Name: culturing and maintaining clostridium difficile in an anaerobic environment
Uploaded: 2013-09-14T21:25:00
Description: Watch this Scientific Journal Video about Culturing and Maintaining Clostridium difficile in an Anaerobic Environment at JoVE.com

Wir möchten Coy Laboratories für die freundliche Bereitstellung von Bildern der anaeroben Kammer danken. Diese Arbeit wurde vom National Institutes of Health Zuschuss DK087763 (SMM) und einem STEP / HHMI Lehrplanentwicklung Fellowship (ANE) unterstützt.

Hinweis: C. difficile ist ein Mensch und Tier Krankheitserreger, der Magen-Darm-Erkrankung verursachen können. Experimente mit C. difficile, müssen mit geeigneten Vorsichtsmaßnahmen zur Biosicherheit (BSL-2) durchgeführt werden. 1. Anaerobe Kammer Betriebs-und Wartungs C. difficile ist eine strenge anaerobe und ist extrem empfindlich auf niedrige Sauerstoffkonzentrationen in der Atmosphäre. Daher wird eine gesteuerte, anaeroben Umgeb...

<ol>
	<li>Hall, I. C., O'Toole, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+flora+in+new-borin+infants+-+With+a+description+of+a+new+pathogenic+anaerobe,+Bacillus+difficilis.">Intestinal flora in new-borin infants - With a description of a new pathogenic anaerobe, Bacillus difficilis.</a> Am. J. Dis. Child. 49, 390-402 (1935).</li><li>Tedesco, F. J., Barton, R. W., Alpers, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clindamycin-Associated+Colitis+-+Prospective+Study.">Clindamycin-Associated Colitis - Prospective Study.</a> Ann. Intern. Med. 81, 429-433 (1974).</li><li>Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+30+years+on:+what+has,+or+has+not,+changed+and+why.">Clostridium difficile 30 years on: what has, or has not, changed and why.</a> Int. J. Antimicrob. Agents. 33, 2-8 (2009).</li><li>Gerding, D. N., Muto, C. A., Owens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+Clostridium+difficile+infection.">Treatment of Clostridium difficile infection.</a> Clin. Infect. Dis. 46, 32-42 (2008).</li><li>Dubberke, E. R., Olsen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+Clostridium+difficile+on+the+healthcare+system.">Burden of Clostridium difficile on the healthcare system.</a> Clin. Infect. Dis. 55, 88-92 (2012).</li><li>Bouza, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+Clostridium+difficile+infection:+understanding+the+healthcare+burden.">Consequences of Clostridium difficile infection: understanding the healthcare burden.</a> Clin. Microbiol. Infect. 18, 5-12 (2012).</li><li>Peery, A. F., 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=Burden+of+gastrointestinal+disease+in+the+United+States:+2012+update.">Burden of gastrointestinal disease in the United States: 2012 update.</a> Gastroenterology. 143, 1171-1173 (2012).</li><li>Deakin, L. 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=The+Clostridium+difficile+spo0A+gene+is+a+persistence+and+transmission+factor.">The Clostridium difficile spo0A gene is a persistence and transmission factor.</a> Infect. Immun. 80, 2704-2711 (2012).</li><li>Drasar, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+anaerobic+intestinal+bacteria.">Cultivation of anaerobic intestinal bacteria.</a> J. Pathol. Bacteriol. 94, 417-427 (1967).</li><li>Leach, P. A., Bullen, J. J., Grant, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+CO+2+cabinet+for+the+cultivation+of+strict+anerobes.">Anaerobic CO 2 cabinet for the cultivation of strict anerobes.</a> Appl. Microbiol. 22, 824-827 (1971).</li><li>Killgore, G. E., Starr, S. E., Del Bene, ., Whaley, V. E., N, D., Dowell, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+three+anaerobic+systems+for+the+isolation+of+anaerobic+bacteria+from+clinical+specimens.">Comparison of three anaerobic systems for the isolation of anaerobic bacteria from clinical specimens.</a> Am. J. Clin. Pathol. 59, 552-559 (1973).</li><li>Bacic, M. K., Smith, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+maintenance+and+cultivation+of+bacteroides+species.">Laboratory maintenance and cultivation of bacteroides species.</a> Curr. Protoc. Microbiol. Chapter 13, Unit 13C 11 (2008).</li><li>Doan, N., Contreras, A., Flynn, J., Morrison, J., Slots, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proficiencies+of+three+anaerobic+culture+systems+for+recovering+periodontal+pathogenic+bacteria.">Proficiencies of three anaerobic culture systems for recovering periodontal pathogenic bacteria.</a> J. Clin. Microbiol. 37, 171-174 (1999).</li><li>Socransky, S., Macdonald, J. B., Sawyer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cultivation+of+Treponema+microdentium+as+surface+colonies.">The cultivation of Treponema microdentium as surface colonies.</a> Arch. Oral. Biol. 1, 171-172 (1959).</li><li>George, W. L., Sutter, V. L., Citron, D., Finegold, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+and+differential+medium+for+isolation+of+Clostridium+difficile.">Selective and differential medium for isolation of Clostridium difficile.</a> J. Clin. Microbiol. 9, 214-219 (1979).</li><li>Wilson, K. H., Silva, J., Fekety, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+Clostridium+difficile+by+normal+hamster+cecal+flora+and+prevention+of+antibiotic-associated+cecitis.">Suppression of Clostridium difficile by normal hamster cecal flora and prevention of antibiotic-associated cecitis.</a> Infect. Immun. 34, 626-628 (1981).</li><li>Wilson, K. H., Kennedy, M. J., Fekety, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sodium+taurocholate+to+enhance+spore+recovery+on+a+medium+selective+for+Clostridium+difficile.">Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile.</a> J. Clin. Microbiol. 15, 443-446 (1982).</li><li>Bliss, D. Z., Johnson, S., Clabots, C. R., Savik, K., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cycloserine-cefoxitin-fructose+agar+(CCFA)+and+taurocholate-CCFA+for+recovery+of+Clostridium+difficile+during+surveillance+of+hospitalized+patients.">Comparison of cycloserine-cefoxitin-fructose agar (CCFA) and taurocholate-CCFA for recovery of Clostridium difficile during surveillance of hospitalized patients.</a> Diagn. Microbiol. Infect. Dis. 29, 1-4 (1997).</li><li>Marler, L. M., 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=Comparison+of+five+cultural+procedures+for+isolation+of+Clostridium+difficile+from+stools.">Comparison of five cultural procedures for isolation of Clostridium difficile from stools.</a> J. Clin. Microbiol. 30, 514-516 (1992).</li><li>Sorg, J. A., Dineen, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+maintenance+of+Clostridium+difficile.">Laboratory maintenance of Clostridium difficile.</a> Curr. Protoc. Microbiol. Chapter 9, Unit 9A 1 (2009).</li><li>Bouillaut, L., McBride, S. M., Sorg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+Clostridium+difficile.">Genetic manipulation of Clostridium difficile.</a> Curr. Protoc. Microbiol. Chapter 9, Unit 9A 2 (2011).</li><li>Lemee, L., Pons, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilocus+sequence+typing+for+Clostridium+difficile.">Multilocus sequence typing for Clostridium difficile.</a> Methods. Mol. Biol. 646, 77-90 (2010).</li><li>Shanholtzer, C. J., Peterson, L. R., Olson, M. N., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+study+of+gram-stained+stool+smears+in+diagnosis+of+Clostridium+difficile+colitis.">Prospective study of gram-stained stool smears in diagnosis of Clostridium difficile colitis.</a> J. Clin. Microbiol. 17, 906-908 (1983).</li><li>Smith, C. J., Markowitz, S. M., Macrina, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferable+tetracycline+resistance+in+Clostridium+difficile.">Transferable tetracycline resistance in Clostridium difficile.</a> Antimicrob. Agents Chemother. 19, 997-1003 (1981).</li><li>Permpoonpattana, 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=Surface+layers+of+Clostridium+difficile+endospores.">Surface layers of Clostridium difficile endospores.</a> J. Bacteriol. 193, 6461-6470 (2011).</li><li>Putnam, E. E., Nock, A. M., Lawley, T. D., Shen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpoIVA+and+SipL+are+Clostridium+difficile+spore+morphogenetic+proteins.">SpoIVA and SipL are Clostridium difficile spore morphogenetic proteins.</a> J. Bacteriol. , (2013).</li><li>Burns, D. A., Minton, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+studies+in+Clostridium+difficile.">Sporulation studies in Clostridium difficile.</a> J. Microbiol. Methods. 87, 133-138 (2011).</li><li>Perez, J., Springthorpe, V. S., Sattar, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clospore:+a+liquid+medium+for+producing+high+titers+of+semi-purified+spores+of+Clostridium+difficile.">Clospore: a liquid medium for producing high titers of semi-purified spores of Clostridium difficile.</a> J. AOAC Int. 94, 618-626 (2011).</li><li>Sorg, J. A., Sonenshein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+the+initiation+of+Clostridium+difficile+spore+germination+using+analogs+of+chenodeoxycholic+acid,+a+bile+acid.">Inhibiting the initiation of Clostridium difficile spore germination using analogs of chenodeoxycholic acid, a bile acid.</a> J. Bacteriol. 192, 4983-4990 (2010).</li><li>O'Connor, J. R., 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=Construction+and+analysis+of+chromosomal+Clostridium+difficile+mutants.">Construction and analysis of chromosomal Clostridium difficile mutants.</a> Mol. Microbiol. 61, 1335-1351 (2006).</li><li>Buggy, B. P., Wilson, K. H., Fekety, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+methods+for+recovery+of+Clostridium+difficile+from+an+environmental+surface.">Comparison of methods for recovery of Clostridium difficile from an environmental surface.</a> J. Clin. Microbiol. 18, 348-352 (1983).</li><li>Koch, C. J., Kruuv, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+release+of+oxygen+from+polystyrene+Petri+dishes.">The release of oxygen from polystyrene Petri dishes.</a> Br. J. Radiol. 45, 787-788 (1972).</li><li>Ethapa, 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=Multiple+factors+modulate+biofilm+formation+by+the+anaerobic+pathogen+Clostridium+difficile.">Multiple factors modulate biofilm formation by the anaerobic pathogen Clostridium difficile.</a> J. Bacteriol. , (2012).</li><li>Speers, A. M., Cologgi, D. L., Reguera, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+cell+culture.">Anaerobic cell culture.</a> Curr. Protoc. Microbiol. Appendix 4, Appendix 4F (2009).</li><li>Lyras, D., 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=Toxin+B+is+essential+for+virulence+of+Clostridium+difficile.">Toxin B is essential for virulence of Clostridium difficile.</a> Nature. 458, 1176-1179 (2009).</li></ol>

Die hier beschriebenen Methoden ermöglichen eine einfache und schnelle Wiederherstellung von C difficile aus einer Vielzahl von Stuhlproben, einschließlich Menschen, Mäuse und Hamster, sowie die Langzeitlagerung von C difficile Glycerin oder Sporen Aktien. C difficile kann eine schwierige Organismus zu kultivieren, aber sorgfältige Wartung einer anaeroben Umgebung und die Anwendung aseptischer Techniken können für robustes Wachstum und eine Verringerung der Verschmutzung bieten. <p c...

Clostridium difficile ist ein gram-positives, sporenbildendes Bakterium, das ein obligat anaerobe und eine potenziell tödliche Magen-Darm-Erreger von Mensch und Tier ist. Ursprünglich im Jahre 1935 beschrieben, als Kommen Organismus in Stuhlproben von Neugeborenen 1, C. gefunden difficile wurde später gezeigt, dass der Erreger der Pseudomembrankolik mit Antibiotika-Behandlung 2 zugeordnet werden. C difficile-Infektionen (CDI) in der Regel durch Antibiotika-Behandlung, die in der Störung der normalen Darmflora führt, die Schaffung einer Nische für C voraus difficile zu gedeihen 2. C difficile als ruhende Sporen fäkal-oralen Weg übertragen und anschließend keimt innerhalb des Magen-Darm-Trakt, wodurch vegetativen Zellen fähig ist, mehrere Toxine und verursachen schwere Krankheit und Colitis 3. CDI sind oft resistent gegen herkömmliche Behandlungen und diese inInfektionen werden häufig wiederkehrende 4. Als Ergebnis sind CDI für bis zu 4,8 Milliarden Kosten im Gesundheitswesen in den USA 07.05 $ verantwortlich. C. difficile ist sehr empfindlich auf geringe Mengen an Sauerstoff in der Umgebung. Für C. difficile in der Umwelt verbleiben und effizient von Host zu Host übertragen werden kann, ist die Bildung einer metabolisch inaktiven Sporen kritischen 8. Da der Labor Wartung und Manipulation von C. difficile erfordert einen kontrollierten anaeroben Umgebung, diese Techniken erfordern den Einsatz einer anaeroben Kammer. Verwendung von anaeroben Kammern hat in erhöhte Gewinnung und Isolierung von obligaten Anaerobiern 9-11 geführt und hat eine Reihe von molekularen Techniken, um in einer anaeroben Atmosphäre durchgeführt werden dürfen. Zusätzlich zu C. difficile, die anaerobe Kammer Gebrauch und die Wartung hier beschriebenen geltenzu anderen obligat anaerobe Bakterien wie Clostridien anderen Spezies (zB C. perfringens), andere Magen-Darm-Arten (z. B. Bacteroides 12) und parodontalen Krankheitserreger (zB Peptostreptococcus Art 13).

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Proteose Peptone no. 2</td>
			<td>BD</td>
			<td>212120</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher</td>
			<td>S373</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>BP362</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S27</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4 (anhydrous)</td>
			<td>Fisher</td>
			<td>M65</td>
			<td></td>
		</tr>
		<tr>
			<td>&#7429;-Fructose</td>
			<td>Fisher</td>
			<td>L96</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Sigma</td>
			<td>T4009</td>
			<td></td>
		</tr>
		<tr>
			<td>&#7429;-cycloserine</td>
			<td>Sigma</td>
			<td>C6880</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefoxitin</td>
			<td>Fluka</td>
			<td>C4786</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion medium</td>
			<td>BD</td>
			<td>237300</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteose Peptone</td>
			<td>BD</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Sigma</td>
			<td>A5132</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C7755</td>
			<td></td>
		</tr>
		<tr>
			<td>BactoPeptone</td>
			<td>BD</td>
			<td>211684</td>
			<td></td>
		</tr>
		<tr>
			<td>Columbian sheep blood agar</td>
			<td>Fisher</td>
			<td>L21928</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S27</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher</td>
			<td>P217</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>BP2291</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile inoculating loops</td>
			<td>Fisher</td>
			<td>22363596</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile swabs</td>
			<td>Fisher</td>
			<td>1495990</td>
			<td></td>
		</tr>
		<tr>
			<td>Coy Vinyl Anaerobic Chamber and Accessories</td>
			<td>Coy Laboratory Products, Inc</td>
			<td>Customer Specified</td>
			<td>These items are custom ordered per laboratory needs</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>TCCFA agar 
			 
			Proteose peptone no. 2 (Difco) 40 g 
			Na2HPO4 5 g 
			KH2PO4 1 g 
			NaCl 2 g 
			MgSO4 (anhydrous) 0.1 g 
			Fructose 6 g 
			Agar 20 g 
			 
			Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. 
			 
			After autoclaving, add: 
			10 ml of 10% (w/v) sodium taurocholate, filter-sterilized (dissolve in water; final concentration, 0.1%) 
			25 ml of 10 mg/ml &#7429;-cycloserine, filter-sterilized (dissolve in water; final concentration, 250 &#956;g/ml) 
			1.6 ml of 10 mg/ml cefoxitin, filter-sterilized (dissolve in water; final concentration, 16 &#956;g/ml) 
			 
			BHIS Medium 
			 
			Brain heart infusion 37 g 
			Yeast extract 5 g 
			 
			For plates, add 15 g agar. Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. 
			 
			Optional (add after autoclaving): 
			 
			3 ml of 10% (w/v) L-cysteine (dissolve in water; final concentration, 0.03%) 
			10 ml of 10% (w/v) sodium taurocholate (dissolve in water; final concentration, 0.1%) 
			 
			SMC Sporulation Medium 
			 
			BactoPeptone 90 g 
			Protease peptone 5 g 
			(NH4)2SO4 1 g 
			Tris base 1.5 g 
			Agar 15 g 
			 
			Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. 
			 
			Optional (add after autoclaving): 
			3 ml of 10% (w/v) L-cysteine (dissolve in water; final concentration, 0.03%) 
			 
			70:30 Medium 
			 
			BactoPeptone 63 g 
			Protease peptone 3.5 g 
			Brain heart infusion 11.1 g 
			Yeast extract 1.5 g 
			(NH4)2SO4 0.7 g 
			Tris base 1.06 g 
			 
			For plates, add 15 g agar. Bring to 1 L with deionized water and autoclave at 121 &#176;C for 20 min to sterilize. After autoclaving, add 3 ml of 10% (w/v) L-cysteine (final concentration, 0.03%). 
			 
			Blood agar 
			 
			The use of premade Columbia anaerobic sheep blood agar plates (Fisher Scientific, L21928)35 is recommended. 
			 
			1X Phosphate buffered saline (PBS) 
			 
			NaCl 8.01 g 
			KCl 0.2 g 
			Na2HPO4 1.44 g 
			KH2PO4 0.27 g 
			 
			Bring to 1 L with deionized water and adjust pH to 7.4 with HCl. Filter sterilize before use.</td>
		</tr>
	</tbody>
</table>

culturing and maintaining clostridium difficile in an anaerobic environment

Clostridium difficile ist ein Gram-positive, anaerobe, sporenbild Bakterium, das vor allem für Antibiotika-assoziierten Diarrhoe (AAD) verantwortlich ist und der eine bedeutende nosokomiale Erreger. C difficile ist notorisch schwierig zu isolieren und zu kultivieren und ist extrem empfindlich, auch geringe Mengen an Sauerstoff in der Umgebung. Hier sind Verfahren zur Isolierung von C. difficile aus Stuhlproben und anschließend Kultivierung C. difficile zur Herstellung von Glycerin-Stammlösungen für die Langzeitlagerung dargestellt. Techniken zur Herstellung und Auflisten Sporenvorräte im Labor für eine Vielzahl von Anwendungen, einschließlich Mikroskopie abwärts und Tierstudien werden ebenfalls beschrieben. Diese Techniken erfordern eine anaerobe Kammer, die eine konsistente anaerobe Umgebung zu angemessenen Bedingungen für eine optimale C gewährleisten hält difficile Wachstum. Wir stellen Protokolle zum Übertragen von Materialien in und aus der Kammer ohne Camit erheblichen Sauerstoffkontamination zusammen mit Vorschlägen für die regelmäßige Wartung erforderlich, um die entsprechenden anaeroben Umgebung für die effiziente und konsequente C aufrecht difficile Anbau.

Ein Beispiel C difficile auf BHIS und Columbia anaerobe Schafblut-Agar-Medien gezüchtet in Fig. 2 zu sehen ist. C. difficile bildet unregelmäßige Kolonien, die flach sind und einen Boden-Glas-Optik, die auf beiden Medien ist evident. Hier wird ein Erythromycin-sensitive klinischen Isolat von C. difficile, 630E 30 wird auf BHIS Agar, einer angereicherten, nicht-selektives Medium für 24 Stunden bei 37 ° C (2A) gewachsen. Kolonien auf Columbia ana...

Watch this Scientific Journal Video about Culturing and Maintaining Clostridium difficile in an Anaerobic Environment at JoVE.com

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile ist ein pathogenes Bakterium, das eine strikte Anaerobier ist und bewirkt, dass Antibiotika-assoziierten Diarrhoe (AAD). Hier, Methoden zur Isolierung, Kultivierung und Pflege von C. difficile vegetativen Zellen und Sporen werden beschrieben. Diese Techniken erfordern eine anaerobe Kammer, die regelmäßige Wartung erfordert, um den richtigen Bedingungen sorgen für optimale C. difficile Anbau.

Kultivierung und Pflege<em&gt; Clostridium difficile</em&gt; In einer anaeroben Umgebung

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Research

JoVE Journal

Immunology and Infection

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Culturing Microglia from the Neonatal and Adult Central Nervous System

Kultivierung Mikroglia aus dem Neugeborenen und Erwachsenen Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Forschung

Immunologie und Infektion

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Mit RNA-Interferenz, um die angeborene Immunantwort in Makrophagen der Maus Untersuchen

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Porphyromonas gingivalis als Modellorganismus für die Beurteilung der Interaktion von anaeroben Bakterien mit Wirtszellen

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Einrichtung eines Setup-Hochdurchsatz-Screening für kleine Moleküle, die Modulate c-di-GMP-Signalisierung in<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

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

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 ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Cefoperazon behandelten Maus-Modell der klinisch relevanten<em&gt; Clostridium difficile</em&gt; Dehnungs R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Ein Protokoll zur Charakterisierung der morphologischen Veränderungen von<em&gt; Clostridium difficile</em&gt; In Antwort auf Antibiotika-Behandlung

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

Ein Protein-Microarray-Assay für die serologische Bestimmung der Antigen-spezifische Antikörper Antworten folgende Clostridium Difficile Infektion

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Eine Reinigung und In vitro- Aktivität Assay für ein (p) PpGpp Synthestase von Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

Multicolor Flow Cytometry-basierte Quantifizierung der Mitochondrien, Lysosomen in T-Zellen

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...