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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giving+structure+to+the+biofilm+matrix:+an+overview+of+individual+strategies+and+emerging+common+themes.">Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes.</a> FEMS Microbiol.Rev. 39 (5), 649-669 (2015).</li><li>Katharios-Lanwermeyer, S., Xi, C., Jakubovics, N. S., Rickard, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-review:+Microbial+coaggregation:+ubiquity+and+implications+for+biofilm+development.">Mini-review: Microbial coaggregation: ubiquity and implications for biofilm development.</a> Biofouling. 30 (10), 1235-1251 (2014).</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=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><tbody><tr><td>Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td><td>Thermo Sicher Scientific</td><td>155409</td><td></td></tr><tr><td>Filmtracer Live/Dead Biofilm Viabilty Kit</td><td>Thermo Fisher Scientific</td><td>L10316</td><td></td></tr><tr><td>Blood agar plates</td><td>Thermo Fisher Scientific</td><td>R10215</td><td>Confirming viability via CFU counts or selecting colonies for innoculation</td></tr><tr><td>COMSTAT</td><td>Availble software online</td><td></td><td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&amp;nbsp;</td></tr><tr><td>Millers LB Broth</td><td>Thermo Fisher Scientific</td><td>12780-052</td><td>Standard media for overnight gowth/biofilm growth</td></tr><tr><td>Millex-GV Syringe Filters</td><td>Millipore</td><td>SLGV013SL</td><td>Filtering of sputum supernants</td></tr><tr><td>Phosphate Buffered Saline (Dulbecco A)</td><td>Oxoid</td><td>BR0014G</td><td>Washing of biofilm chambers after media removal</td></tr><tr><td>Zeiss AxioVert 200M</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hamamatsu C9100-13 EM-CCD</td><td>QS Technologies Inc.</td><td></td><td></td></tr><tr><td>Spectral Borealis</td><td>Qs Technologies Inc.</td><td></td><td></td></tr><tr><td>Perkin Elmer Volocity</td><td>QS Technologies Inc.</td><td></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 ...

visualizing-effects-sputum-on-biofilm-development-using-chambered

Universidad de Cartagena UDEC

Research

JoVE Journal

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

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-Kultur Modelle Pseudomonas aeruginosa Biofilme auf lebenden menschlichen Zellen gezüchtet Airway

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Forschung

Immunologie und Infektion

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa und Saccharomyces cerevisiae Biofilm im Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

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 ...

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. ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

Verwendung eines Hochdurchsatz-<em&gt; In-vitro-</em&gt; Mikrofluidik-System auf die mündliche Multi-Spezies-Biofilme entwickeln

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Biotechnik

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Methodologien für das Studium<em&gt; B. subtilis</em&gt; Biofilme als Modell für kleine Molekül-Inhibitoren Biofilm Charakterisieren

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Das WinCF-Modell - Ein preiswerter und tragbarer Mikrokosmos eines Schleims verstopfte Bronchiole zur Untersuchung der Mikrobiologie von Lungeninfektionen

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Visualisierung der Biofilmbildung in Candida Albicans ein automatisiertes mikrofluidischen Gerät

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Visualisierung von Pseudomonas aeruginosa im Sputum von Mukoviszidose-Patienten

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

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

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video