This work was supported by an NSF DBI Biology Integration Institute (BII) grant (award no. 2119968; PI-Ceballos) and by the Arkansas INBRE program, with a grant from the National Institute of General Medical Sciences, (NIGMS), P20 GM103429 from the National Institutes of Health. The authors also appreciate the support of the Patterson Summer Undergraduate Research Program at Ouachita Baptist University.

While this protocol is written for Gordonia terrae, it has also been successfully used for Gordonia lacunae, Gordonia rubripertincta, and Gordonia westfalica.
1. Bacteria preparation
<ol>
	<li>In a biosafety cabinet, use good microbiological practice17 to inoculate a single colony of Gordonia terrae CAG3 into a 1 L sterile baffled flask with 200 mL of peptone yeast calcium broth18(PYCa) containing 0.01 mg/mL cycloheximide (see the Table of Materials).</li>
	<li>Incubate the flask at 30 &#176;C with shaking at 250 rpm until the culture is saturated or for 3 days7.</li>
	<li>Serially dilute the saturated G. terrae culture in PYCa broth, and spread-plate 100 &#181;L each of the 10&#8722;4, 10&#8722;5, and 10&#8722;6 dilutions on PYCa plates19,20. Refrigerate the undiluted saturated culture at 4 &#176;C.</li>
	<li>Incubate the spread plates inverted for 3 days at 30 &#176;C.</li>
	<li>Following incubation, identify a &#34;countable plate&#34;, a plate with 20-200 colonies. Count the number of colonies on that plate, and calculate the colony-forming units per milliliter (cfu/mL)19,20.</li>
	<li>Dilute the saturated culture with PYCa broth to 4.0 x 106 cfu/mL bacteria.</li>
</ol>
2. Phage preparation
NOTE: The representative results reported were obtained with the Gordonia phage DelRio21, a temperate bacteriophage isolated on G. terrae. These methods have also been successfully used with other Gordonia phages.
<ol>
	<li>Beginning with an isolated bacteriophage, serially dilute the phage sample in phage buffer7 to a 1 x 10&#8722;8 dilution. Perform a double-layer agar phage titer assay7 by infecting 0.3 mL of the saturated G. terrae culture with 10 &#181;L of each phage dilution. Following a 5-10 min room temperature incubation, combine the bacteria-phage mixture with 3 mL of PYCa top agar, and pour onto PYCa agar plates.</li>
	<li>Incubate the plates inverted at 30 &#176;C for 3 days or until plaques form7.</li>
	<li>Following incubation, identify a &#34;countable plate&#34;, a plate with 20-200 plaques. Count the number of plaques on that plate, and calculate plaque-forming units per milliliter (pfu/mL)7.</li>
</ol>
3. Microplate preparation
NOTE: Flat-bottomed 96-well microplates (see the Table of Materials) should be used for this method. All the plate preparation and loading must be completed in a biosafety cabinet, and good microbiological practice17should be used.
<ol>
	<li>Prepare the anti-fog lid coating solution by combining 100 &#181;L of Triton-X 100, 40 mL of 100% isopropanol, and 160 mL of deionized water22. Stir to mix, and store at room temperature.</li>
	<li>In a biosafety cabinet, add 6 mL of lid coating solution to the inside surface of a sterile 96-well microplate lid, holding the lid by the edges and rotating it to ensure that it is covered by the solution. Let the solution sit on the lid for 20 s, then pour the solution off, and invert the lid on an autoclaved paper towel at an angle until the lid is completely dry, which typically takes 35-45 min. Be careful to hold the lid by the edges.</li>
	<li>Prepare 20 mL of 0.1% agarose in water for each 96-well microplate, microwaving to melt the agarose.</li>
	<li>Once the agarose has cooled to 50-60 &#176;C, pipet 100 &#181;L of the 0.1% agarose into all of the spaces between the wells of the plate and 200 &#181;L of agarose into the wells in row A and row H and column 1, column 2, column 11, and column 1223 (Figure 1).</li>
</ol>
4. Loading the plate with bacteria and phage
NOTE: All the plate preparation and loading must be completed in a biosafety cabinet, and good microbiological practice17 should be used.
<ol>
	<li>The 96-well plate will contain 75 &#181;L of 2.0 x 106 cfu/mL bacteria in each well9. Dilute the 4.0 x 106 cfu/mL bacterial culture 1:1 with 2x PYCa broth to 2.0 x 106 cfu/mL. Prepare 5 mL per 96-well plate.</li>
	<li>Dilute the phage lysate using phage buffer7 to make 1 mL each of 2.0 x 106 pfu/mL, 2.0 x 105 pfu/mL, and 2.0 x 104 pfu/mL concentrations. This will allow for a multiplicity of infection (MOI) of 1, 0.1, and 0.01 within the microplate9.</li>
	<li>To load the microplate, take a plate that was prepared with anti-fog solution and agarose, and pipet 75 &#181;L of the 2.0 x 106 cfu/mL bacteria into columns 3-10, following Figure 1.</li>
	<li>To column 3 and column 4, add 75 &#181;L of sterile phage buffer to each well to serve as a no-phage positive control, following Figure 1, and pipette up and down to mix after each addition. Add 75 &#181;L of the 2.0 x 106 pfu/mL phage to column 5 and column 6, 75 &#181;L of the 2.0 x 105 pfu/mL phage to column 7 and column 8, and 75 &#181;L of the 2.0 x 104 pfu/mL phage to column 9 and column 10, pipetting up and down to mix after each addition.</li>
	<li>Tape both short sides of the plate with 0.5 in labeling tape to partially seal the plate while allowing gas exchange.</li>
</ol>
5. Incubation and absorbance measurement
<ol>
	<li>Once the plates are loaded with bacteria and phage, place them on a microplate shaker at 250 rpm, and incubate at 30 &#176;C.</li>
	<li>Incubate the plates for 96 h, taking an optical density measurement at 600 nm on a microplate reader every 8 h starting at hour 16. Return the plates to the shaker between measurements. 
	NOTE: Measurement periods of 4, h 6 h, 8 h, and 12 h were assessed, beginning at hour 0, and it was determined that an 8 h sampling period beginning at 16 h post-infection best captured the Gordonia-phage interactions.</li>
	<li>Monitor the lid for condensation throughout the experiment. If condensation is observed, replace the lid with another lid coated according to step 3.2.</li>
	<li>Generate control and infected growth curves following protocol section 6.</li>
</ol>
6. Data analysis
<ol>
	<li>Use a spreadsheet program to calculate the average and standard deviation for each phage dilution, following the spreadsheet layout in the Stacy-Ceballos-Index GitHub repository (https://github.com/eichristenson/Stacy-Ceballos-Index).</li>
	<li>Create control and infected growth curves, and calculate infection metrics using R (see the Table of Materials) with the DescTools24, dplyr25, ggplot226, and readxl27 packages and following the R script in the Stacy-Ceballos-Index GitHub repository (https://github.com/eichristenson/Stacy-Ceballos-Index).
	<ol>
		<li>AUCcon is the area under the uninfected control curve, while AUCinf is the area under the infected curve16. Calculate the AUC, and then calculate percent growth inhibition based on the area under the curve values, PIAUC16, using the following equation: 
		(&#8203;1&#160;- [AUCinf/AUCcon]) &#215; 100</li>
		<li>The dashed horizontal lines on each curve show the peak growth, with the uninfected growth peak labeled Nasymptote(con) and the infected growth peak labeled Nasymptote(inf). Identify the Nasymptote values, and then calculate percent growth inhibition based on these peak growth values, PImax16, using the following equation: 
		(1&#160;- [Nasymptote(inf)/ Nasymptote(con)]) &#215; 100</li>
		<li>Calculate the Stacy-Ceballos index, ISC16, from the PIAUC and PImax values, as follows: 
		(PIAUC &#215; PImax)0.5 
		Calculate relative virulence by integrating the Stacy-Ceballos index over time16.</li>
	</ol>
	</li>
</ol>

Characterizing Actinobacteriophage Infection Using an Adapted Optical Density Assay

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A., Wilpiszeski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+analysis+of+growth+differences+among+phage+strains.">High-throughput analysis of growth differences among phage strains.</a> Journal of Microbiological Methods. 88 (1), 117-121 (2012).</li><li>Storms, Z. J., Teel, M. R., Mercurio, K., Sauvageau, 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+virulence+index:+A+metric+for+quantitative+analysis+of+phage+virulence.">The virulence index: A metric for quantitative analysis of phage virulence.</a> PHAGE. 1 (1), 27-36 (2020).</li><li>. Phage Discovery Guide Available from: <a target="_blank" href="https://seaphagesphagediscoveryguide.helpdocsonline.com/home">https://seaphagesphagediscoveryguide.helpdocsonline.com/home</a> (2012)</li><li>Martinez-Soto, C. 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The authors have nothing to disclose.

This optical density-based microplate method permits investigation into bacteriophage host range and infection dynamics11 and shows the utility of the Stacy-Ceballos index16 as a measure of bacteriophage virulence. While this method could be utilized with any bacteriophage-host system, it was designed specifically to adapt rapid microplate growth assays9,10,11 for use with slower-growing bacteria such as actinomycetes. Rapid microplate assays cannot be used for slow-growing bacteria without modifications to address evaporation and lid condensation. This method describes these necessary modifications and demonstrates, for the first time, the use of the Stacy-Ceballos index and related metrics16 to describe bacteriophage infection.
Evaporation can be a substantial challenge in multi-day 96-well plate growth curve assays; this method resolves that problem by adding agarose to the border wells and the spaces between the wells. The agarose margin, combined with the anti-fog lid treatment22, provides the necessary humidity within the microplate and allows for reliable optical density measurements. Without the added humidity, substantial edge effect evaporation occurs23 during the lengthy incubation period required, leading to artificially high optical density readings. The anti-fog lid treatment is a necessary modification because lid condensation can also artificially elevate the optical density values. Shaking the plates during the incubation period is a recommended modification, as actinomycete bacteria may clump during growth, giving artificially high optical density values and effectively decreasing the multiplicity of infection.
The ratio of bacteria to phage in experiments characterizing infection dynamics is critical, as there must be enough phage to show an infection effect but not so many that the host bacterial population immediately crashes9 or the frequency of lysogeny is dramatically increased28. In this method, the ratio found to be most effective for obtaining consistent results was an MOI of 1, but usable results were also obtained with MOIs of 0.1 and 0.01. When implementing this method, it is recommended to choose one concentration of bacteria and test multiple phage concentrations in the MOI range of 0.01-19,10,11.
This technique described here allows bacteriophage-host interactions to be assessed for slow-growing bacteria in high-throughput microplate assays rather than with sub-sampling from a larger culture flask at each measurement interval29. Further, by demonstrating how microplate growth assays9,10,11 may be adapted, this technique increases the utility of other microplate-based bacteriophage assays for slower-growing bacteria, including phage characterization5,6,12 and evolution studies30,31. Finally, this method demonstrates the use of the Stacy-Ceballos index16 to describe bacteriophage infection. This metric was initially developed with data from an archaeal virus model system and is calculated from optical density values, thus giving it widespread utility across disparate virus systems.

Bacteriophages or phages are viruses that infect bacteria. They are the most numerous biological entities on the planet1, and it is generally accepted that bacteriophages influence bacterial evolution and ecosystem processes2,3,4. Several methods exist to describe, measure, and analyze bacteriophage host range8 and infection dynamics5,6, including agar-based methods such as the double-layer agar method7 and optical density-based microplate methods8,9,10,11,12. Each method has its own advantages and disadvantages. Due to their efficiency, plating tests using the double-layer agar method are the &#34;gold standard&#34; for host range assays, but this method is time- and labor-intensive9. Rapid microplate methods, which return results in 24 h or less, give excellent results for fast-growing bacterial hosts such as Escherichia coli9,10,11,12 but are too brief to showcase bacteriophage infection progression in slower-growing bacterial hosts such as actinomycetes7,13,14,15.
An optical density-based microplate assay designed for fast-growing bacteria cannot be run for the multiple days required to characterize infection dynamics in a slow-growing host bacterium without evaporation occurring and giving artificially high bacterial densities. Thus, obtaining comparable high-throughput data on bacteriophage infection dynamics for slow-growing bacterial species requires specialized techniques adapted for these bacteria.
The microplate method presented here reduces evaporation, thus allowing slow-growing bacteria to be co-cultured with a phage for an extended 96 h period and enabling investigations into phage infection dynamics and host range. This method also showcases the Stacy-Ceballos index16, an optical density-based metric that allows for virulence comparisons among disparate host-virus systems.

<table><tbody><tr><td>Agarose</td><td>Omni-Pur</td><td>2090</td><td>for filling border wells of microplate&amp;nbsp;</td></tr><tr><td>Costar 96 Well Lid Low Evaporation Corner Notch</td><td>Corning</td><td>3931</td><td>replacement microplate lid</td></tr><tr><td>Isopropanol</td><td>Fisher Chemical</td><td>A461-4</td><td>for lid coating</td></tr><tr><td>Microplate reader</td><td>Tecan Spark 20M</td><td></td><td></td></tr><tr><td>Microplate Shaker with 4-Place Platform</td><td>Thermo Fisher Scientific</td><td>88-861-023</td><td>to shake plates during incubation</td></tr><tr><td>Non-Tissue Culture-Treated Plate 96 well</td><td>Falcon (a Corning Brand)</td><td>351172</td><td>microplate for growth curve assay</td></tr><tr><td>Peptone yeast calcium (PYCa) agar</td><td>Homemade</td><td></td><td>1 g peptone&lt;br/&gt; 15 g yeast extract&lt;br/&gt; 15 g agar&lt;br/&gt; 990 mL dd H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 4.5 mL 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 2.5 mL 40% dextrose&lt;br/&gt; 1 mL 10 mg/mL cycloheximide</td></tr><tr><td>Peptone yeast calcium (PYCa) broth</td><td>Homemade, from Reference 16</td><td></td><td>1 g peptone&lt;br/&gt; 15 g yeast extract&lt;br/&gt; 990 mL dd H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 4.5 ml 1 M CaCl&lt;sub&gt;2 &lt;/sub&gt;&lt;br/&gt; 2.5 mL 40% dextrose&lt;br/&gt; 1 mL 10 mg/mL cycloheximide</td></tr><tr><td>Peptone yeast calcium (PYCa) top agar</td><td>Homemade</td><td></td><td>1 g peptone&lt;br/&gt; 15 g yeast extract&lt;br/&gt; 4 g agar&lt;br/&gt; 990 mL dd H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 4.5 mL 1M CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 2.5 mL 40% dextrose</td></tr><tr><td>Petri plates</td><td>Thermo Fisher Scientific</td><td>FB0875713</td><td>for determination of bacterial concentration and phage titer assay</td></tr><tr><td>Phage Buffer</td><td>Homemade, from Reference 7</td><td></td><td>10 mL 1 M Tris, pH 7.5&lt;br/&gt; 10 mL 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt; 4 g NaCl&lt;br/&gt; 980 ml dd H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>R software</td><td>https://www.r-project.org/</td><td>version 4.3.0</td><td></td></tr><tr><td>Sterile Disposable PETG Flask Baffled Bottom w/Vented Closure</td><td>Thermo Fisher Scientific</td><td>4116-1000</td><td>for bacterial culture</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>9036-19-5</td><td>for lid coating</td></tr></tbody></table>

an adapted optical density-based microplate assay for characterizing actinobacteriophage infection

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual phages interact with slow-growing bacterial hosts such as actinomycetes, an easy and reliable method for quantifying long-term bacterial growth in the presence of phages is needed. Spectrophotometric microplate readers allow for high-throughput repeated measurements, but incubating a small volume for an extended time can present technical challenges. This procedure adapts a standard 96-well microplate to allow for the co-culturing of phages and bacteria without sub-sampling for 96 h, with the bacterial growth recorded every 8 h using spectrophotometric absorbance values. These optical density values are analyzed using R to yield infection metrics, including the percent growth inhibition, relative virulence, and the Stacy-Ceballos index. The methods outlined here provide an effective way to conduct and analyze extended-duration microplate growth curve experiments and includes modifications to reduce evaporation and lid condensation. These protocols facilitate microplate-based assays of interactions between slow-growing bacterial hosts and their bacteriophages.

An experiment is successful if the resulting growth curve shows an increase in the positive control bacterial population over time with no sudden fluctuation in absorbance. Examples of a successful experiment at an MOI of 1 with and without a productive phage infection are shown in Figure 2 and Figure 3, respectively. A productive infection at an MOI of 0.01 is represented in Figure 4. The positive control growth pattern (green curve) seen in all three figures indicates that the bacteria are growing, they are not clumping during growth, and no contaminants are present. Clumping and contamination are indicated by unusually high absorbance at a single time point. Standard deviations typically increase over the time course of an experiment; however, a drastic increase or standard deviations overlapping between the control and infected curves may indicate contamination or clumping in one or more wells.
The growth curve depicting a productive phage infection, represented by Figure 2, shows reduced bacterial absorbance over time in wells with the phage added. This reduction in bacterial density will not be seen if the bacterium is outside of the phage&#39;s host range, as shown in Figure 3.
Infection metrics are shown for all the representative experiments, with a relatively large ISC for the productive infections in Figure 2 and Figure 4 and a very small ISC in Figure 3 for the phage that did not efficiently infect the host bacterium.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65482/65482fig01.jpg" /> 
Figure 1: Microplate layout. The gray areas are filled with 0.1% agarose. The blank wells in column 3 and column 4 are no-phage positive control wells containing only phage buffer and 2.0 x 106 cfu/mL bacteria. The dotted wells contain phage and 2.0 x 106 cfu/mL bacteria; the large dots in column 5 and column 6 indicate an MOI of 1 with 2.0 x 106 pfu/mL phage; the medium dots in column 7 and column 8 indicate an MOI of 0.1 with 2.0 x 105 pfu/mL phage; and the small dots in column 9 and column 10 indicate an MOI of 0.01 with 2.0 x 104 pfu/mL phage. <a href="https://www.jove.com/files/ftp_upload/65482/65482fig01large.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/65482/65482fig02.jpg" /> 
Figure 2: A successful growth curve experiment with a phage at an MOI of 1 that productively infects the host bacterium. The average absorbance values (&#177; standard deviation) are shown for uninfected bacteria (green) and bacteria with the phage added (blue). Abbreviations: AUC = area under the curve; PIAUC = percent growth inhibition calculated from the area under the curve; Nasymptote = peak growth value; PImax = percent growth inhibition calculated from the peak growth values; ISC = Stacy-Ceballos index. This graph represents the temperate Gordonia phage DelRio infecting G. terrae, the bacterium it was isolated on. <a href="https://www.jove.com/files/ftp_upload/65482/65482fig02large.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/65482/65482fig03.jpg" /> 
Figure 3: A successful growth curve experiment with a phage at an MOI of 1 that does not efficiently infect the host bacterium. The average absorbance values (&#177; standard deviation) are shown for uninfected bacteria (green) and bacteria with the phage added (blue). Abbreviations: AUC = area under the curve; PIAUC = percent growth inhibition calculated from the area under the curve; Nasymptote = peak growth value; PImax = percent growth inhibition calculated from the peak growth values; ISC = Stacy-Ceballos index. This graph represents G. rubripertincta infection by DelRio. <a href="https://www.jove.com/files/ftp_upload/65482/65482fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65482/65482fig04.jpg" /> 
Figure 4: A successful growth curve experiment with a phage at an MOI of 0.01. The average absorbance values (&#177; standard deviation) are shown for uninfected bacteria (green) and bacteria with the phage added (blue). Abbreviations: AUC = area under the curve; PIAUC = percent growth inhibition calculated from the area under the curve; Nasymptote = peak growth value; PImax = percent growth inhibition calculated from the peak growth values; ISC = Stacy-Ceballos index. This graph represents G. terrae infection by DelRio. <a href="https://www.jove.com/files/ftp_upload/65482/65482fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection at JoVE.com

Author Spotlight: An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection  

An optical density-based microplate method is described to quantify long-term bacterial growth in the presence of bacteriophages, suitable for actinomycetes and other slow-growing bacteria. This method includes modifications to reduce evaporation and lid condensation and R code to calculate virus infection metrics, including the area under the curve, growth maximum, and relative virulence.

An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual ...

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2.0K Views.  Ouachita Baptist University. An optical density-based microplate method is described to quantify long-term bacterial growth in the presence of bacteriophages, suitable for actinomycetes and other slow-growing bacteria. This method includes modifications to reduce evaporation and lid condensation and R code to calculate virus infection metrics, including the area under the curve, growth maximum, and relative virulence.

Video: Author Spotlight: An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection  

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 ...

Immunology and Infection

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

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 ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...

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 ...

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.

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 ...

Exploring Gut Phage-Bacteria Dynamics in Murine Models Using T4 Phage

T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host&#39;s health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli,&#160;in the context of the murine gastrointestinal tract. The methods described here outline&#160;how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels.&#160;This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

Author Spotlight: Investigating Bacteriophage-Induced Immune Responses in Gnotobiotic Mice

Bacteriophages (phages), viruses that infect bacteria, are an integral component of the gut microbiome. Though these symbiotic inhabitants drive bacterial fitness and population dynamics, little is understood about how they impact gut homeostasis and disease. This protocol studies isolated T4 phages within a mouse model, adaptable to other phage-bacterial pairs.

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across ...

Visualizing Antibiotic Persistence in Bacterial Pathogens

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

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

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

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

Plaque Assay: A Method to Determine Viral Titer as Plaque Forming Units (PFU)

Source: Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Viruses that infect prokaryotic organisms, called bacteriophages or simply phages, were identified in the early 20th century by Twort (1) and d&#39;H&#233;relle (2) independently. Phages have since been widely recognized for their therapeutic value (3) and their influence on human (4), as well as global, ecosystems (5). Current concerns have fueled a renewed interest in the use of phages as an alternative to modern antibiotics in treatment of infectious disease (6). Essentially all phage research relies on the ability to purify and quantify viruses, also known as a viral titer. Initially described in 1952, this was the purpose of the plaque assay (7). Decades and multiple technological advancements later, the plaque assay remains one of the most reliable methods for determination of viral titer (8).
Bacteriophages subsist by injecting their genetic material into host cells, hijacking the machineries for production of new phage particles, and eventually causing the host to release numerous progeny virions through cell lysis. Because of their minute size, bacteriophages cannot be observed using solely light microscopy; therefore, scanning electron microscopy is required (Figure 1). Additionally, phages cannot be cultivated on nutritional agar plates like bacteria, as they need host cells to prey on.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10514/10514fig1.jpg" /> 
Figure 1: The morphology of a bacteriophage, here exemplified by an E. coli phage, can be studied using scanning electron microscopy. Most bacteriophages belong to Caudovirales (tailed bacteriophages). This particular phage has a very short tail structure and an icosahedral head, placing it in the family of Podoviruses.
The plaque assay (Figure 2) is based on incorporation of host cells, preferentially in log-phase growth, into the medium. This creates a dense, turbid layer of bacteria able to sustain viral growth. An isolated phage can subsequently infect, replicate within, and lyse one cell. With each lysed cell, multiple adjacent ones become immediately infected. Several cycles in, a clear zone (a plaque) can be observed in the otherwise turbid plate (Figure 2B/Figure 3A), indicating the presence of what was initially a single bacteriophage particle. The number of plaque forming units per volume (i.e. PFU/mL) of a sample, can thus be determined from the number of plaques generated.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10514/10514fig2.jpg" /> 
Figure 2: Testing for plaque forming units (PFU) is a common method for determining the number of bacteriophages in a sample. (A) The base of a sterile Petri dish is covered with an appropriate solid nutrient medium, followed by a mixture of soft media, susceptible host cells and a dilution of the original bacteriophage sample. Note that the phage-suspension could, in some cases, also be evenly spread-out across the surface of already solidified soft agar. (B) Growth of the host bacteria forms a lawn of cells in the top agar layer. Bacteriophage replication generates clear zones, or plaques, caused by host cell lysis.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10514/10514fig3.jpg" /> 
Figure 3: Results from PFU-testing show multiple plaques generated by bacteriophages. Due to lysis of susceptible host cells, plaques can be seen as clearing zones in the bacterial lawn, either with (A) full clearance, or (B) partial re-growth caused by generation of resistant bacteria (or possibly by temperate phages in the lysogenic cycle).
Certain temperate phages can adopt what is referred to as a lysogenic lifecycle, in addition to the formerly described lytic growth. In lysogeny, the virus assumes a latent state through incorporation of its genetic material into the genome of the host cell (9), often conferring resistance to further phage infections. This is sometimes revealed through a slight clouding of the plaque (Figure 3B). It is worth noting however, that plaques can also appear blurred due to re-growth of bacteria that have evolved resistance to the phage independent of previous phage infections.
Viruses can attach, or adsorb, to only a limited range of host bacteria (10). Host ranges are further limited by intracellular anti-viral strategies such as the CRISPR-Cas system (11). The resistance/sensitivity towards specific phages displayed by bacterial subgroups has historically been used to categorize bacterial strains into different phage types (Figure 4). Although the effectiveness of this method has now been surpassed by novel sequencing techniques, phage typing can still provide valuable information about bacteria-phage interactions, for instance, facilitating the design of a phage cocktail for clinical usage.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/10514/10514fig4.jpg" /> 
Figure 4: Phage sensitivity of different bacterial strains. Soft agar plates with Cutibacterium acnes strain (A) AD27 and (B) AD35, were spotted with 21 different C. acnes bacteriophages. Only phage 11 were able to infect and kill AD27 while strain AD35 showed sensitivity towards all phages. This technique, termed phage typing, can be used to divide bacterial species and strains into different subgroups based on phage susceptibility.

Determining Viral Titer as Plaque Forming Units PFU

Source: Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 ...

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Advanced Biology

Microbiology

Detection of Bacteriophages in Environmental Samples

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona 
Demonstrating Author: Alex Wassimi
Viruses are a unique group of biological entities that infect both eukaryotic and prokaryotic organisms. They are obligate parasites that have no metabolic capacity, and in order to replicate, rely on host metabolism to produce viral parts that self-assemble inside host cells.
Viruses are ultramicroscopic&#8212;too small to be viewed with the light microscope, visible only with the greater resolution of the electron microscope. A viral particle consists of a nucleic acid genome, either DNA or RNA, surrounded by a protein coat, known as a capsid, composed of protein subunits or capsomers. In some more complex viruses, the capsid is surrounded by an additional lipid envelope, and some have spike-like surface appendages or tails.
Viruses that infect the intestinal tract of humans and animals are known as enteric viruses. They are excreted in feces and can be isolated from domestic wastewater. Viruses which infect bacteria are known as bacteriophages, and those which infect coliform bacteria are called coliphages (Figure 1). The phages of coliform bacteria are found anywhere coliform bacteria are found.
<img alt="Figure 1" src="/files/ftp_upload/10190/10190fig1.jpg" /> 
Figure 1. Coliphage T2.

Plaque Assay: Phage Enumeration in Environmental Samples

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Alex Wassimi
Viruses are a unique group of ...