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 st...

<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 v...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>LB Broth powder</td>
			<td>Fisher</td>
			<td>BP9722-2</td>
			<td>It&#39;s best to order these in bulk.</td>
		</tr>
		<tr>
			<td>Granulated Agar</td>
			<td>Fisher</td>
			<td>BP1423-2</td>
			<td>It&#39;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>.22 um buchner filters</td>
			<td>Fisher</td>
			<td align="right">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 align="right">76002844</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Fisher</td>
			<td align="right">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 ...

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 ...

an-optimized-enrichment-technique-for-isolation-arthrobacter

Research

JoVE Journal

Environment

19.2K Görüntüleme.  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.