TB erkennt ein Forschungsstipendium von Cystic Fibrosis Kanada.

Beachten Sie, dass Forschungsethik Board (REB) erforderlich zu sammeln und zu speichern, Sputum-Proben von menschlichen Probanden. Diese Studien wurden von der Klinik zugelassen für kranke Kinder REB # 1000019444. 1. Vorbereiten CF Sputum-Proben <ol><li> Sammeln Sie Sputum Probe von Patienten während der Routine Abfragen der Mukoviszidose-Klinik und auf Eis halten. </li><li> Transport Sputumprobe auf Eis innerhalb der ersten Stunde nach der Entnahme, zum Forschungslabor, die Verarbeitung zu unterziehen. </li></ol> 2. Sputum Verarbeitung <ol><li> Aufzeichnen des Volumens des Sputum-Probe erhalten. Fügen Phosphat - gepufferter Salzlösung (PBS) , um das Volumen der Probe auf 2x (dh 2 Teile PBS, 1 Teil Probe). </li><li> Mischen Sie die Probe gut mit einer Transferpipette. Vortex die Probe auf die höchste Einstellung für 1 min vollständig zu mischen. </li><li> Aliquot 1 ml der obigen Mischung in die entsprechende Anzahl von 1,5 ml Mikrozentrifugenröhrchen und Spin bei 5000 × g nach unten für20 min bei 4 ° C. </li><li> Nach der Zentrifugation den Überstand und das Pellet verwerfen. </li><li> Filter sterilisieren den Überstand durch ein 0,22 um Filter und sammeln sich in einem sauberen Reaktionsgefäß. HINWEIS: Sterility des Filtrats durch Ausplattieren auf LB-Agar getestet und flüssigen Medien Animpfen. </li><li> Speichern von Sputum Überstand bei -80 ° C für zukünftige Verwendung. HINWEIS: Sputum von mehreren Patienten können auch nach der Filtration gesammelt werden. </li><li> Vor der Verwendung verdünnt Sputum Filtrat 1/10 v / v (100 &amp; mgr; l Sputum, 900 ul Medium) in gewünschte Medien. HINWEIS: Hier Standard LB-Medium (LB) Medien verwendet wurde. </li></ol> 3. Kammerdeckgläser Verfahren zur Bildung von Biofilmen <ol><li> Wachsen Bakteriumisolat von Interesse über Nacht in das gewünschte Medium bei 37 ° C unter Schütteln (200 rpm). Hinweis: Eine Anzahl verschiedener Bakterien verwendet wurden, einschließlich der klinischen Isolate von Pseudomonas aeruginosa, Staphylococcus aureus,Burkholderia cepacia und Achromobacter xylosoxidans. Wahl der Medien hängt von Stämmen und Bedingungen von Interesse kann jedoch LB-Medien für erste Experimente verwendet werden. </li><li> Von Übernachtkultur, legen 40 &amp; mgr; l der Kultur in die 4 ml frisches Medium wachsen und für 3-4 h bei 37 ° C unter Schütteln (200 Upm) , um eine Kultur mit einer optischen Dichte bei 600 nm (OD 600) von ungefähr 0,5 zu erhalten , -0.6. </li><li> Verdünnen Sie die Kultur aus Schritt 3,2 bis 1/5 in das gewünschte Medium mit 10% Sputum Filtrat oder ohne Auswurf Filtrat (als Kontrolle). Andere Konzentration von Sputum Filtrat kann (dh 50% oder 100%) getestet werden. </li><li> Verwenden Sie 200 ul der Verdünnung Vertiefungen Schiebekammern auf Saatgut. </li><li> Lassen Bakterien für 4 Stunden bei 37 ° C zu befestigen ohne Schütteln. </li><li> Nach 4 Stunden, um die Medien zu entfernen und sanft den Biofilm mit 1x frisches Medium waschen. Ersetzen mit 200 &amp; mgr; l frisches Medium. Hinweis: Um die Auswirkungen von Sputum auf dem Biofilm studieren, die fresh Medien sollten Sputum Überstand enthalten. </li><li> Erlauben Biofilmen für gewünschte Zeitdauer bei 37 ° C zu wachsen, ohne Schütteln, Medien alle 12 Stunden ersetzt, ohne Waschen, bis die Zeit für die Mikroskopie. ANMERKUNG: Um die Wirkung von Sputum Überstände auf Biofilm antibiotischen Empfänglichkeit untersuchen, werden Antibiotika zum Medium hinzugefügt folgenden 24 h Biofilmwachstum und werden in den Medien bis zum Anfärben und Abbildung von Biofilmen gehalten. </li></ol> 4. Die Färbung Biofilme und konfokale Mikroskopie <ol><li> gewünschte Wachstumszeit verfolgt (24-48 h funktioniert am besten), entfernen Sie Medien aus der Kammer Brunnen und sanft jede Kammer zweimal mit 300 ul sterilem PBS waschen. </li><li> Bereiten Sie Färbemischung für Biofilm durch Mischen von 1 ul jeder Farbstoff (sofern in der Lebensfähigkeit Kit) für jeden ml Lösung benötigt. Machen Farbstoff in Wasser oder Media-Lösung. HINWEIS: Das Wasser wird vom Hersteller empfohlen. </li><li> Mit 200 &amp; mgr; l der Farbstoffmischung in jede Vertiefung chambered coverglass und für 45 min bei Raumtemperatur im Dunkeln inkubieren. </li><li> Entfernen Färbung Mischung aus Kammern und waschen Sie jede Vertiefung mit 300 ul sterilem PBS. Entfernen PBS und ersetzen mit frischem Wasser oder Medien. </li><li> Fahren Sie mit der Visualisierung von Biofilmen durch konfokale Mikroskopie. </li></ol> 5. Visualizing Biofilme mit konfokale Mikroskopie <ol><li> Lesen gefärbten Biofilmen in Kammern unmittelbar nach dem Färben (innerhalb von 1 h). Minimieren Verzögerung in Visualisierung der Objektträger durch Anfärben August 1-02-well Kammern zu einer Zeit. </li><li> Führen Bildgebung mit konfokalen Mikroskop mit Lasern für die Anregung und Filtersätze für die Erfassung. HINWEIS: Hier wird die sich drehende Scheibe konfokalen System mit spektraler borealis Laser (Grün: 491nm, Rot: 561 nm) zur Anregung verwendet wurden. Emissionsfiltersätze von 515/40 und 624/40 wurden verwendet, um die Flecken aus dem Biofilm Lebensfähigkeit Kit zu visualisieren. </li><li> Nehmen Sie Bilder mit einem 25X Wasser Ziel auf konfokalen Mikroskop mit Kamera. </li><li&gt; Verwenden Z-Stacks des Biofilms zu modellieren. Nehmen Sie Bilder jede 0,5-1 &amp; mgr; m ausgehend von der ersten in-Fokusebene auf den letzten im Fokus befindlichen Rahmen des Biofilms (typischerweise Spanning 30-80 &amp; mgr; m für 48 Stunden Biofilme) </li><li> Nehmen Sie 3-5 Bilder von jedem gut. HINWEIS: So für einen 8 gut chambered Abdeckglas, 24-40 Bilder erzeugt werden. </li><li> Speichern Sie die Bilder für die Analyse. HINWEIS: Bilder gespeichert werden sollen als OME-TIFF - Dateien mit COMSTAT 18,19 analysiert werden. Hinweise zur Biofilmbildanalyse kann bei http://www.comstat.dk/ finden. Sobald Bilder importiert werden, können Parameter wie die durchschnittliche Dicke, Biomasse und Flächendeckung für jeden Kanal (rot und grün) analysiert werden. </li></ol>

<ol>
	<li>Beaudoin, T., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+with+biofilm+formation:+selection+of+antimicrobials+and+role+of+prolonged+antibiotic+therapy.">Infections with biofilm formation: selection of antimicrobials and role of prolonged antibiotic therapy.</a> Pediatr.Infect.Dis.J. , (2016).</li><li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+microbial+life+on+surfaces.">Biofilms: microbial life on surfaces.</a> Emerg.Infect.Dis. 8 (9), 881-890 (2002).</li><li>Hobley, L., Harkins, C., MacPhee, C. E., Stanley-Wall, N. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation:+a+clinically+relevant+microbiological+process.">Biofilm formation: a clinically relevant microbiological process.</a> Clin.Infect.Dis. 33 (8), 1387-1392 (2001).</li><li>Bjarnsholt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+biofilm.">The in vivo biofilm.</a> Trends Microbiol. 21 (9), 466-474 (2013).</li><li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+resistance.">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li><li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm-specific+antibiotic+resistance+gene+ndvB+is+important+for+expression+of+ethanol+oxidation+genes+in+Pseudomonas+aeruginosa+biofilms.">The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li><li>Beaudoin, T., Aaron, S. D., Giesbrecht-Lewis, T., Vandemheen, K., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+clonal+strains+of+Pseudomonas+aeruginosa+isolated+from+cystic+fibrosis+patients+in+Ontario,+Canada.">Characterization of clonal strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Ontario, Canada.</a> Can. J. Microbiol. 56 (7), 548-557 (2010).</li><li>Vidya, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+infection+phenotypes+of+Pseudomonas+aeruginosa+are+associated+with+failure+of+eradication+in+children+with+cystic+fibrosis.">Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis.</a> Eur.J.Clin.Microbiol.Infect.Dis. , (2015).</li><li>Beaudoin, T., Lafayette, S., Nguyen, D., Rousseau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucoid+Pseudomonas+aeruginosa+caused+by+mucA+mutations+result+in+activation+of+TLR2+in+addition+to+TLR5+in+airway+epithelial+cells.">Mucoid Pseudomonas aeruginosa caused by mucA mutations result in activation of TLR2 in addition to TLR5 in airway epithelial cells.</a> Biochem.Biophys.Res.Commun. 428 (1), 150-154 (2012).</li><li>Beaudoin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+level+of+p38alpha+mitogen-activated+protein+kinase+activation+in+airway+epithelial+cells+determines+the+onset+of+innate+immune+responses+to+planktonic+and+biofilm+Pseudomonas+aeruginosa.">The level of p38alpha mitogen-activated protein kinase activation in airway epithelial cells determines the onset of innate immune responses to planktonic and biofilm Pseudomonas aeruginosa.</a> J.Infect.Dis. 207 (10), 1544-1555 (2013).</li><li>LaFayette, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis-adapted+quorum+sensing+mutants+cause+hyperinflammatory+responses.">Cystic fibrosis-adapted quorum sensing mutants cause hyperinflammatory responses.</a> Sci.Adv. 1 (6), e1500199 (2015).</li><li>Staudinger, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+associated+with+the+cystic+fibrosis+defect+promote+chronic+Pseudomonas+aeruginosa+infection.">Conditions associated with the cystic fibrosis defect promote chronic Pseudomonas aeruginosa infection.</a> Am.J.Respir.Crit.Care Med. 189 (7), 812-824 (2014).</li><li>Kennedy, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+Tobramycin+against+Cystic+Fibrosis+Isolates+of+Burkholderia+cepacia+Complex+Grown+as+Biofilms.">Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms.</a> Antimicrob.Agents Chemother. 60 (1), 348-355 (2015).</li><li>Tom, S. K., Yau, Y. C., Beaudoin, T., LiPuma, J. J., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+High-Dose+Antimicrobials+on+Biofilm+Growth+of+Achromobacter+Species+Isolated+from+Cystic+Fibrosis+Patients.">Effect of High-Dose Antimicrobials on Biofilm Growth of Achromobacter Species Isolated from Cystic Fibrosis Patients.</a> Antimicrob.Agents Chemother. 60 (1), 650-652 (2015).</li><li>Heydorn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (Pt 10), 2395-2407 (2000).</li><li>Vorregaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Informatics and Mathematical Modelling. , (2008).</li><li>Jurcisek, J. A., Dickson, A. C., Bruggeman, M. E., Bakaletz, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Biofilm+Formation+in+an+8-well+Chamber+Slide.">In vitro Biofilm Formation in an 8-well Chamber Slide.</a> J. Vis. Exp. (47), e2481 (2011).</li></ol>

Die hierin beschriebenen Verfahren ermöglichen die Visualisierung von bakteriellen Biofilmen in Gegenwart von exogenen Produkten gewachsen. Es überrascht nicht, die Herstellung der exoproducts ist von Bedeutung, wenn diese Art von System. Zum Beispiel Dithiothreitol (DTT), wird häufig auf die menschliche Sputum-Proben verwendet, um die Proben helfen verflüssigen. Jedoch allein der Wirkung von DTT Biofilmentwicklung verringern kann und die Lebensfähigkeit (Daten nicht gezeigt). Somit sind geeignete Kontrollen für a...

Bakterielle Biofilme sind Gruppen von Mikroorganismen, die miteinander und umhüllt in einer selbst sezerniert Matrix gebunden sind. 1,2 Classically stellen sie Bakterien physisch an einen abiotischen oder biotischen Oberfläche unter den Bedingungen der Strömung gebildet angebracht. Biofilme sind auch gezeigt worden, unter statischen Bedingungen (keine Strömung) und distal von Oberflächen, beispielsweise an der Luft-Flüssigkeit-Grenzfläche der thermischen Pools oder Häutchen gebildet in Reagenzgläsern zu wachsen. Diese Biofilme sind seit langem in der Umwelt erkannt und sind eine große Nachteil für industrielle Prozesse, wie sie in Wasserbehältern oder in Rohren bilden können, was zu Biofouling, Korrosion und Verstopfung. 3,4 Biofilme sind auch im Gesundheitsbereich kritisch, da sie in Katheter bedingten Infektionen, Lungeninfektionen bei Mukoviszidose-Patienten, sowie in zahlreichen anderen Infektionen beteiligt haben gezeigt werden. 5,6 Eines der Markenzeichen von Biofilm - Infektionen ist die deerhöhte Empfindlichkeit von Bakterien gegen Antibiotika und die Beeinträchtigung der Clearance durch das angeborene Immunsystem. 7-9 Die gut untersucht, klinisch relevante Szenarien Biofilm-basierten Infektion beteiligt tritt bei Patienten mit zystischer Fibrose (CF), die chronisch infiziert sind , mit Pseudomonas aeruginosa Biofilme. P. aeruginosa kann eine Reihe von Änderungen bei Etablierung einer chronischen Infektion zu unterziehen , die es sehr schwierig zu behandeln , machen. 10,11 Biofilme können unterschiedlich angeborenen Immunität aktivieren und Entzündung fahren. 12-14 Da diese Infektionen zu einer erhöhten Morbidität und Mortalität bei CF - Patienten führen, ist es wichtig , Faktoren zu verstehen , die Biofilmbildung in diesem Zusammenhang beeinflussen können. Eine neue Studie schlägt vor , dass Host-Faktoren bei der Bildung von P. aeruginosa Biofilm Aggregate kritisch sind. 15 Diese Biofilme tragen zu einer reduzierten Empfindlichkeit gegenüber Antibiotika und Abwehrmechanismen des Wirts. Die Presenz von Wirt-abgeleiteten Faktoren, wie Neutrophilen-Elastase, sowie sekretierte Produkte von Mikroorganismen, die in der CF-Lunge, haben das Potential stark zu Biofilmbildung und Entwicklung modulieren. 16 Darüber hinaus interagieren Biofilmen mit dem Host - Expression zahlreicher Wege zu modulieren und Entzündungen auslösen. Während Hochdurchsatzverfahren, wie zum Beispiel die Standardkristallviolett-Assay, einige Informationen in Bezug auf die Biofilmverfahren zur Verfügung stellen kann, die Visualisierung des Biofilms in Reaktion auf diese Faktoren liefern weitergehende Informationen. In diesem Manuskript beschreiben wir ein Verfahren zur Faktoren aus dem Sputum von Patienten mit CF mit der Entwicklung von Biofilmen in vitro zu studieren. Dieses Verfahren ermöglicht eine schnelle Visualisierung von auf Sputum enthaltenden Wirtsfaktoren ausgesetzt Biofilmen Kommerzielle Biofilm Lebensfähigkeit Kit. Diese Technik kann verwendet werden, um visuell Veränderungen zu identifizieren, die während der Biofilmwachstum in Gegenwart von exogeno auftretenuns Produkte und stellt ein verbessertes Verfahren der Änderungen in Biofilmentwicklung unter verschiedenen Bedingungen zu untersuchen.

<table border="0" cellpadding="0" cellspacing="0" width="1270">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td>
			<td style="width:159px;">Thermo Sicher Scientific</td>
			<td style="width:136px;">155409</td>
			<td style="width:729px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Filmtracer Live/Dead Biofilm Viabilty Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">L10316</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Blood agar plates</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">R10215</td>
			<td>Confirming viability via CFU counts or selecting colonies for innoculation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">COMSTAT</td>
			<td style="width:159px;">Availble software online</td>
			<td style="width:136px;"></td>
			<td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Millers LB Broth</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>12780-052</td>
			<td style="width:729px;">Standard media for overnight gowth/biofilm growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex-GV Syringe Filters</td>
			<td style="width:159px;">Millipore</td>
			<td>SLGV013SL</td>
			<td>Filtering of sputum supernants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Phosphate Buffered Saline (Dulbecco A)</td>
			<td style="width:159px;">Oxoid</td>
			<td style="width:136px;">BR0014G</td>
			<td>Washing of biofilm chambers after media removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss AxioVert 200M</td>
			<td style="width:159px;">Carl Zeiss</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamamatsu C9100-13 EM-CCD</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectral Borealis</td>
			<td style="width:159px;">Qs Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perkin Elmer Volocity</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td>
		</tr>
	</tbody>
</table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

Biofilme bestehen aus Gruppen von in einem selbst sezerniert Matrix eingeschlossen Bakterien. Sie spielen eine wichtige Rolle in der industriellen Verschmutzung sowie in der Entwicklung und Persistenz von vielen gesundheitsbezogenen Infektionen. Eines der am besten beschrieben und untersucht Biofilmen in der menschlichen Erkrankung tritt bei chronischen Lungeninfektion von Mukoviszidose-Patienten. Wenn Biofilme im Rahmen des Host studieren, können viele Faktoren die Biofilmbildung und Entwicklung beeinflussen. Um zu ermitteln, wie Wirtsfaktoren die Bildung von Biofilmen und Entwicklung beeinflussen können, haben wir eine statische chambered Abdeckglas Methode Biofilmen in Gegenwart von Wirt abgeleiteten Faktoren in Form von Sputum Stände zu wachsen. Die Bakterien werden in die Kammern ausgesät und Sputum Filtrate ausgesetzt. mit einem handelsüblichen Biofilm Lebensfähigkeit Kit vor der konfokalen Mikroskopie und Analyse folgenden 48 h des Wachstums werden Biofilme gefärbt. Nach der Bildaufnahme können Biofilm Eigenschaften mit Hilfe Plattformen unterschiedlicher Software bewertet werden.Dieses Verfahren ermöglicht es uns, Schlüsseleigenschaften von Biofilmwachstum in Gegenwart verschiedener Substanzen einschließlich Antibiotika zu visualisieren.

Das Gesamtdesign des Experiments ist in Abbildung 1 dargestellt. Die Verwendung dieses Protokoll bietet eine bequeme Methode , die Veränderungen in Biofilmen für verschiedene Zeiträume (beispielsweise 24, 48 oder 72 Stunden) gezüchtet zu visualisieren. Wichtig ist, dass exogene Signale, wie Sputum Filtrate können hinzugefügt werden, um die Veränderungen in der Biofilmentwicklung zu visualisieren. Wie in Figur 2 zu sehen ist , kann die Geg...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualisierung der Auswirkungen von Sputum auf Biofilm-Entwicklung eines Modells mit Kammerdeckgläser

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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Immunology and Infection

8.3K Aufrufe.  Hospital for Sick Children. This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Serie: Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Mikrotiterschale Biofilmbildung Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Forschung

Immunologie und Infektion

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

In-vitro-Bildung von Biofilmen in einem 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Ein Mikroplatten Assay Chemical Auswirkungen auf die RBL-2H3 Mastzelldegranulation bewerten: Effekte von Triclosan ohne Verwendung eines organischen Lösungsmittels

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Mit Klick-Chemie, um die Wirkung von Viral Infection auf Host-Zell-RNA Synthese Messen

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Untersuchung der Effekte von Probiotika auf Pneumokokken-Kolonisation Mit ein<em&gt; In-vitro-</em&gt; Einhaltung Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Analyse der Auswirkungen von Stromazellen an der Rekrutierung von Leukozyten von Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Durchflusszytometrische Analyse von partikelgebundenen Bet v 1 Allergens in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Echtzeit-Bildgebung und Quantifizierung der Pilz Biofilm-Entwicklung mit einem Zweiphasensystem rezirkulierenden Flow

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

Ein In-Vitro-Batch-Kulturmodell zur Abschätzung der Auswirkungen interventionaler Regimen auf humane fäkale Mikrobiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Mechanistische Einblicke in die Entwicklung von TNBS-vermittelter Darmfibrose und Bewertung der hemmenden Wirkungen von Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...