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., Drulis-Kawa, 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+life+cycles+behind+bacterial+biodiversity.">Phage life cycles behind bacterial biodiversity.</a> Current Medicinal Chemistry. 24 (36), 3987-4001 (2017).</li><li>Weitz, J. S., 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=Phage-bacteria+infection+networks.">Phage-bacteria infection networks.</a> Trends in Microbiology. 21 (2), 82-91 (2013).</li><li>Turner, P. E., Draghi, J. 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. E., 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=PHIDA:+A+high+throughput+turbidimetric+data+analytic+tool+to+compare+host+range+profiles+of+bacteriophages+isolated+using+different+enrichment+methods.">PHIDA: A high throughput turbidimetric data analytic tool to compare host range profiles of bacteriophages isolated using different enrichment methods.</a> Viruses. 13 (11), 2120-2137 (2021).</li><li>Rajnovic, D., Mu&#241;oz-Berbel, X., Mas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+phage+detection+and+quantification:+An+optical+density-based+approach.">Fast phage detection and quantification: An optical density-based approach.</a> PLoS One. 14 (5), e0216292 (2019).</li><li>Xie, Y., Wahab, L., Gill, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+microtiter+plate-based+assay+for+determination+of+bacteriophage+host+range+and+virulence.">Development and validation of a microtiter plate-based assay for determination of bacteriophage host range and virulence.</a> Viruses. 10 (4), 189-204 (2018).</li><li>S&#248;rensen, P. 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American Society for Microbiology Available from: <a target="_blank" href="https://asm.org/ASM/media/Protocol-Images/Preparing-Spread-Plates-Protocols.pdf">https://asm.org/ASM/media/Protocol-Images/Preparing-Spread-Plates-Protocols.pdf</a> (2006)</li><li>Mathes, H. N., 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=Complete+genome+sequences+of+Chop,+DelRio,+and+GrandSlam,+three+Gordonia+phages+isolated+from+soil+in+Central+Arkansas.">Complete genome sequences of Chop, DelRio, and GrandSlam, three Gordonia phages isolated from soil in Central Arkansas.</a> Microbiology Resource Announcements. 12 (5), e0002323 (2023).</li><li>Krishnamurthi, V. R., Niyonshuti, I. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Omni-Pur</td>
			<td>2090</td>
			<td>for filling border wells of microplate&#160;</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 
			15 g yeast extract 
			15 g agar 
			990 mL dd H2O 
			4.5 mL 1 M CaCl2 
			2.5 mL 40% dextrose 
			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 
			15 g yeast extract 
			990 mL dd H2O 
			4.5 ml 1 M CaCl2 
			2.5 mL 40% dextrose 
			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 
			15 g yeast extract 
			4 g agar 
			990 mL dd H2O 
			4.5 mL 1M CaCl2 
			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 
			10 mL 1 M MgSO4 
			4 g NaCl 
			980 ml dd H2O</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 ...

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

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Biology

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  

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...