et al., but suffers from drawbacks and has not been widely adopted16. Specifically, this technique requires observing broth samples at time intervals that can be burdensome, and does not allow for the discrimination between bacteriophage-derived and bacteriocin-derived killing activities.
The main limitations of this technique is that it is not well suited for organisms that do not grow robustly (and thus will lack easily discernable clearing zones) or that harbor prophages or bacteriocins that are not robustly produced under laboratory conditions. Adaption of this technique to detect bacteriocins produced in environmental samples would both expand the utility of this technique and facilitate greater insight into the role of bacteriocins within natural settings.

Recently, there has been significant interest in deepening our understanding of microbial ecology (particularly the microbiomes of various environments), as well as new antibacterial compounds to use in combating antibiotic resistant pathogens1,2. An interconnecting theme between these interests is understanding antagonistic interactions among bacterial strains in their natural environment. There are numerous ways in which bacteria antagonize competitors3. Bacteriocins, a diverse group of proteinaceous, antibacterial compounds, have long been studied for their role in mediating interbacterial antagonism, with two of the most studied species being Pseudomonas aeruginosa4,5 and Escherichia coli6, important human pathogens. In addition to bacteriocins, induced prophages can also act as anticompetitor agents, allowing a strain to invade into a niche that is already colonized7. Pseudomonas syringae is a plant pathogen known to produce an array of antimicrobial agents, including single protein bacteriocins8, bacteriophage tail-derived bacteriocins9 (termed tailocins), as well as non-proteinaceous secondary metabolites10. Recently there has been interest in understanding how these antimicrobials influence the ecology of this organism, as well as how they can be harnessed to control plant disease11.
A widely utilized method for studying both bacteriocins and bacteriophage is the soft-agar overlay technique. This method was first described by Gratia in 1936 to aid in enumerating bacteriophage12,13.
Here we describe the application of the soft-agar overlay method, in combination with targeted genetic manipulation and polyethylene glycol (PEG) precipitation, in distinguishing among three different antimicrobial agents (a bacteriophage, a high molecular weight bacteriocin, and a low molecular weight bacteriocin) produced by a single bacterial strain. The benefit of this approach is that it is relatively simple and cheap, which is why, despite it being decidedly &#39;low-tech&#39;, it is still widely utilized.

<table><tbody><tr><td>Mitomycin C</td><td>Santa Cruz Biotechnology</td><td>sc-3514</td><td>Carcinogenic. use appropriate PPE (&lt;em&gt;i.e.&lt;/em&gt; gloves, eye protection, and face mask), particularly when preparing the stock solution.</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>34854</td><td>Acutely toxic, respiratory hazard. Use appropriate PPE (glove and eye shield), handle open containers within a chemical fume hood.&amp;nbsp;</td></tr><tr><td>Polyethylene Glycol (PEG)</td><td>Amresco</td><td>0159</td><td></td></tr><tr><td>Bacteriological grade agar</td><td>Genesee Scientific</td><td>20-274</td><td></td></tr></tbody></table>

use of the soft-agar overlay technique to screen for bacterially produced inhibitory compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

The combination of the soft agar overlay and PEG precipitation can be used to identify and characterize different antimicrobial agents produced by the same strain. Figure 1 shows two strains of P. syringae (A and B) that are inhibited by a third strain of the same species. The two strains, however, are inhibited by different bacteriocins. The clearing zone on strain A exhibits a sharp edge, whereas the clearing zone on strain B is larger, and exhibits a non-sharp border. Genetic manipulations within the producing strain that specifically inactivate either the tailocin (tailocin deficient) or low molecular weight bacteriocin (bacteriocin deficient) confirm that strains A and B are sensitive to distinct bacteriocins. Figure 2 shows that bacteriophage-mediated killing can be distinguished from other non-replicative antimicrobials by comparing a dilution series. Because both bacteriocins and prophage can be induced by mitomycin C treatment, activities of these two agents can closely resemble each other (particularly if the activated phage is abundant). In the case of bacteriophage-mediated clearing, dilution of the supernatant will resolve into individual plaques (clearing zones) while bacteriocin-mediated clearing will not resolve into such plaques.
<img alt="Figure 1" src="/files/ftp_upload/55064/55064fig1.jpg" /> 
Figure 1: Phenotypic distinction of multiple antimicrobial agents produced by the same strain. Strain A is sensitive to a high molecular weight bacteriocin (tailocin) but not an alternative low molecular weight bacteriocin, as evidenced by lack of clearing in the tailocin deficient strain, but not the bacteriocin deficient strain. Conversely, strain B is insensitive to the tailocin, but is sensitive to the low molecular weight bacteriocin. The tailocin is efficiently recovered following PEG precipitation, while lower molecular weight bacteriocins are not. WT = wild type. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55064/55064fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55064/55064fig2.jpg" /> 
Figure 2: Distinguishing between non-replicative antimicrobials and bacteriophages. (A) Dilution of a high titer of bacteriophages results in individual plaques that become less numerous (high titer top, low titer bottom). (B) Dilution of bacteriocins results in uniformly less clearing that does not resolve into individual plaques. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55064/55064fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds at JoVE.com

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

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Immunology and Infection

32.3K Views.  University of Arizona. We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria. 

Video: Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Aseptic Laboratory Techniques: Plating Methods

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) as well as Material Safety Data Sheets (MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection (ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to: 
 
&#9679; Perform plating procedures without contaminating media. 
&#9679; Isolate single bacterial colonies by the streak-plating method. 
&#9679; Use pour-plating and spread-plating methods to determine the concentration of bacteria. 
&#9679; Perform soft agar overlays when working with phage. 
&#9679; Transfer bacterial cells from one plate to another using the replica-plating procedure. 
&#9679; Given an experimental task, select the appropriate plating method.

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

Biology

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. They are an important health concern as they have been associated with recurrent chronic infections. Although stochastic and deterministic dynamics of stress-related mechanisms are known to play a significant role in persistence, mechanisms underlying the phenotypic switch to/from the persistence state are not completely understood. While persistence factors triggered by environmental signals (e.g., depletion of carbon, nitrogen and oxygen sources) have been extensively studied, the impacts of osmolytes on persistence are yet to be determined. Using microarrays (i.e., 96 well plates containing various chemicals), we have designed an approach to elucidate the effects of various osmolytes on Escherichia coli persistence in a high throughput manner. This approach is transformative as it can be readily adapted for other screening arrays, such as drug panels and gene knockout libraries.

In this method paper, we present a high-throughput screening strategy to identify chemical compounds, such as osmolytes, that have a significant impact on bacterial persistence.

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Soft Agar Colony Formation Assay: A Method to Test Effects of Novel Compounds on Cancer Cell Proliferation

Source: Horibata A. et. al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/52727/utilization-soft-agar-colony-formation-assay-to-identify-inhibitors"> Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells.</a> J. Vis. Exp. (2015).
This video describes a soft agar colony formation assay to test the effects of a PADI enzyme inhibitor and BB-Cl-amidine on breast cancer tumorigenicity in vitro. This assay also enables the quantitative assessment of cell transformation potential within the context of genetic perturbations.

Source: Horibata A. et. al.,  Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells. J. Vis. ...

JoVE Encyclopedia of Experiments

Cancer Research

Breast Cancer

In vitro studies

A Peg Plate Biofilm Assay for Screening Antibacterial Agents

Source: Dalecki, A. G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53925">Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay.</a> J. Vis. Exp. (2016)
This video demonstrates an assay for screening antimicrobial compounds that efficiently eliminate biofilms using a peg-plate. The methodology monitors the biofilm&#39;s metabolic state after treatment with antimicrobial compounds, facilitating the determination of the minimum biofilm eradication concentration.

1. Biofilm Initiation

	Grow a culture of biofilm-producing organisms in a nutrient-rich medium. Inoculate&#160;Staphylococcus aureus&#160;strain Newman ...

Immunology

Immunodiagnostics

Specialized Assays

A Deferred Growth Inhibition Assay to Evaluate the Effect of Bacteria-Derived Antimicrobial Compounds

Source: Moran, J. C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54437">Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition.</a> J. Vis. Exp.&#160;(2016)
This video showcases the deferred growth inhibition assay, used to investigate how antimicrobial compounds produced by one type of bacteria affect another competing bacterial strain.

The following method is adapted&#160;to test competitor strains with inhibitor-producing strains.
NOTE: This protocol involves aerosol generation with ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

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