Le financement pour le développement de ce protocole a été fourni par le Southeastern Pennsylvania Consortium pour l&#39;enseignement supérieur et le ministère de la Science Cabrini College. Financement et de soutien supplémentaires sont venus de l&#39;Université Arcadia et l&#39;Université Immaculata. Nous remercions tout particulièrement le Dr Karen Snetselaar à l&#39;Université Saint-Joseph pour prendre de bien vouloir les images microscope électronique de nos phages isolés. Un soutien supplémentaire a été fourni par les chasseurs Howard Hughes Medical Institute Science Education Alliance phages Advancing génomique et le programme (SEA-PHAGES) évolutive Science.

1. Préparation des cellules pour Arthobacter Phage Isolement <ul><li> Culture Arthrobacter sp. KY3901 colonies striées sur une Luria Bertaini (LB) plaque de gélose incubées à 30 ° C pendant 2-3 jours. Choisissez une colonie et utiliser une boucle stérile pour l&#39;ajouter à 250 ml de bouillon LB dans un flacon de culture perplexe et incuber dans un incubateur sous agitation à 225 tours par minute à 30 ° C. </li><li> Laisser environ 24 h de la croissance pour obtenir fin des cellules en phase stationnaire exponentielles / début pour des expériences d&#39;infection de phage. Surveiller l&#39;état de la croissance bactérienne étroitement pour empêcher les cellules de pénétrer dans le milieu à la fin l&#39;état stationnaire de croissance. NOTE: La croissance cellulaire devrait être composé d&#39;une densité optique OD 600 de 0,5-.0.7 pour les procédures d&#39;isolement et de purification de phages / étapes décrites ci-dessous. </li></ul> 2. Collecte des phages d&#39;échantillons de sol <ol><li> Rassemblez 200-400 g de sol de l&#39;emplacement souhaité. Remarque: Comme les bactéries dans le Arthrobagenre Cter sont des bactéries du sol et les communes qu&#39;ils phages qui les infectent peuvent être trouvés dans et sont omniprésents dans la plupart des types de sol. </li><li> Ajouter au moins 400 ml de tampon de phage (PB) [mM de NaCl 68 mM MgSO4 10, 10 mM de Tris-Cl (pH 7,5)] pour le sol et les mélanger dans un grand ballon suffisamment pour qu&#39;au moins 200 ml de PB est dans le surnageant et est apte à être extrait. </li><li> Mélanger par agitation ou remuant doucement jusqu&#39;à ce que le sol est suspendu et laisser les sédiments du sol pour se installer, généralement 30 min. </li><li> Passez le &quot;extrait de sol&quot; à travers un papier filtre par filtration par gravité pour éliminer les débris sédiments du sol. </li><li> Ajouter la poudre LB et mélanger pour obtenir une solution à 2% (4 g de poudre LB dans 200 ml d&#39;extrait sol). </li><li> Filtrer la solution à travers un filtre de 0,22 um par filtration sous vide pour obtenir un filtrat stérile. Si possible, passez à l&#39;étape 2.7 immédiatement. Si besoin est, magasin filtré échantillons à 4 ° C pendant jusqu&#39;à une semaine avant de continuer, cependant, le nombre de particules de phages viablessera réduite. </li><li> Aliquote multiples portions de 50 ml du mélange d&#39;extrait LB / sol stérilisée par filtration dans individuels de 250 ml stérilisés secouant flacons de culture dérouté aide de techniques aseptiques. Ajouter stérile 2,0 M CaCl 2 solution aux concentrations finales souhaitées (0-50 mm) depuis différents phages Arthrobacter croissent de manière optimale dans différentes conditions de CaCl 2. REMARQUE: On constate que la majorité des phages Arthrobacter sont identifiés par nous dans la plage de 1 mM à 2,5 mM, CaCl 2. Cependant, plusieurs des phages isolés ont augmenté par nous exclusivement à 0 mM CaCl2 .ou à de très hautes concentrations de CaCl 2. Procéder immédiatement à l&#39;étape 3.1. </li></ol> 3. Isolation Phage <ol><li> Ajouter 1 à 2,5 ml de retard fixe culture de bactéries en phase exponentielle / début de chacun des flacons. Pour une DO 600 de 0,5 à 0,7, utiliser 1 ml de cellules. Pour une DO 600 supérieure à une DO de 0,7 utiliser 2,5 ml decellules. REMARQUE: Les cellules en phase de croissance exponentielle donnent généralement plus de phages et des titres plus élevés que les cellules de la croissance de la phase stationnaire. </li><li> Secouez flacons à 250 tours par minute à 30 ° C pendant environ 24 heures dans un incubateur à agitation. </li><li> Après une période d&#39;incubation de 24 h retirer flacons enrichissement de l&#39;incubateur secouant et diluer les échantillons d&#39;enrichissement de dix fois dans le tampon de phage. </li><li> Mettre en place des tubes de culture avec 0,5 ml de retard fixe culture de bactéries en phase exponentielle / début et ajouter diverses quantités de la culture d&#39;enrichissement (5 ul à 500 ul d&#39;une dilution à 10 -1 PB) aux tubes de culture. NOTE: Il est conseillé de tester une large gamme de concentrations puisque chaque échantillon de sol génère différents titres de phages après enrichissement. </li><li> Ajouter CaCl 2 pour des tubes de culture pour rendre une concentration finale égale à la concentration présente dans le ballon de l&#39;enrichissement initial. Utilisez une gamme de différents CaCl 2 concentrations de sélectionner pour nombreux phages wCaCl 2 ième variable dépendances. REMARQUE: La plupart des phages seront isolés dans la plage de 1 à 2,5 CaCl 2 mm, mais il ya des phages isolés plus optimale partout 0-50 mM CaCl 2 concentrations. Pour cette raison, il est préférable de vérifier une gamme de CaCl 2 concentrations de maximiser le nombre de phages uniques Arthrobacter isolés. </li><li> Mélanger 4,5 ml d&#39;agar LB dans le tube de culture et versez le mélange sur une plaque de gélose LB. Agiter doucement pour répartir uniformément sur la plaque et permettre top agar se solidifier pendant environ 15 min. REMARQUE: Top agar peut être préparé en lots plus importants (250 ml) et top agar supplémentaire peut être conservé à température ambiante et réutilisé pour des expériences ultérieures pendant au moins deux semaines. Bien qu&#39;il existe différentes formules utilisées pour faire agar dessus, nous faisons top agar en utilisant 7g / L d&#39;agar mélangé avec LB. agar Placez dans un bain-marie à 60 ° pour conserver l&#39;état liquide au cours d&#39;une expérience. </li><li> Inverser til plaque et incuber à la température souhaitée (température à 30 ° C) O / N à 48 h. Vary ces conditions pour optimiser les phages présents. Phages plus isolées proviennent de plaques ont été incubées à 30 ° C à 1 à 2,5 mM CaCl concentration 2 après une période d&#39;incubation de 24 h. </li></ol> 4. Phage Purification <ol><li> Après un contrôle d&#39;incubation de 24 heures pour la formation de plaque sur des plaques. Continuer incubation jusqu&#39;à 72 heures si aucune plaques sont évidentes ou pour déterminer si des plaques supplémentaires apparaîtront. Si nécessaire, des plaques de magasins avec plaques putatifs pour jusqu&#39;à une semaine à 4 ° C avant de procéder. NOTE: Il ne est pas rare de trouver une variété de différentes morphologies de la plaque sur une plaque. Trouver plaques après 3 jours d&#39;incubation est possible, mais rare. </li><li> Purifier phages souhaités de plaques clairement isolé en utilisant la méthode de la plaque de série. Touchez une plaque avec un bâton en bois stérile. Streak la plaque en exécutant un bâton en bois à travers une flamme, allowing le bâton pour refroidir brièvement, puis frotter le bâton sur la plaque de gélose dans le mouvement doux comme le montre la figure 1. Répétez cette aide d&#39;un bâton propre pour chaque passage. </li><li> Vous pouvez également utiliser le dosage traditionnelle plaque de titre pour isoler plaques de phages individuels. La plaque de dosage titre exige l&#39;utilisation de plusieurs réactifs tels que l&#39;agar LB et LB agar supérieur et des matériaux tels que des boîtes de Pétri pour l&#39;isolement de la plaque de phage. Nous avons eu beaucoup de succès en utilisant la méthode de la plaque de série, mais il est techniquement plus facile à isoler plaques individuelles en utilisant le test de la plaque titre. </li><li> Une fois que la plaque de gélose LB est striée au moins trois fois, marquer la zone avec la plus faible concentration de particules de phage putatifs et appliquer doucement 0,5 ml d&#39;Arthrobacter et 4,5 ml de top agar fondu LB (contenant la même concentration de chlorure de calcium comme la plaque mère ) et lui permettre de se répandre uniformément sur la plaque. </li><li> Inversez et incuber la plaque O / N. Répétez la plaque de sérietechnique pendant au moins trois itérations pour assurer une espèce de phage pur est isolé. plaques de conserver à 4 ° C pour jusqu&#39;à une semaine selon les besoins entre les stries sans perte significative de la viabilité du phage. </li><li> Une fois plaques souhaités ont été cultivées et isolé sur la plaque de série finale, touchez une plaque bien isolée avec une pointe de micropipette et remettre en suspension dans 100 pi de PB. Série diluer cette solution à 10 0 à la dilution 10 -8 en passant 20 pi de l&#39;échantillon dans 180 pl de PB frais. </li><li> Ajouter 10 ul de chaque dilution de 0,5 ml de cultivé Arthrobacter dans un tube de culture pendant 5 à 10 min à la température ambiante et la plaque en mélangeant les bactéries infectées avec 4,5 ml de top agar LB fondu contenant la même concentration de CaCl 2 utilisé pour isoler les phages de l&#39;échantillon de sol d&#39;origine. Enregistrer toutes les dilutions faites à 4 ° C jusqu&#39;au lendemain. </li><li> Déterminer la dilution en série de phage nécessaire pour faire un modèle Web en utilisant le titre de plaque traditionnelledire. Le but de l&#39;essai de plaque de titrage est de déterminer le titre de phage nécessaire pour obtenir une plaque-modèle Web. Identifier une plaque de motif de forme Web comme la plupart du temps mais dépourvu de bactéries contenant des restes de bactéries qui ne ont pas encore été lysées par les phages. Ajouter 5 ml de la culture exponentielle / début fin Arthrobacter fixe à une culture de 50 ml flacon stérile. <ol><li> Ajouter 100 ul de la dilution (qui a donné un motif de bande de la culture de Arthrobacter) dans le flacon de culture et de permettre aux phages pour infecter les cellules bactériennes pendant 5 à 10 min à température ambiante. Mélanger avec le dessus de la gélose LB fondu contenant la même concentration de CaCl 2 comme initialement utilisé pour identifier le phage et la plaque 5 ml de ce mélange sur 10 plaques LB frais. Laisser solidifier et incuber les plaques inversées à 30 ° C. </li></ol></li><li> Le lendemain, inonder les plaques modèles Web avec 5 ml de PB et stocker O / N à 4 ° C ou 4 heures à température ambiante. </li><li> Filtrer le lysat de phage à travers un filtre à seringue de 0,22 umet stocker à 4 ° C. Utilisez ce phage finale stock pour des expériences supplémentaires tels que des images de microscopie électronique de particules de phage, phage isolement de l&#39;ADN génomique pour l&#39;analyse de restriction enzyme ADN et le séquençage génomique en utilisant des procédures standard. Pour le stockage à long terme du stock de phages, ajouter un montant égal du lysat de phage glycérol stérile dans de petites fioles pour le stock archivage à -80 ° C. NOTE: Pour ceux qui ne expérience avec ces techniques de phage, une grande ressource est le site de www.phagesdb.org accessible en ligne. </li></ol>

<ol>
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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. 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Auteurs déclarent aucun intérêt financier concurrentes.

Malgré de nombreuses tentatives antérieures afin d&#39;isoler les phages capables d&#39;infecter des hôtes Arthrobacter, nous avons eu peu de succès en utilisant des procédures d&#39;enrichissement standard. La méthode généralisée de l&#39;enrichissement bactérienne développé et adapté par van Twest et Kropinski 10 pour enrichir phages partir d&#39;échantillons environnementaux demeure la base de la majorité des procédures d&#39;enrichissement. Les résultats d&#39;études précédent...

L&#39;omniprésence des espèces Arthrobacter dans des environnements de sol offre un grand nombre et la diversité des phages susceptibles d&#39;être isolé de cette espèce de bactéries hôtes. Bactériennes membres de la famille Acintobacteriaceae sont les plus notables pour leurs voies cataboliques de dégrader des composés récalcitrants comme l&#39;atrazine et d&#39;autres pesticides et herbicides 1,2,3. Bien que la plupart des recherches ont été faites en utilisant des souches environnementales de Arthrobacter, isolats cliniques de ce genre se trouve dans le sang, l&#39;urine, les yeux, et de nombreuses autres sources humaines tout une hétérogénéité phylogénétique 4. Bien qu&#39;il existe un corps plutôt vaste de la recherche sur les bactéries Arthrobacter, seules quelques études signalent sur ​​les phages capables d&#39;infecter les membres de ce genre diversifié. Fait intéressant cependant, le travail fait précédemment sur ​​Arthrobacter phages touches sur plusieurs sujets distincts clés tels que le typage du sol &lt;em&gt; espèces Arthrobacter 5, les utilisations industrielles dans le but de réduire la mousse délétère dans les usines de traitement des boues activées 6, et le travail en soulignant recombinaison spécifique de site et les gènes de l&#39;intégrase 7. Divers protocoles de technique d&#39;enrichissement ont été employées pour générer des isolats de phage pur dans les espèces Arthrobacter. Les premières procédures comprennent incubations de sol avec des agents toxiques ajoutés comme les sels de nicotine pour des périodes de plus d&#39;un an donnant lieu à 8 phages capables de ne infecter A. globiformis. Des études effectuées en utilisant le sol percole avec des matières organiques labiles semblent produire des phages détectables par des techniques d&#39;analyse plaque, en omettant longues périodes d&#39;incubation 8. Fait intéressant cependant, une technique qui ressemble à ensemencement direct a été utilisé dans le passé donnant lieu à plusieurs phages tout en ayant un taux de réussite particulièrement faible par les enquêteurs 5, citant des études passées avec faibles taux de réussite <sjusqu&#39;à&gt; 8. Dans l&#39;ensemble, les techniques d&#39;isolation utilisés dans le passé ont été remarquables pour avoir peu d&#39;efficacité dans la pratique malgré les genre Arthrobacter représentant le sol aérobie la plus courante isoler dans la nature 4,9, Van Twest et Kropinski 10 méthodes d&#39;enrichissement présents pour isoler les phages de l&#39;eau et du sol adapté de techniques antérieures utilisées pour enrichir isolats bactériens environnementaux, mais ces techniques d&#39;enrichissement se est avéré inefficace à isoler phages Arthrobacter. Le but de la méthode décrite ici est de montrer &quot;preuve de concept&quot; que les méthodes d&#39;enrichissement début peuvent être adaptés pour isoler cohérente et efficace phages Arthrobacter, surmonter les défis techniques associés à isoler précédentes phages de ce genre bactérien.

<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.

Pour démontrer la reproductibilité de la technique d&#39;enrichissement amélioré pour phages Arthrobacter, 30 échantillons de sol différents ont été utilisés à différents moments et les lieux au cours du printemps et de l&#39;été 2014. Parmi ces échantillons 30 de sol phages Arthrobacter uniques ont été obtenus à partir de 22 échantillons de sol prélevés en utilisant cet enrichissement procédure. La procédure d&#39;enrichissement donné norme phages uniques à partir de trois de c...

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.

Une technique d&#39;enrichissement Optimisé pour l&#39;isolement de<em&gt; Arthrobacter</em&gt; Bactériophage Espèces de l&#39;échantillon du sol Isole

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

19.3K vues.  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.

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

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

Conception et l&#39;exploitation d&#39;un continu<sup&gt; 13</sup&gt; C et<sup&gt; 15</sup&gt; N Chambre pour uniforme ou différentiel étiquetage, métabolique et structurale, usine de production d&#39;isotopes étiquetage

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

Recherche

Environnement

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

Conception et construction d&#39;une installation de recherche de ruissellement urbain

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

L&#39;échantillonnage du sol et isolement de nématodes entomopathogènes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

Un CO<sub&gt; 2</sub&gt; Facilité Concentration de Gradient pour les essais CO<sub&gt; 2</sub&gt; Enrichissement et du sol Effets sur l&#39;écosystème de prairie Fonction

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Isolement et analyse des communautés microbiennes dans le sol, rhizosphère et racines dans des expériences de graminée vivace

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Un protocole de Rhizobox optimisé pour visualiser la croissance racinaire et la réceptivité aux nutriments localisées

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Utilisation de Caenorhabditis elegans pour étudier les effets trans et multigénérationnels des substances toxiques

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

Conception de microplots et préparation d’échantillons de plantes et de sols pour 15analyses d’azote

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Electrostatic Method to Remove Particulate Organic Matter from Soil

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of roots and plant material from soil can result in highly variable carbon measurements. Methods to remove the plant material are often limited to the largest, most visible plant materials. In this manuscript we describe how electrostatic attraction can be used to remove plant material from a soil sample. An electrostatically charged surface passed close to dry soil naturally attracts both undecomposed and partially decomposed plant particles, along with a small quantity of mineral and aggregated soil. The soil sample is spread in a thin layer on a flat surface or a soil sieve. A plastic or glass Petri dish is electrostatically charged by rubbing with polystyrene foam or nylon or cotton cloth. The charged dish is passed repeatedly over the soil. The dish is then brushed clean and recharged. Re-spreading the soil and repeating the procedure eventually results in a diminishing yield of particulates. The process removes about 1 to 5% of the soil sample, and about 2 to 3 times that proportion in organic carbon. Like other particulate removal methods, the endpoint is arbitrary and not all free particulates are removed. The process takes approximately 5 min and does not require a chemical process as do density flotation methods. Electrostatic attraction consistently removes material with higher than average C concentration and C:N ratio, and much of the material can be visually identified as plant or faunal material under a microscope.

Removing recently deposited and incompletely decomposed plant material from soil samples reduces the influence of temporary seasonal inputs on soil organic carbon measurements. Attraction to an electrostatically charged surface can be used to quickly remove a substantial amount of particulate organic matter.

Procédé électrostatique pour éliminer les matières organiques particulaires du sol

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of ...

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a significant role in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. The cell wall is primarily composed of cellulose, lignin, hemicellulose and pectin. Recently plant cell walls have been targeted for the second-generation biofuel and bioenergy production. Specifically, the cellulose component of the plant cell wall is used for the production of cellulosic ethanol. Estimation of cellulose content of biomass is critical for fundamental and applied cell wall research. The Updegraff method is simple, robust, and the most widely used method for the estimation of crystalline cellulose content of plant biomass. The alcohol insoluble crude cell wall fraction upon treatment with Updegraff reagent eliminates the hemicellulose and lignin fractions. Later, the Updegraff reagent resistant cellulose fraction is subjected to sulfuric acid treatment to hydrolyze the cellulose homopolymer into monomeric glucose units. A regression line is developed using various concentrations of glucose and used to estimate the amount of the glucose released upon cellulose hydrolysis in the experimental samples. Finally, the cellulose content is estimated based on the amount of glucose monomers by colorimetric anthrone assay.

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

Estimation de la teneur en cellulose cristalline de la biomasse végétale à l’aide de la méthode Updegraff

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...