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.

<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>

Keine Interessenskonflikte erklärt.

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 ...

culturing-maintaining-clostridium-difficile-an-anaerobic

Research

JoVE Journal

Immunology and Infection

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

38.3K Aufrufe.  Emory University School of Medicine.  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. 

Serie: Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Culturing Microglia from the Neonatal and Adult 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 pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

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

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

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

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

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

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

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

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

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

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

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

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 ...

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

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

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

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

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

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

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

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

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

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

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

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

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

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

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

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

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

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

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 ...