Funding for the development of this protocol was provided by the Southeastern Pennsylvania Consortium for Higher Education and the Cabrini College Science Department. Additional funding and support came from Arcadia University and Immaculata University. We especially thank Dr. Karen Snetselaar at St. Joseph University for kindly taking the electron microscopic images of our isolated phages.&#160;Additional support was provided by the Howard Hughes Medical Institute Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program.

1. Preparation of Arthobacter Cells for Phage Isolation
<ul>
	<li>Culture Arthrobactersp. KY3901 colonies streaked on an Luria Bertaini (LB) agar plate incubated at 30 &#176;C for 2-3 days. Pick a colony and use a sterile loop to add it to 250 ml of LB broth in a baffled culture flask and incubate in a shaking incubator at 225 rpm at 30 &#176;C.</li>
	<li>Allow approximately 24 hr of growth to obtain late exponential/early stationary phase cells for phage infection experiments. Monitor the state of bacterial growth closely to prevent cells from entering the middle to late stationary growth state. 
	NOTE: Cell growth should consist of an optical density OD600 between 0.5-.0.7 for the phage isolation and purification procedures/steps described below.</li>
</ul>
2. Collection of Phage from Soil Samples
<ol>
	<li>Gather 200-400 g of soil from desired location. Note: Since bacteria in the Arthrobacter genus are common soil bacteria they and the phages that infect them can be found in and are pervasive in most types of soil.</li>
	<li>Add at least 400 ml of Phage Buffer (PB) [68 mM NaCl, 10 mM MgSO4,10 mM Tris-CL (pH 7.5)] to the soil and mix in a large enough flask so that at least 200 ml of PB is in the supernatant and is able to be extracted.</li>
	<li>Mix by gently stirring or swirling until the soil is suspended and allow the soil sediment to settle, typically 30 min.</li>
	<li>Pass the &#8220;soil extract&#8221; through filter paper by gravity filtration to remove soil sediment debris.</li>
	<li>Add LB powder and mix to make a 2% solution (4 g LB powder in 200 ml soil extract).</li>
	<li>Pass the solution through a 0.22 &#181;m filter by vacuum filtration to obtain a sterile filtrate. If possible, continue on to step 2.7 immediately. If need be, store filtered samples at 4 &#176;C for up to a week before proceeding, however, the number of viable phage particles will be reduced.</li>
	<li>Aliquot multiple 50 ml portions of the filter-sterilized LB/soil extract mixture into individual 250 ml sterilized shaking baffled culture flasks using aseptic techniques. Add sterile 2.0 M CaCl2 solution to the final desired concentrations (0-50 mM) since different Arthrobacter phages grow optimally under different CaCl2 conditions. 
	NOTE: We find that the majority of the Arthrobacter phages&#160;identified by us are within the range of 1 mM-2.5 mM CaCl2. However, several of the isolated phages grew by us exclusively at 0 mM CaCl2.or at very high CaCl2 concentrations. Proceed immediately to step 3.1.</li>
</ol>
3. Phage Isolation
<ol>
	<li>Add 1 to 2.5 ml of late exponential/early stationary phase bacteria culture to each of the flasks. For an OD600 from 0.5-0.7, use 1 ml of cells. For an OD600 higher than an OD of 0.7 use 2.5 ml of cells. 
	NOTE: Cells in exponential phase growth typically yield more phages and higher titers than cells in stationary phase growth.</li>
	<li>Shake flasks at 250 rpm at 30 &#176;C for approximately 24 hr in a shaking incubator.</li>
	<li>After a 24 hr incubation period remove enrichment flasks from the shaking incubator and dilute the enrichment samples ten-fold in phage buffer.</li>
	<li>Set up culture tubes with 0.5 ml of late exponential/early stationary phase bacteria culture and add various amounts of the enrichment culture (5 &#181;l to 500 &#181;l of a 10-1 dilution in PB) to the culture tubes. 
	NOTE: It is advisable to test a wide range of concentrations since each soil sample generates different phage titers after enrichment.</li>
	<li>Add CaCl2 to culture tubes to make a final concentration equal to the concentration present in the original enrichment flask. Use a range of different CaCl2 concentrations to select for many phages with varying CaCl2 dependencies. 
	NOTE: Most phages will be isolated within the CaCl2 range of 1-2.5 mM but there are phages isolated most optimally anywhere from 0-50 mM CaCl2 concentrations. Because of this it is best to check a range of CaCl2 concentrations to maximize the number of unique isolated Arthrobacter phages.</li>
	<li>Mix 4.5 ml&#160;of LB top agar in the culture tube and pour the mixture onto an LB agar plate. Swirl gently to distribute across the plate evenly and allow for top agar to solidify for approximately 15 min. 
	NOTE: Top agar can be prepared in larger batches (250 ml) and extra top agar can be stored at RT and reused for subsequent experiments for at least two weeks. Although there are different formulas used to make top agar, we make top agar using 7g/L of agar mixed with LB. Place top agar in a 60 &#176;C water bath to retain the liquid state during the course of an experiment.</li>
	<li>Invert the plate and incubate at desired temperature (temperature to 30 &#176;C) O/N to 48 hr. Vary these conditions to optimize for the phages present. Most isolated phages come from plates incubated at 30 &#176;C at 1-2.5 mM CaCl2 concentration after a 24 hr incubation period.</li>
</ol>
4. Phage Purification
<ol>
	<li>After a 24 hr incubation check for plaque formation on plates. Continue incubation for up to 72 hr if no plaques are evident or to determine if additional plaques will appear. If needed, store plates with putative plaques for up to a week at 4 &#176;C prior to proceeding. 
	&#8203;NOTE: It is not uncommon to find a variety of different plaque morphologies on one plate. Finding plaques after 3 days of incubation is possible but rare.</li>
	<li>Purify desired phages from clearly isolated plaques using the streak plaque method. Touch a plaque with a sterile wooden stick. Streak the plaque by running a wooden stick through a flame, allowing the stick to cool briefly, and then rubbing the stick on the agar plate in the gentle motion as shown in Figure 1. Repeat this using a clean stick for each pass.</li>
	<li>Alternatively, use the traditional plaque titer assay to isolate individual phage plaques. The plaque titer assay does require the use of more reagents such as LB agar and LB top agar and materials such as petri plates for phage plaque isolation. We have been highly successful using the streak plaque method but it is technically easier to isolate individual plaques using the plaque titer assay.</li>
	<li>Once the LB agar plate is streaked at least three times, mark the area with the lowest concentration of putative phage particles and GENTLY apply 0.5 ml of Arthrobacter and 4.5 ml of molten LB top agar (containing the same concentration of calcium chloride as the parent plate) and allow it to spread evenly across the plate.</li>
	<li>Invert and incubate plate O/N. Repeat the streak plaque technique for at least three iterations to ensure a pure phage species is isolated. Store plates at 4 &#176;C for up to a week as needed between streaks without significant loss of phage viability.</li>
	<li>Once desired plaques have been grown and isolated on the final streak plate, touch one well isolated plaque with a micropipette tip and re-suspend in 100 &#181;l PB. Serially dilute this 100 solution out to the 10-8 dilution by passing 20 &#181;l of the sample into 180 &#181;l of fresh PB.</li>
	<li>Add 10 &#181;l of each dilution to 0.5 ml of grown Arthrobacter in a culture tube for 5 to 10 min at RT and plate by mixing the infected bacteria with 4.5 ml of molten LB top agar containing the same concentration of CaCl2 used to isolate the phage from the original soil sample. Save all dilutions made at 4 &#176;C until the next day.</li>
	<li>Determine the phage serial dilution needed to make a web pattern using the traditional plaque titer assay. The purpose of the plaque titer assay is to determine the phage titer needed to obtain a web pattern plate. Identify a web pattern plate as mostly devoid of bacteria but containing remnants of bacteria that have not yet been lysed by phages. Add 5 ml of late exponential/ early stationary Arthrobacter culture to a sterile 50 ml culture flask.
	<ol>
		<li>Add 100 &#181;l of the dilution (that gave a web pattern to the Arthrobacter culture) in the culture flask and allow the phages to infect the bacteria cells for 5 to 10 min at RT. Mix with molten LB top agar containing the same concentration of CaCl2 as used to initially identify the phage and plate 5 ml of this mixture onto 10 fresh LB plates. Allow to solidify and incubate inverted plates at 30 &#176;C.</li>
	</ol>
	</li>
	<li>The next day, flood the web pattern plates with 5 ml of PB and store O/N&#160;at 4 &#176;C or 4 hr at RT.</li>
	<li>Filter the phage lysate through a 0.22 &#181;m syringe filter and store at 4 &#176;C. Use this final phage stock for additional experiments such as electron microscopy images of phage particles, phage genomic DNA isolation for DNA restriction enzyme analysis and genomic sequencing using standard procedures. For long term storage of the phage stock, add an equal amount of the phage lysate to sterile glycerol in small vials for stock archiving at -80 &#176;C. 
	NOTE: For those not experienced with these phage techniques,&#160;a great resource is the online accessible www.phagesdb.org website.</li>
</ol>

<ol>
	<li>Shapir, N., Mongodin, E. F., Sadowsky, M. J., Daugherty, S. C., Nelson, K. E., Wackett, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Catabolic+Pathways:+Genomic+Insights+into+Microbial+s-Triazine+Metabolism.">Evolution of Catabolic Pathways: Genomic Insights into Microbial s-Triazine Metabolism.</a> J Bacteriol. 189 (3), 674-682 (2007).</li><li>Qingyan, L., Ying, L., Xikun, Z., Baoli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Atrazine+Degrading+Arthrobacter+sp.+AD26+and+Use+of+this+Strain+in+Bioremediation+of+Contaminated+Soil.">Isolation and Characterization of Atrazine Degrading Arthrobacter sp. AD26 and Use of this Strain in Bioremediation of Contaminated Soil.</a> J Environ Sci. 20, 1226-1230 (2008).</li><li>Wang, J., Zhu, L., Liu, A., Ma, T., Wang, Q., Xie, H., Wang, J., Jiang, T., Zhao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+an+Arthrobacter+sp.+Strain+HB-5+that+Transforms+Atrazine.">Isolation and characterization of an Arthrobacter sp. Strain HB-5 that Transforms Atrazine.</a> Environ Geochem Hlth. 33, 259-266 (2011).</li><li>Mages, I. S., Frodl, R., Bernard, K. A., Funke, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identities+of+Arthrobacter+spp.+And+Arthrobacter-Like+Bacteria+Encountered+in+Human+Clinical+Specimens.">Identities of Arthrobacter spp. And Arthrobacter-Like Bacteria Encountered in Human Clinical Specimens.</a> J Clin Microbiol. 46 (9), 2980-2986 (2008).</li><li>Brown, D. R., Holt, J. G., Pattee, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Arthrobacter+Bacteriophages+and+Their+Application+to+Phage+Typing+of+Soil+Arthrobacters.">Isolation and Characterization of Arthrobacter Bacteriophages and Their Application to Phage Typing of Soil Arthrobacters.</a> Appl Environ Microbiol. 35 (1), 185-191 (1978).</li><li>Petrovski, S., Seviour, R. J., Tillett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+Gordonia+and+Nocardia+Stabilized+Foam+Formation+by+Using+Bacteriophage+GTE7.">Prevention of Gordonia and Nocardia Stabilized Foam Formation by Using Bacteriophage GTE7.</a> Appl Environ Microbiol. 77 (21), 7864-7867 (2011).</li><li>Le Marrec, C., Moreau, S., Loury, S., Blanco, C., Trautwetter, a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Characterization+of+Site-specific+Integration+Functions+of+phi+AAU2+infecting+&#8220;Arthrobacter+aureus&#8221;+C70.">Genetic Characterization of Site-specific Integration Functions of phi AAU2 infecting &#8220;Arthrobacter aureus&#8221; C70.</a> J Bacteriology. 178 (7), 1996-2004 (1996).</li><li>Casida, L. E., Liu, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthrobacter+globiformis+and+Its+Bacteriophage+in+Soil.">Arthrobacter globiformis and Its Bacteriophage in Soil.</a> J Appl Microbiol. 28 (6), 951-959 (1974).</li><li>Einck, K. H., Pattee, P. A., Holt, J. G., Hagedorn, C., Miller, J. A., Berryhill, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+a+Bacteriophage+of+Arthrobacter+globiformis.">Isolation and Characterization of a Bacteriophage of Arthrobacter globiformis.</a> J Virol. 12 (5), 1031-1033 (1973).</li><li>Twest, R., Kropinski, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+Enrichment+from+Soil+and+Water.">Bacteriophage Enrichment from Soil and Water.</a> Methods Mol Bio. 501, 15-21 (2009).</li><li>Fullner, K. J., Hatfull, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacteriophage+L5+Infection+of+Mycobacterium+bovis+BCG:+Implications+for+Phage+Genetics+in+the+Slow-growing+Mycobacteria.">Mycobacteriophage L5 Infection of Mycobacterium bovis BCG: Implications for Phage Genetics in the Slow-growing Mycobacteria.</a> Mol Microbiol. 26 (4), 755-766 (1997).</li><li>Robb, S. M., Woods, D. R., Robb, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+Growth+Characteristics+on+Stationary+Phase+Achromobacter+cells.">Phage Growth Characteristics on Stationary Phase Achromobacter cells.</a> J Gen Virol. 41 (2), 265-272 (1978).</li><li>Bolger-Munro, M., Cheung, K., Fang, A., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T4+Bacteriophage+Average+Burst+Size+Varies+with+Escherichia+coli+B23+Cell+Culture+Age.">T4 Bacteriophage Average Burst Size Varies with Escherichia coli B23 Cell Culture Age.</a> Journal of Experimental Microbiology and Immunology. 17, 115-119 (2013).</li></ol>

Authors declare no competing financial interests.

Despite many previous attempts to isolate phages capable of infecting Arthrobacter hosts, we had little success using standard enrichment procedures. The generalized method of bacterial enrichment developed and adapted by van Twest and Kropinski10 to enrich phages from environmental samples remains the basis for the majority of enrichment procedures. Evidence from previous studies suggests that methods of direct plating have produced detectable plaques on strains of Arthrobacter albeit with very low success rates of isolation5. With the direct plating method, phages are extracted from soil samples in a phage buffer and filtered to remove contaminating bacteria. The filtered extract containing phage particles are added to the desired bacteria host to allow host cell infection and plated without additional rounds of bacteria growth and phage amplification.
Our enrichment technique is robust and optimized for the relatively easy isolation of Arthrobacter phages from Arthrobactersp. KY3901. With traditional enrichment procedures, phages, along with many different types of bacteria present in the soil samples, are extracted from soil in phage buffer and added to fresh growth media with desired bacterial cells used as the host for phage infection. With the enrichment method, all soil bacteria are removed from the soil extracted phage samples by filtration through a 0.22 &#181;M filter. The sterile filtered lysate containing phage particles, but not soil bacteria, are diluted in 2x LB broth with the desired bacteria used as the phage propagation host. The main difference between our enrichment procedure and others is that there are NO contaminating soil bacteria competing for growth with the host bacteria during the enrichment incubation time period. The only host the soil extracted phage can infect is that of the desired host bacteria and therefore maximizes the growth potential of the host bacteria and amplification of the desired host-specific phages.
In the development of the procedure described here we attempted to account for selective bias inherent in any enrichment procedure by testing a range of CaCl2 concentrations11. What became apparent is that some phage isolates show physiological differences dependent on parameters like calcium ion concentration and temperature. For future Arthrobacter phage isolations we plan on testing key factors such as like pH, temperature, salinity, nutrients, divalent metal ions to isolate phages capable of reproducing in their hosts optimally under various environmental conditions.
We use freshly grown Arthrobacter cells in this experiment. We do not use cells that reach middle to late stationary phase of growth since we have had difficulty isolating phage or having decent titers with known phages at this growth period. Others have shown an inhibition of phage infection in stationary phase Achromobacter cells12 which may explain the lack of success of isolating phages using bacterial cells in middle to late stationary phase of growth. We have had success using cultures for these experiments with a cell density anywhere from an OD600 of 0.5 to 0.9. Grown cultures are stored at 4 &#176;C and can be used for phage isolation up to seven days. However, the longer the Arthrobacter cells are stored at 4 &#176;C the lower the titer and burst size of infectious phage particles. Similar results were found with using Escherichia coli for the infection of phageT413. Therefore using bacterial cells as soon as possible after they reach the desired growth maximizes chances of obtaining a novel phage from a soil sample.
Broadly speaking, the use of this enrichment technique with LB media should prove to be hardly exclusive to the isolation of Arthrobacter phages. While LB has been the industry standard for culturing E.&#160;coli strains and other members of the Enterobacteriaceae, it supports the growth of a wide variety of bacteria in aerobic conditions14. The nutrient rich composition of LB media can likely be used in this method to propagate phages in an incredibly diverse group of aerobic bacteria preventing technical challenges from hindering phage discovery.

The ubiquity of Arthrobacter species in soil environments offers a vast number and diversity of phages capable of being isolated from this species of host bacteria. Bacterial members of the Acintobacteriaceae family are most notable for their catabolic pathways of degrading recalcitrant compounds like atrazine and various other pesticides and herbicides1,2,3. Though most research has been done using environmental strains of Arthrobacter, clinical isolates of this genus is found in blood, urine, eyes, and many other human sources all displaying phylogenetic heterogeneity4.
While there is a rather extensive body of research on Arthrobacter bacteria, only a few studies report on the phages capable of infecting members of this diverse genus. Interestingly though, work done previously on Arthrobacter phages touches on several key distinct topics such as the typing of soil Arthrobacter species5, industrial uses with the purpose of reducing deleterious foam in activated sludge treatment plants 6, and work highlighting site specific recombination and integrase genes7.
Various enrichment technique protocols have been employed to generate pure phage isolates in Arthrobacter species. Early procedures include incubations of soil with added toxic agents like nicotine salts for periods of over one year8 giving rise to phages capable of only infecting A. globiformis. Studies done using soil percolated with labile organics appeared to produce detectable phages via plaque assay techniques, omitting lengthy incubation periods8. Interestingly though, a technique resembling direct plating was used in the past giving rise to several phages while still having a notably low success rate by the investigators5, citing past studies with low success rates8.
Overall, the isolation techniques used in the past were notable for having little efficacy in practice despite the Arthrobacter genus representing the most common aerobic soil isolate in nature4,9,,Van Twest and Kropinski10 present enrichment methods for isolating phages from water and soil adapted from earlier techniques used to enrich environmental bacterial isolates but these enrichment techniques proved inefficient in isolating Arthrobacter phages. The purpose of the method described here is to show &#8220;proof of concept&#8221; that the early enrichment methods can be adapted to consistently and effectively isolate Arthrobacter phages, overcoming previous technical challenges associated with isolating phages from this bacterial genus.

<table><tbody><tr><td>LB Broth powder</td><td>Fisher</td><td>BP9722-2</td><td>It&#039;s best to order these in bulk.</td></tr><tr><td>Granulated Agar</td><td>Fisher</td><td>BP1423-2</td><td>It&#039;s best to order these in bulk.</td></tr><tr><td>0.22 um syringe filters</td><td>Fisher</td><td>09-719A</td><td></td></tr><tr><td>0.22 um buchner filters</td><td>Fisher</td><td>430320</td><td>More than 50 ml of liquid can be obtained by carefully swapping the receiving tube.</td></tr><tr><td>Eppendorf Tubes</td><td>Fisher</td><td>05-408-129</td><td></td></tr><tr><td>5 ml pipets individual</td><td>Fisher</td><td>13-678-11D</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Fisher</td><td>76002844</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Fisher</td><td>76002845</td><td></td></tr><tr><td>10 ml pipets individual</td><td>Fisher</td><td>13-676-10J</td><td></td></tr><tr><td>25 ml pipets individual</td><td>Fisher</td><td>13-676-10K</td><td></td></tr><tr><td>Whatman qualitative filter paper</td><td>Fisher</td><td>1001-824</td><td></td></tr></tbody></table>

an optimized enrichment technique for the isolation of arthrobacter bacteriophage species from soil sample isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

To demonstrate reproducibility of the improved enrichment technique for Arthrobacter phages,&#160;30 different soil samples were used at different times and locations during the spring and summer of 2014. Of these 30 soil samples unique Arthrobacter phages were obtained from 22 of collected soil samples using this enrichment procedure. The standard enrichment procedure yielded unique phages from 3 of the same soil samples. The enrichment samples can have a very high phage titer needing initial dilution to isolate plaques or a relatively low phage titer requiring a larger volume of enriched Arthrobacter for successful detection of plaques. We use the plaque streak method to isolate pure phage populations after initial enrichment and plating on LB agar (Figure 2). Alternatively, a traditional standard plaque titer assay may be used to isolate a pure phage isolate though this method takes more time, reagents, and materials. As mentioned earlier, we do use the plaque titer assay to empirically determine phage titer numbers and the number of plaque forming units (PFUs) to form a webbing pattern on a plate. Figure 3 depicts a set of serial dilutions for phage Banana and demonstrates for one of the plates what a good web pattern should look like. Figure 4 depicts electron micrographic images of 20 Arthrobacter phages isolated using the enrichment technique. Of the 25 phages currently isolated, 23 of them are siphoviruses with rather long tails. Two of the isolated phages are myoviruses containing head diameters similar in length to that of their tails.
<img alt="Figure 1" src="/files/ftp_upload/52781/52781fig1.jpg" /> 
Figure 1.&#160;Streak plate technique schematic used for the isolation of individual phage plaques. The yellow star represents the area on the plate that the LB top agar plus bacteria solution should be carefully poured onto the plate after the phage plaque streaking has been completed.
<img alt="Figure 2" src="/files/ftp_upload/52781/52781fig2.jpg" /> 
Figure 2.&#160;Examples of the streak plate technique used for the isolation of plaques for one phage (Dylan) at three different temperatures. Note that phage Dylan produces similar plaques at RT, 30 &#176;C and 37 &#176;C. The red circles on two of the plates surround clearly isolated plaques.
<img alt="Figure 3" src="/files/ftp_upload/52781/52781fig3.jpg" /> 
Figure 3.&#160;Plaque titer assay. Arthrobacter phage Banana was serial diluted in PB and plated on agar plates as described in sections 4.6 &#38; 4.7 of the Protocol Text. Shown are serial dilutions T-4 to T-8 (from the top). Note that the T-5 dilution (from bottom to top) gave a web pattern with just a small amount of the bacterial lawn still remaining. Lower serial dilutions (T-1 to T-3) created clear plates with all of the bacteria lysed by phage particles demonstrated by the lack of any bacterial growth.
<img alt="Figure 4" src="/files/ftp_upload/52781/52781fig4.jpg" /> 
Figure 4.&#160;Electron micrographic images of 20 Arthrobacter phages isolated using the enrichment technique. The images were taken using a FEI Morgagni TEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52781/52781fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates at JoVE.com

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

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Nanyang Technological University

Research

JoVE Journal

Environment

19.4K Views.  University of the Sciences. We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

Video: An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

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 ...

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

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

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

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

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Biology

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Efficient Technique for Assessing Environmental AMR by Enriching Low-MW DNA

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water,&#160;isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.

Author Spotlight: Understanding and Detecting Environmental Antimicrobial Resistance by Combining Culture-Based Techniques and Genomics

Here, we present a simple technique to assess environmental antimicrobial resistance (AMR) by enhancing the proportion of low-molecular-weight extracellular DNA. Prior treatment with 20%-30% PEG and 1.2 M NaCl allows detection of both genomic and horizontally transferred AMR genes. The protocol lends itself to a kit-free process with additional optimization.

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has ...

Aseptic Technique in Environmental Science

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona 
Demonstrating Author: Luisa Ikner
Aseptic technique is a fundamental skill widely practiced in the field of environmental microbiology that requires a balance of mindfulness and practice in the laboratory. Proper use of this technique reduces the likelihood of bacterial or fungal contamination of reagents, culture media, and environmental samples. Aseptic technique is also vital to ensure data integrity and maintain the purity of culture libraries that may be comprised of very rare and difficult to culture isolates. Sources of contamination in the laboratory environment include airborne microorganisms (including those adhering to dust and lint particles), microbes present on the laboratory bench workspace or on unsterilized glassware or equipment, and microbes transferred from the body and hair of the researcher. The use of aseptic technique is also a safety measure that lowers the potential for the transmission of microorganisms to researchers, which is particularly important when working with pathogens.

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
Aseptic technique is a ...

Education

JoVE Science Education

Environmental Sciences

Environmental 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 ...

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

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