Name: visualizing the effects of sputum on biofilm development using a chambered coverglass model
Uploaded: 2016-12-14T14:00:00
Description: Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

TB anerkender et forskningsprojekt stipendium fra cystisk fibrose Canada.

Bemærk, at Research Ethics Board (REB) er forpligtet til at indsamle og opbevare spytprøver fra forsøgspersoner. Disse undersøgelser blev godkendt af Hospital for Sick Children REB # 1000019444. 1. Forberedelse CF spytprøver <ol><li> Saml spyt prøve fra patienter under rutinemæssige besøg på cystisk fibrose klinik og holde på is. </li><li> Transport spyt prøve på is inden for den første time af indsamling, til forskningslaboratoriet, at undergå behandling. </li></ol><p class=...

<ol>
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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. 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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. 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De heri beskrevne fremgangsmåder tillader visualisering af bakterielle biofilm dyrket i nærvær af exogene produkter. Ikke overraskende, at produktionen af ​​de exoproducts er af betydning, når der anvendes denne type system. For eksempel dithiothreitol (DTT), anvendes ofte på menneskelige spytprøver til at hjælpe flydende prøverne. virkningen af ​​DTT alene, kan imidlertid falde biofilm udvikling og levedygtighed (data ikke vist). Således ordentlig kontrol til alle betingelser er nødvendige. Desuden ka...

Bakterielle biofilm er grupper af mikroorganismer, der er knyttet til hinanden og indkapslet i en selvstændig udskilles matrix. 1,2 Klassisk, de repræsenterer bakterier fysisk knyttet til en abiotisk eller biotisk overflade dannet under forhold med strøm. Biofilm har også vist sig at vokse i statiske forhold (fravær af flow) og distalt fra overflader, såsom ved luft-væske-grænsefladen af ​​termiske puljer eller pellikeler dannet i reagensglas. Disse biofilm har længe været anerkendt i miljøet og er en vigtig ulempe for industrielle processer, som de kan dannes i vandreservoirer eller i rør, hvilket resulterer i biologisk forurening, korrosion og blokeringer. 3,4 Biofilm er også kritiske i sundhedsvæsenet, da de har vist være involveret i Kateter-relaterede infektioner, lungeinfektioner i patienter med cystisk fibrose, såvel som i mange andre infektioner. 5,6 Et af kendetegnene ved biofilm infektioner er deøget følsomhed af bakterier mod antibiotika og nedsat clearance af det medfødte immunsystem. 7-9 Den mest velundersøgte, klinisk relevante scenarier involverer biofilm-baserede infektion forekommer hos patienter med cystisk fibrose (CF), som er kronisk inficeret med Pseudomonas aeruginosa biofilm. P. aeruginosa kan undergå en række ændringer under etablering af kronisk infektion, der gør det meget vanskeligt at behandle. 10,11 Biofilm kan forskelligt aktivere medfødte immunitet og drive inflammation. 12-14 Da disse infektioner fører til øget sygelighed og dødelighed i CF-patienter, er det afgørende at forstå faktorer, der kan påvirke biofilm udvikling i denne sammenhæng. En nylig undersøgelse viser, at værten-faktorer er kritiske i dannelsen af P. aeruginosa biofilm aggregater. 15 Disse biofilm bidrage til nedsat følsomhed over for antibiotika og vært forsvarsmekanismer. Den presence af værtsafledte faktorer, såsom neutrofil elastase, samt udskilte produkter fra mikroorganismer til stede i CF lungen, har potentiale til i høj grad at modulere biofilm dannelse og udvikling. 16 Derudover biofilm interagerer med værten til at modulere ekspression af mange veje og initiere inflammation. Mens høje throughput metoder, såsom standard krystalviolet assay, kan give nogle oplysninger med hensyn til biofilmen proces, visualisering af biofilmen som reaktion på disse faktorer giver mere dybdegående information. I dette manuskript beskriver vi en fremgangsmåde til anvendelse faktorer fra spyt af patienter med CF at undersøge udviklingen af biofilm in vitro. Denne fremgangsmåde tillader hurtig visualisering af biofilm udsat for sputum indeholdende værtsfaktorer bruger kommercielt biofilm levedygtighed kit. Denne teknik kan anvendes til visuelt at identificere forandringer, der sker i løbet af biofilm vækst i nærvær af exogenoos produkter, og repræsenterer en forbedret metode til at analysere ændringerne i biofilm udvikling under forskellige forhold.

<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

Biofilm består af grupper af bakterier indkapslet i en selvstændig udskilles matrix. De spiller en vigtig rolle i den industrielle forurening samt i udviklingen og vedvarende mange sundhedsrelaterede infektioner. En af de mest velbeskrevne og studeres biofilm i human sygdom opstår i kronisk lungeinfektion af patienter med cystisk fibrose. Når man undersøger biofilm i forbindelse med værten, kan mange faktorer påvirker biofilmdannelse og udvikling. For at identificere hvordan værtsfaktorer kan påvirke biofilm dannelse og udvikling, vi anvendte en statisk kamre dækglas metode til at vokse biofilm i nærværelse af værtsafledte faktorer i form af sputum supernatanter. Bakterier podes i kamre og udsat for opspyt filtrater. Efter 48 timers vækst, er biofilm farves med et kommercielt biofilm levedygtighed kit før konfokal mikroskopi og analyse. Efter overtagelsen billede, kan biofilm egenskaber vurderes ved hjælp af forskellige softwareplatforme.Denne fremgangsmåde tillader os at visualisere centrale egenskaber af biofilm vækst i nærværelse af forskellige stoffer, herunder antibiotika.

Den overordnede udformning af forsøget er vist i figur 1. Brugen af denne protokol giver en bekvem metode til at visualisere ændringer i biofilm dyrket i forskellige perioder (fx, 24, 48 eller 72 timer). Vigtigere, exogene signaler, såsom opspyt filtrater, kan tilsættes for at visualisere ændringer i biofilm udvikling. Som det ses i figur 2, kan tilstedeværelsen af 10% sputum filtrater ændre arkitekturen i biofilmen (figur 2A,</st...

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.

Visualisere virkningerne af sputum på Biofilm Development Ved hjælp af en Chambered dækglas Model

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 ...

The overall goal of this procedure is to visualize and quantify changes to biofilms in response to sputum and antibiotics. This method can be used by researchers to understand key questions, such as how the efficacy of various antibiotics against bacterial biofilms can be impacted by the presence of sputum. The main advantage of this methodology is it allows individuals to see changes occurring in biofilms in response to different factors.To begin the experiment, record the volume of the previously obtained sputum sample. Add phosphate-buffered saline, or PBS, add two times the volume of the sample. Next, use a transfer pipette to thoroughly mix the sample.Vortex the sample on the highest setting for one minute to mix it completely. Aliquot one milliliter of the mixture into 1.5 milliliter microcentrifuge tubes. Then spin down the tubes.Following centrifugation, save the supernatant and discard the pellet. Filter sterilize the supernatant through a 0.22 micron filter and collect it in a clean microcentrifuge tube. Store the sputum supernatant at 80 degrees Celsius for future use.Place 40 microliters of previously prepared culture into four milliliters of fresh Luria broth, or LB media, and grow the bacteria to obtain a culture with an optical density of 0.55 at 600 nanometers. Next, dilute 100 microliters of 0.5 OD culture into 400 microliters of prepared 10%sputum filtrates in LB.Use 200 microliters of the dilution to seed the wells of the slide chambers. Allow the bacteria to attach for four hours at 37 degrees Celsius without shaking.After four hours, remove the media and gently wash the biofilm with 1X fresh LB.Replace the remaining media with 200 microliters of fresh LB.Allow the biofilms to grow at 37 degrees Celsius without shaking. Replace the media every 12 hours without washing until the biofilm is ready for microscopy. It's very important to gently wash the slides so as not to disrupt any attached bacteria.Following growth, remove the media from the chamber wells and gently wash each chamber two times with 300 microliters of sterile PBS. Next, to prepare the biofilm staining mixture, add one microliter of each dye provided in the viability kit to each milliliter of solution needed and mix the solution. The optimal amount of dye and staining time is determined empirically for each organism.Add 200 microliters of the staining mixture to each well of the chambered cover glass. Incubate the cover glass. After incubation, remove the staining mixture from the chambers and wash each well with 300 microliters of sterile PBS.Replace the PBS with fresh media. Finally, proceed with visualization via confocal microscopy. Representative images of Burkholderia cepacia complex, or BCC clinical isolates, following 48 hours of growth in chambered cover glass with media alone or in the presence of 10%sputum filtrates demonstrate that their presence can change the architecture of the biofilm.The images were analyzed with Comstat software to obtain key biofilm parameters, including the average thickness of the biofilm, which was increased when treated with 10%sputum. In order to appreciate the different factors'effects on biofilm growth, clinical isolates were grown in media only or with sputum and either was treated with or without antibiotic. After watching this video, you should have a good understanding of how to form and treat biofilms on chambered cover glass and prepare them for staining and confocal analysis.

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

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