Nous tenons à remercier Ann Grimm et Michael Zimmerman, pour examen critique de ce manuscrit et Doug Hamilton pour son soutien technique.

<ol>
	<li>DiGiorgio, C. L., Gonzalez, D. A., Huitt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptosporidium+and+Giardia+recoveries+in+natural+waters+by+using+Environmental+Protection+Agency+Method+1623.">Cryptosporidium and Giardia recoveries in natural waters by using Environmental Protection Agency Method 1623.</a> Appl. Environ. Microbiol. 68, 5952 (2002).</li><li>Hill, V. R., Kahler, A. M., Jothikumar, N., Johnson, T. B., Hahn, D., Cromeans, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistate+evaluation+of+an+ultrafiltration-based+procedure+for+simultaneous+recovery+of+enteric+microbes+in+100-liter+tap+water+samples.">Multistate evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in 100-liter tap water samples.</a> Appl. Environ. Microbiol. 73, 4218-4225 (2007).</li><li>Hill, V. R., Polaczyk, A. L., Hahn, D., Narayanan, J., Cromeans, T. L., Roberts, J. M., Amburgey, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+rapid+method+for+simultaneous+recovery+of+diverse+microbes+in+drinking+water+by+ultrafiltration+with+sodium+polyphosphate+and+surfactants.">Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants.</a> Appl. Environ. Microbiol. 71, 6878-6884 (2005).</li><li>Hill, V. R., Polaczyk, A. L., Kahler, A. M., Cromeans, T. L., Hahn, D., Amburgey, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+hollow-fiber+ultrafiltration+to+the+USEPA+VIRADEL+technique+and+USEPA+method+1623.">Comparison of hollow-fiber ultrafiltration to the USEPA VIRADEL technique and USEPA method 1623.</a> J. Environ. Qual. 38, 822-825 (2009).</li><li>Holowecky, P. M., James, R. R., Lorch, D. P., Straka, S. E., Lindquist, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+ultrafiltration+cartridges+for+a+water+sampling+apparatus.">Evaluation of ultrafiltration cartridges for a water sampling apparatus.</a> J. Appl. Microbiol. 106, 738-7347 (2009).</li><li>Lindquist, H. D., Harris, S., Lucas, S., Hartzel, M., Riner, D., Rochele, P., Deleon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+ultrafiltration+to+concentrate+and+detect+Bacillus+anthracis,+Bacillus+atrophaeus+subspecies+globigii,+and+Cryptosporidium+parvum+in+100-liter+water+samples.">Using ultrafiltration to concentrate and detect Bacillus anthracis, Bacillus atrophaeus subspecies globigii, and Cryptosporidium parvum in 100-liter water samples.</a> J. Microbiol. Methods. 70, 484-492 (2007).</li><li>Polaczyk, A. L., Roberts, J. M., Hill, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+1MDS+electropositive+microfilters+for+simultaneous+recovery+of+multiple+microbe+classes+from+tap+water.">Evaluation of 1MDS electropositive microfilters for simultaneous recovery of multiple microbe classes from tap water.</a> J. Microbiol. Methods. 68, 260-266 (2007).</li><li>Rhodes, E. R., Hamilton, D. W., See, M. J., Wymer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+hollow-fiber+ultrafiltration+primary+concentration+of+pathogens+and+secondary+concentration+of+viruses+from.">Evaluation of hollow-fiber ultrafiltration primary concentration of pathogens and secondary concentration of viruses from.</a> 176, 38-45 (2011).</li><li>Shaw, N. J., Villegas, L. F., Eldred, B. J., Gaynor, D. H., Warden, P. S., Pepich, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+to+EPA+Method+1623+to+address+a+unique+seasonal+matrix+effect+encountered+in+some+U.S.+source+waters.">Modification to EPA Method 1623 to address a unique seasonal matrix effect encountered in some U.S. source waters.</a> J. Microbiol. Methods. 75, 445-448 (2008).</li><li>Simmons, O. D., Sobsey, M. D., Heaney, C. D., Schaefer, F. W., Francy, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+detection+of+Cryptosporidium+oocysts+in+surface+water+samples+by+method+1622+using+ultrafiltration+and+capsule+filtration.">Concentration and detection of Cryptosporidium oocysts in surface water samples by method 1622 using ultrafiltration and capsule filtration.</a> Appl. Environ. Microbiol. 67, 1123-1127 (2001).</li><li>Sobsey, M. D., Glass, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+water+quality+on+enteric+virus+concentration+by+microporous+filter+methods.">Influence of water quality on enteric virus concentration by microporous filter methods.</a> Appl. Environ. Microbiol. 47, 956-9560 (1984).</li><li>USEPA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+1623:+Cryptosporidium+and+Giardia+in+Water+by+Filtration/IMS/FA.">Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA.</a> Office of Water. 815-R-05-002, (2005).</li><li>Ware, M. W., Wymer, L., Lindquist, H. D., Schaefer, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+alternative+IMS+dissociation+procedure+for+use+with+Method+1622:+detection+of+Cryptosporidium+in+water.">Evaluation of an alternative IMS dissociation procedure for use with Method 1622: detection of Cryptosporidium in water.</a> J. Microbiol. Methods. 55, 575-583 (2003).</li><li>Zuckerman, U., Tzipori, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portable+continuous+flow+centrifugation+and+method+1623+for+monitoring+of+waterborne+protozoa+from+large+volumes+of+various+water+matrices.">Portable continuous flow centrifugation and method 1623 for monitoring of waterborne protozoa from large volumes of various water matrices.</a> J. Appl. Microbiol. 100, 1220-1227 (2006).</li></ol>

L&#39;Agence américaine de protection de l&#39;environnement à travers son Bureau de la recherche et le développement en collaboration avec l&#39;Office des eaux souterraines et le Centre de l&#39;eau potable d&#39;assistance technique financé la recherche décrite ici. Tous les travaux ont été soutenus sur le site de l&#39;EPA des États-Unis, Cincinnati, Ohio. Bien que les informations décrites dans cet article a été financé en tout ou en partie par l&#39;Agence américaine de protection de l&#39;environnement sous contrat (contrat EP-C-06-031) à Shaw de l&#39;environnement et de l&#39;Infrastructure, Inc, il ne reflète pas nécessairement les vues de la Agence et aucune approbation officielle doit être déduite. Il a été soumis à examen de l&#39;Agence et approuvé pour publication.

Tangentielle débit à fibres creuses d&#39;ultrafiltration est une technique alternative et efficace pour la concentration initiale des oocystes de Cryptosporidium et des kystes de Giardia dans l&#39;eau. Fibres creuses d&#39;ultrafiltration est moins cher que les filtres traditionnels. Depuis il a la capacité de se concentrer des oocystes de Cryptosporidium et des kystes de Giardia à partir d&#39;une variété de matrices d&#39;eau différents, il est une alternative utile aux techniques de filtration actuelles utilisées pour la méthode EPA 1623. Comme avec la plupart des autres méthodes de filtration, d&#39;ultrafiltration à fibres creuses est sujette à l&#39;encrassement des échantillons très turbides. Eau à haute pression qui découlerait de l&#39;encrassement du filtre, c&#39;est pourquoi il est recommandé de surveiller la pression pendant la course de filtration. En plus de oocystes de Cryptosporidium et des kystes de Giardia, fibres creuses d&#39;ultrafiltration a été montré qu&#39;il était capable de se concentrer les bactéries et les virus 1-3,5,8. Fibres creuses d&#39;ultrafiltration outlined dans cette méthode peut être utilisée pour concentrer des organismes multiples dans un seul échantillon. Il est à noter que l&#39;obtention d&#39;un volume final compris entre 200 et 250 ml est l&#39;étape finale critique dans la procédure de concentration, afin que les étapes de centrifugation supplémentaires, qui peuvent entraîner une perte de kyste (oo), sont évités (étape 2.2). Cependant, ce qui permet le volume dans la bouteille d&#39;abandonner trop faible peut avoir des effets défavorables sur les recouvrements car il ne sera pas un volume suffisant de liquide de forcer tous les oocystes ou des kystes dans la bouteille rétentat. Par conséquent, il est recommandé de maintenir un volume final compris entre 200 et 250 ml. La chaleur de dissociation est une alternative à l&#39;étape de dissociation acide dans la méthode 1623. Cette autre étape a été montré pour améliorer la récupération des oocystes de Cryptosporidium et de réduire la variation méthode lorsqu&#39;il est isolé de la rivière soit ou 9 eau à réactif. Une comparaison côte à côte de l&#39;acide et la chaleur de dissociation démontré que les méthodes utilisant la chaleur pour dissociationte les organismes des billes immunomagnétiques produit des recouvrements plus élevés pour les deux moyennes Cryptosporidium et Giardia. En outre, la précision des recouvrements Cryptosporidium et Giardia était meilleure dans les échantillons traités à la chaleur de dissociation par rapport à la dissociation acide 9. L&#39;incorporation de HFUF que l&#39;étape de concentration permet une plus grande souplesse en offrant la possibilité de se concentrer de multiples organismes. En outre, il est une alternative moins coûteuse à la méthode de filtration options actuelles de 1623.

<table border="1"><tbody><tr><td> Equipement / Réactif </td><td> Vendeur </td><td> Catalogue # </td></tr><tr><td> Asahi Kasei Rexeed 25 S / R humides à fibres creuses ultrafiltres </td><td> Composez médicale </td><td> REXEED25S / R </td></tr><tr><td> I / P 73 (Masterflex R-3603), ou équivalent </td><td> Cole Parmer </td><td> EW-06408-73 </td></tr><tr><td> L / S 24 (Masterflex platinum), ou équivalent </td><td> Cole Parmer </td><td> EW-96410-24 </td></tr><tr><td> L / S 15 (Masterflex platinum), ou équivalent </td><td> Cole Parmer </td><td> EW-96410-15 </td></tr><tr><td> L / S 36 (Masterflex platinum), ou équivalent </td><td> Cole Parmer </td><td> EW-96410-36 </td></tr><tr><td> I / P Entraînement Brushless de précision </td><td> Cole Parmer </td><td> EW-77410-10 </td></tr><tr><td> I / P Chef Facile pompe de charge </td><td> Cole Parmer </td><td> EW-77601-10 </td></tr><tr><td> Noir PEHD Tee, 1/4 &quot;x 3/8&quot; x 3/8 &quot; </td><td> Plastiques US </td><td> 62064 </td></tr><tr><td> Masterflex connecteur en T L / S 15-25 </td><td> Cole Parmer </td><td> Ex.-30613-12 </td></tr><tr><td> Nalgene lourde bouteille pp L 1 </td><td> Cole Parmer </td><td> EW-06257-10 </td></tr><tr><td> 10 ml Pipettes </td><td> Fisher Scientific </td><td> 13-678-11C </td></tr><tr><td> Nalgene remplissage / purge bouchon pour 1/4 &quot;tube, 53B </td><td> Cole Parmer </td><td> EW-06258-10 </td></tr><tr><td> Manomètre </td><td> Cole Parmer </td><td> A-680-46-10 </td></tr><tr><td> Raccord droit, NPT (F), 1/4 &quot; </td><td> Cole Parmer </td><td> EW-06469-18 </td></tr><tr><td> TNP branches de tés, pp naturelle </td><td> Cole Parmer </td><td> A-30610-75 </td></tr><tr><td> Attaches de pincement, 1/2 &quot; </td><td> Cole Parmer </td><td> EW-06833-00 </td></tr><tr><td> Custom ajustement adaptateurs DIN </td><td> Moulé Products Corp </td><td> MPC-855NS.250 </td></tr><tr><td> Anneau support </td><td> Fisher Scientific </td><td> 14-670b </td></tr><tr><td> Pinces support annulaire </td><td> Fisher Scientific </td><td> 05-769-6Q </td></tr><tr><td> Keck rampe de serrage, 14mm </td><td> Cole Parmer </td><td> EW-06835-10 </td></tr><tr><td> Polyphosphate de sodium </td><td> Sigma-Aldrich </td><td> 305553 </td></tr><tr><td> Thiosulfate de sodium pentahydraté </td><td> Sigma-Aldrich </td><td> 72050 </td></tr><tr><td> Antimousse Y-30 émulsion </td><td> Sigma-Aldrich </td><td> A5758 </td></tr><tr><td> Tween-80 </td><td> Sigma-Aldrich </td><td> P1754 </td></tr><tr><td> 10 L cubitainer pliable en polyéthylène haute densité </td><td> VWR </td><td> IR314-0025 </td></tr><tr><td> Centrifuger porte-bouteilles </td><td> Fisher Scientific </td><td> 05-663-103 </td></tr><tr><td> 250 ml tubes à centrifuger coniques </td><td> Corning </td><td> 430776 </td></tr><tr><td> Entonnoir jetable </td><td> Cole Parmer </td><td> U 6122-10 </td></tr><tr><td> Pissette </td><td> Cole Parmer </td><td> U-06252-40 </td></tr><tr><td> Centrifuger </td><td> Beckman Coulter </td><td> Allegra X-15R </td></tr><tr><td> Rotor swing-out </td><td> Beckman Coulter </td><td> ARIES SX4750 </td></tr><tr><td> Centrifuger adaptateurs baquets pour les tubes de 250 ml coniques </td><td> Beckman Coulter </td><td> 349849 </td></tr><tr><td> 200 ul de gros calibre des pointes de pipette </td><td> Fisher Scientific </td><td> 02-707-134 </td></tr><tr><td> VacuShield filtre </td><td> Gelman </td><td> 629-4402 </td></tr><tr><td> 5 pipettes ml </td><td> Fisher Scientific </td><td> 13-678-11D </td></tr><tr><td> Dynabeads: Cryptosporidium / Giardia combo kit </td><td> JeDexx </td><td> 73002 </td></tr><tr><td> Tubes de 50 ml à centrifuger coniques </td><td> Faucon </td><td> 352098 </td></tr><tr><td> Dynal L10 plats tubes face </td><td> IDEXX </td><td> 74003 </td></tr><tr><td> Minuteur </td><td> VWR </td><td> 23609-202 </td></tr><tr><td> Dynal MPC-6 aimant </td><td> IDEXX </td><td> 12002D </td></tr><tr><td> 1 ml Pipettes </td><td> VWR </td><td> 53283-700 </td></tr><tr><td> 1,5 ml à faible adhérence microtubes </td><td> Fisher Scientific </td><td> 02-681-320 </td></tr><tr><td> 1000 pipette ul &amp; conseils barrière correspondantes </td><td> Gilson </td><td> P1000/DF1000ST </td></tr><tr><td> 100 pipette ul &amp; conseils barrière correspondantes </td><td> Gilson </td><td> P100/DF100ST </td></tr><tr><td> 9 pouces pipettes Pasteur </td><td> VWR </td><td> 14672-412 </td></tr><tr><td> Dynal MPC-S aimant </td><td> IDEXX</td><td> 12020D </td></tr><tr><td> Vortex </td><td> VWR </td><td> 14216-188 </td></tr><tr><td> Mélangeur rotateurs Dynabeads </td><td> IDEXX </td><td> 94701 </td></tr><tr><td> Bloc thermique </td><td> Fisher Scientific </td><td> 11-718-2 </td></tr><tr><td> Perles Armure de laboratoire </td><td> Lab Armure </td><td> 42370-750 </td></tr><tr><td> Thermomètre digital </td><td> Fisher Scientific </td><td> 15-077-60 </td></tr><tr><td> Phosphate-tampon salin 1X pH 7,4 (PBS 1X) </td><td> Sigma </td><td> P4417 </td></tr><tr><td> Diapositives seul endroit </td><td> IDEXX </td><td> 30201 </td></tr><tr><td> Couverture de verre </td><td> Corning </td><td> 287018 </td></tr><tr><td> EasyStain kit directe </td><td> BTF </td><td> - </td></tr><tr><td> 10 pipette ul &amp; conseils barrière correspondantes </td><td> Gilson </td><td> P10 &amp; DF10ST </td></tr><tr><td> 4 &#39;, 6&#39;-Diamidino-2-phényl-indole dichlorhydrate (DAPI) </td><td> Sigma </td><td> D9542 </td></tr><tr><td> Vernis à ongles </td><td> Fisher Scientific </td><td> S30697 </td></tr><tr><td> Le méthanol </td><td> Fisher Scientific </td><td> L6815 </td></tr><tr><td> Kimwipes </td><td> Kimberly Clark </td><td> 34155 </td></tr><tr><td> Incubateur </td><td> Boekel scientifique </td><td> 133000 </td></tr><tr><td> glisser plus chaud </td><td> Fisher Scientific </td><td> 11-474-521 </td></tr><tr><td> Huile à immersion, type A ND = 1,515 </td><td> Nikon </td><td> MXA20234 </td></tr><tr><td> Nikon 90i microscope avec des capacités de DIC </td><td> Nikon </td><td> MBA 77000 </td></tr><tr><td> Plan d&#39;APO objectif 100x </td><td> Nikon </td><td> MRD01901 </td></tr><tr><td> Planifiez Achro 20X </td><td> Nikon </td><td> MRL00202 </td></tr><tr><td> Filtre FITC </td><td> Nikon </td><td> 96302 </td></tr><tr><td> DAPI filtre </td><td> Nikon </td><td> 96301 </td></tr><tr><td> X-Cite illuminateur de fluorescence </td><td> Nikon </td><td> 87540 </td></tr><tr><td> Papier pour lentilles </td><td> Nikon </td><td> 76997 </td></tr><tr><td> Biohazard sac jetable </td><td> Fisher Scientific </td><td> 01-829D </td></tr><tr><td> Biohazard récipient pour objets tranchants </td><td> Fisher Scientific </td><td> 14-827-117 </td></tr><tr><td> Peroxyde d&#39;hydrogène à 3% </td><td> VWR </td><td> BDH3540-2 </td></tr><tr><td> Javellisant </td><td> Fisher Scientific </td><td> 1952030 </td></tr><tr><td> Wypall </td><td> Kimberly Clark </td><td> 34790 </td></tr></tbody></table>

a modified epa method 1623 that uses tangential flow hollow-fiber ultrafiltration and heat dissociation steps to detect waterborne cryptosporidium and giardia spp.

Espèces Cryptosporidium et Giardia sont deux des plus répandus des protozoaires qui causent des épidémies d&#39;origine hydrique diarrhéiques dans le monde. Afin de mieux caractériser la prévalence de ces agents pathogènes, la méthode EPA 1623 a été élaboré et utilisé pour surveiller les niveaux de ces organismes dans l&#39;eau potable aux États-Unis fournit 12. Procédé comporte trois parties principales, la première est la concentration de l&#39;échantillon dans lequel au moins 10 litres d&#39;eau de surface brute est filtrée. Les organismes et les débris piégés sont ensuite élue du filtre et centrifugé pour concentrer davantage l&#39;échantillon. La deuxième partie de la méthode utilise une procédure de séparation immunomagnétique où l&#39;échantillon d&#39;eau concentrée est appliquée à des billes immunomagnétiques qui se lient spécifiquement aux oocystes de Cryptosporidium et des kystes de Giardia permettant l&#39;élimination spécifique des parasites des débris concentré. Ces kystes (oo) sont ensuite détachés des billes magnétiques par un procé dissociation acidecédure. La dernière partie de la méthode est la coloration par immunofluorescence et le dénombrement où (oo) kystes sont appliqués à une diapositive, taché, et énuméré par microscopie. Méthode 1623 a quatre cotés systèmes concentration de l&#39;échantillon pour capturer les oocystes de Cryptosporidium et des kystes de Giardia dans l&#39;eau: filtres Envirochek (Pall Corporation, Ann Arbor, MI), les filtres Envirochek HT (Pall Corporation), les filtres Filta-Max (IDEXX, Westbrook, MA), ou centrifugation à flux continu (Haemonetics, Braintree, MA). Toutefois, le Cryptosporidium et Giardia recouvrements kystes et d&#39;oocystes) ont beaucoup varié en fonction de la matrice des sources d&#39;eau et les filtres utilisés 1,14. Un nouveau flux tangentiel à fibres creuses d&#39;ultrafiltration (HFUF) système a récemment été montré pour être plus efficace et plus robuste à la récupération des oocystes de Cryptosporidium et les kystes de Giardia à partir de matrices d&#39;eau différents, d&#39;ailleurs, il est moins coûteux que l&#39;option autre capsule de filtres et peut se concentrer de multiples pathogènes simultanément 1-3,5-8,10,11. En outre, des études antérieures par Hill et ses collègues ont démontré que le HFUF considérablement amélioré Cryptosporidium recouvrements des oocystes lors directement comparés avec les filtres HT Envirochek 4. D&#39;autres modifications aux méthodes actuelles ont également été signalés à améliorer les performances des méthodes. Remplacement de la procédure de dissociation acide avec la chaleur de dissociation a été montré pour être plus efficace pour séparer Cryptosporidium des billes magnétiques dans certaines matrices 9,13. Ce protocole décrit une méthode de modification de 1623 qui utilise le système de filtration HFUF nouvelle étape avec la chaleur de dissociation. L&#39;utilisation de HFUF avec cette méthode modifiée est une alternative moins chère à l&#39;APE actuels options Méthode de filtration 1623 et offre plus de souplesse en permettant la concentration des organismes multiples.

Watch this Scientific Journal Video about A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Ce protocole décrit l&#39;utilisation d&#39;un flux de fibres creuses système d&#39;ultrafiltration tangentielle concentration de l&#39;échantillon et une dissociation de chaleur étapes alternatives pour la détection de aqueuse<em&gt; Cryptosporidium</em&gt; Et<em&gt; Giardia</em&gt; Espèces en utilisant la méthode EPA 1623.

Une méthode mise à jour EPA 1623 qui utilise tangentielle débit à fibres creuses d&#39;ultrafiltration et de chaleur étapes de dissociation pour la détection de voie d&#39;eau<em&gt; Cryptosporidium</em&gt; Et<em&gt; Giardia spp.</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

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Research

JoVE Journal

Immunology and Infection

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

20.3K vues.  Office of Research and Development, US Environmental Protection Agency. Ce protocole décrit l'utilisation d'un flux de fibres creuses système d'ultrafiltration tangentielle concentration de l'échantillon et une dissociation de chaleur étapes alternatives pour la détection de aqueuse Cryptosporidium Et Giardia Espèces en utilisant la méthode EPA 1623.

Vidéo: A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sept étapes pour Stellate Cellules

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Recherche

Immunologie et infection

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Un protocole Chelex simple pour l&#39;extraction d&#39;ADN à partir de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

Écrans à haut débit et faible avec Les nématodes à galles Meloidogyne spp.</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Verrouillé flux Nucleic Acid cytométrie de fluorescence In situ</emHybridation> (LNA flux FISH): une méthode d&#39;bactérienne détection de l&#39;ARN petits

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium1, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses2. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.

Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.

Filtres laine de verre pour concentration des virus d&#39;origine hydrique et agricole des agents pathogènes zoonotiques

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Une méthode simple et efficace pour détecter l&#39;activation du facteur nucléaire dans les neutrophiles humains par cytométrie en flux

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Basée sur l&#39;image de cytométrie en flux Technique pour évaluer les changements de granulocytes Fonction<em&gt; In Vitro</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Un protocole simple et rapide aux non enzymatique dissocier frais tissus humains pour l&#39;analyse des lymphocytes infiltrant

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Une méthode de référence de microdilution de bouillon pour dalbavancine<em&gt; In Vitro</em&gt; Tests de sensibilité des bactéries qui se développent Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Une cytométrie en flux simple méthode pour mesurer l&#39;absorption du glucose et de glucose Expression Transporter pour monocytes dans les sous-populations de sang total

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...