Biz nazik anaerobik odasının fotoğraflarını sağlamak için Coy Laboratuvarları teşekkür etmek istiyorum. Bu çalışma Sağlık hibe DK087763 (SMM) ve STEP / HHMI Müfredat Geliştirme Bursu (ANE) Ulusal Sağlık Enstitüleri tarafından desteklenmiştir.

<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. 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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=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>

Çıkar çatışması ilan etti.

Burada açıklanan yöntemler, C, basit ve hızlı bir iyileşme sağlar insan, fare ve hamster, hem de C, uzun süreli depolama içeren dışkı örnekleri, çeşitli difficile gliserol veya spor hisse senetleri gibi difficile. C. difficile yetiştirmek için zor bir organizma olabilir, ancak dikkatli bir anaerobik ortam bakım ve aseptik tekniklerin uygulanması güçlü büyüme ve kirlenme bir azalma sağlayabilir. Anaerobik odaları: Hususlar ve Bakım <...

Clostridium difficile bir zorunlu anaerob ve insanlarda ve hayvanlarda bir potansiyel olarak ölümcül bir gastrointestinal patojen olan bir Gram-pozitif, spor oluşturucu bakteridir. Başlangıçta yenidoğanlarda 1, C. dışkısında bulunan bir ortakçı organizma olarak 1935 yılında açıklanan difficile daha sonra antibiyotik tedavisi ile ilişkili 2 psödomembranöz kolit neden olan ajan olduğu gösterilmiştir. C&#39;yi difficile enfeksiyonları (CDI), genellikle C için bir niş oluşturmak, normal kolonik floranın bozulmasına sonuçları antibiyotik tedavisi ile takip edilir difficile 2 gelişmek için. C. difficile fekal-oral yolla bir atıl sporun olarak iletilir ve daha sonra çeşitli toksinleri üreten ve ciddi hastalık ve kolit 3 neden yeteneğine vejetatif hücreleri üreten, gastrointestinal sistem içinde filizlenir edilir. CDI, genellikle geleneksel tedavilere ve bu &#39;de refrakterFections sık sık 4. tekrarlamış edilir. Sonuç olarak, CDI Amerika Birleşik Devletleri 5-7 sağlık maliyetlerinde kadar 4800000000 $ sorumludur. C. difficile ortamında oksijen düşük seviyelerde bile çok hassastır. C. için difficile ortamda hayatta kalmaya ve verimli bir konağa ana bulaşabilir, metabolik olarak aktif olmayan sporun oluşumu kritik 8&#39;dir. Çünkü C laboratuvar bakım ve manipülasyon difficile Bu teknikler, bir anaerobik odanın kullanımını gerektiren, kontrollü, oksijensiz bir ortam gerektirir. Anaerobik bölmelerin kullanılması artan geri kazanım ve zorunlu anaeroblar 9-11 izolasyonu ile sonuçlandı ve anaerobik atmosferde gerçekleştirilebilir moleküler teknikler sağladı. C ek olarak, difficile, burada açıklanan anaerobik odası kullanımı ve bakımı uygulanabilirBu diğer Clostridial türleri (örneğin, C. perfringens), diğer mide-bağırsak türler (örneğin, Bacteroides türleri, 12) ve periodontal patojenlere (örneğin, Peptostreptococcus türlerinin 13) gibi diğer zorunlu anaeroblar için.

<table><tbody><tr><td>Proteose Peptone no. 2</td><td>BD</td><td>212120</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>S373</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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>MgSO&lt;sub&gt;4&lt;/sub&gt; (anhydrous)</td><td>Fisher</td><td>M65</td><td></td></tr><tr><td>á´…-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>á´…-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>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</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 colspan="4">&lt;strong&gt;Materials&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;TCCFA agar&lt;/strong&gt;&lt;br/&gt; &lt;br/&gt; Proteose peptone no. 2 (Difco) 40 g&lt;br/&gt; Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; 5 g&lt;br/&gt; KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; 1 g&lt;br/&gt; NaCl 2 g&lt;br/&gt; MgSO&lt;sub&gt;4&lt;/sub&gt; (anhydrous) 0.1 g&lt;br/&gt; Fructose 6 g&lt;br/&gt; Agar 20 g&lt;br/&gt; &lt;br/&gt; Bring to 1 L with deionized water and autoclave at 121 &amp;deg;C for 20 min to sterilize.&lt;br/&gt; &lt;br/&gt; After autoclaving, add:&lt;br/&gt; 10 ml of 10% (w/v) sodium taurocholate, filter-sterilized (dissolve in water; final concentration, 0.1%)&lt;br/&gt; 25 ml of 10 mg/ml á´…-cycloserine, filter-sterilized (dissolve in water; final concentration, 250 &amp;mu;g/ml)&lt;br/&gt; 1.6 ml of 10 mg/ml cefoxitin, filter-sterilized (dissolve in water; final concentration, 16 &amp;mu;g/ml)&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;BHIS Medium&lt;/strong&gt;&lt;br/&gt; &lt;br/&gt; Brain heart infusion 37 g&lt;br/&gt; Yeast extract 5 g&lt;br/&gt; &lt;br/&gt; For plates, add 15 g agar. Bring to 1 L with deionized water and autoclave at 121 &amp;deg;C for 20 min to sterilize.&lt;br/&gt; &lt;br/&gt; Optional (add after autoclaving):&lt;br/&gt; &lt;br/&gt; 3 ml of 10% (w/v) L-cysteine (dissolve in water; final concentration, 0.03%)&lt;br/&gt; 10 ml of 10% (w/v) sodium taurocholate (dissolve in water; final concentration, 0.1%)&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;SMC Sporulation Medium&lt;/strong&gt;&lt;br/&gt; &lt;br/&gt; BactoPeptone 90 g&lt;br/&gt; Protease peptone 5 g&lt;br/&gt; (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&lt;sub&gt; &lt;/sub&gt; 1 g&lt;br/&gt; Tris base 1.5 g&lt;br/&gt; Agar 15 g&lt;br/&gt; &lt;br/&gt; Bring to 1 L with deionized water and autoclave at 121 &amp;deg;C for 20 min to sterilize.&lt;br/&gt; &lt;br/&gt; Optional (add after autoclaving):&lt;br/&gt; 3 ml of 10% (w/v) L-cysteine (dissolve in water; final concentration, 0.03%)&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;70:30 Medium&lt;/strong&gt;&lt;br/&gt; &lt;br/&gt; BactoPeptone 63 g&lt;br/&gt; Protease peptone 3.5 g&lt;br/&gt; Brain heart infusion 11.1 g&lt;br/&gt; Yeast extract 1.5 g&lt;br/&gt; (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt; 0.7 g&lt;br/&gt; Tris base 1.06 g&lt;br/&gt; &lt;br/&gt; For plates, add 15 g agar. Bring to 1 L with deionized water and autoclave at 121 &amp;deg;C for 20 min to sterilize. After autoclaving, add 3 ml of 10% (w/v) L-cysteine (final concentration, 0.03%).&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;Blood agar&lt;/strong&gt;&lt;br/&gt; &lt;br/&gt; The use of premade Columbia anaerobic sheep blood agar plates (Fisher Scientific, L21928)&lt;sup&gt;35&lt;/sup&gt; is recommended.&lt;br/&gt; &lt;br/&gt; &lt;strong&gt;1X Phosphate buffered saline (PBS)&lt;/strong&gt;&lt;br/&gt; &lt;br/&gt; NaCl 8.01 g&lt;br/&gt; KCl 0.2 g&lt;br/&gt; Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; 1.44 g&lt;br/&gt; KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; 0.27 g&lt;br/&gt; &lt;br/&gt; 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 antibiyotik ilişkili ishal (AAD) için öncelikle sorumlu ve önemli bir nozokomiyal patojen olan bir Gram-pozitif, anaerobik, sporogenic bakteridir. C. difficile izole etmek ve geliştirmek için çok zordur ve çevre oksijen düşük seviyelerde bile son derece duyarlıdır. Burada, C izole edilmesi için yöntemler dışkı örnekleri difficile ve daha sonra kültür C. , uzun süreli depolama için gliserol stoklarının hazırlanması için difficile sunulmaktadır. Mikroskopi ve hayvan çalışmaları dahil olmak üzere alt çeşitli uygulamalar için laboratuarda spor stoklarının hazırlanması ve numaralandırma için teknikler de tarif edilmiştir. Bu teknikler en iyi C için uygun koşulları temin etmek üzere tutarlı oksijensiz bir ortamın oksijensiz odası, gerektirecek difficile büyüme. Biz ca olmadan odasının içinde ve dışında malzeme aktarımı için protokolleri sağlamakverimli ve tutarlı C için uygun anaerobik ortam sürdürmek için gerekli düzenli bakım önerileriyle birlikte önemli oksijen kirlenmeyi kullanarak difficile ekimi.

C. bir örneği BHIS ve Columbia anaerobik koyun kan agar ortamı üzerinde büyütülmüş difficile, Şekil 2&#39;de görülebilir. C. difficile düz ve hem de medya üzerinde belirgin bir buzlu cam görünümü sahip düzensiz koloniler oluşturur. Burada, C&#39;lik bir eritromisin duyarlı klinik izolat difficile, 630E 30, 37 ° C &#39;(Şekil 2A), 24 saat boyunca, BHIS ağar, bir zenginleştirilmiş ve seçici olmayan ortam ü...

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 sıkı anaerob ve antibiyotik ilişkili ishal (AAD) neden olan bir patojen bakteridir. Burada, izole edilmesi ve kültür C muhafaza edilmesi için yöntemler difficile bitkisel hücreleri ve sporlar açıklanmıştır. Bu teknikler optimum C için uygun koşulları sağlamak için düzenli bakım gerektirir, bir anaerobik odasını gerektiren difficile ekimi.

Kültürleme ve bakımı<em&gt; Clostridium difficile</em&gt; Bir Anaerobik Çevre

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

38.4K Görüntüleme.  Emory University School of Medicine.  Clostridium difficile sıkı anaerob ve antibiyotik ilişkili ishal (AAD) neden olan bir patojen bakteridir. Burada, izole edilmesi ve kültür C muhafaza edilmesi için yöntemler difficile bitkisel hücreleri ve sporlar açıklanmıştır. Bu teknikler optimum C için uygun koşulları sağlamak için düzenli bakım gerektirir, bir anaerobik odasını gerektiren difficile ekimi. 

Video: Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

Kesinlikle Anaerobik/Anoksik Koşullarda Mikroorganizmaların Yetiştirilmesi için Orta Hazırlık

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

Biyokimya

Çevresel mikropların kültürlenmesi için yerinde simüle edilmiş anaerobik yöntem

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

Yazar Spotlight: Yerinde Kültürleme ile Keşfedilmemiş Ortamlarda Karasal Anaerobik Mikroorganizmaların Gizemlerini Çözmek

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

Çevresel Olarak Edinilmiş Anaerobik Mikroorganizmaların Kültürlenmesi için Bir Dizi Yerinde Bilgilendirilmiş Simüle Edilmiş Ortam Formatı

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Çevre

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.

ve Cefoperazone-tedavi edilmiş fare modeli klinik olarak ilgili<em&gt; Clostridium difficile</em&gt; Gerilme R20291

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

İmmünoloji ve Enfeksiyon

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.

Morfolojik Değişiklikleri Karakterize Eden Bir Protokol<em&gt; Clostridium difficile</em&gt; Antibiyotik Tedavisine Yanıt Vermek

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

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

Yapay Olarak Yetiştirilen Bakteriler Tarafından Murin Vajinal Kolonizasyon Modelleri

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and inflammatory mediators, which dynamically alter community composition and microbial metabolic output. Understanding how physiological culture conditions affect microbial communities and diversity and their metabolic capacity will contribute important knowledge of their impact on health and diseases. Here, we present a protocol adapted from the template published by Auchtung et al. to create mini-bioreactor arrays that can cultivate complex fecal communities, define bacterial consortiums or single strains under precise conditions, including nutrient availability, temperature, flow rate, pH, and oxygen content. We describe the process for building the mini-bioreactor system, including adaptations to improve limitations in the published protocol. We also discuss the setup of the mini-bioreactor system under anaerobic conditions with the use of MEGA media (adapted from Han et al.), which is a rich media supporting the growth of diverse bacteria. We describe the inoculation of gut humanized mouse fecal samples into the mini-bioreactor system, followed by the establishment of complex community cultures within the mini-bioreactor system, which can be grown under continuous flow with aseptic sampling to monitor community composition. This system is adaptable to dietary changes and other cultural modifications. The techniques described here allow for the characterization of diverse, fastidious fecal communities under dynamic conditions or in response to perturbation in isolation from host-derived factors.

This protocol details the assembly of mini-bioreactor arrays to be utilized for continuous flow culture of complex fecal communities under anaerobic conditions. We also discuss the methods for assembly, inoculation, and sampling of the reactors for further analysis.

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Anaerobik büyüme ve bakım memeli hücre hatları

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

Biyoloji

Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 &#181;m filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.

We demonstrate a method for isolating difficult-to-grow members of the novel bacterial phylum, Saccharibacteria, by filtering dental plaque and co-culturing with host bacteria.

Ağız Boşluğundan Simbiyotik Sakkarakterinin Kararlı İkili Kültürlerinin Kurulması

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial ...

Kültürleme ve bakımı

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Kesinlikle Anaerobik/Anoksik Koşullarda Mikroorganizmaların Yetiştirilmesi için Orta Hazırlık

Çevresel Olarak Edinilmiş Anaerobik Mikroorganizmaların Kültürlenmesi için Bir Dizi Yerinde Bilgilendirilmiş Simüle Edilmiş Ortam Formatı

Çevresel Olarak Edinilmiş Anaerobik Mikroorganizmaların Kültürlenmesi için Bir Dizi Yerinde Bilgilendirilmiş Simüle Edilmiş Ortam Formatı

Çevresel Olarak Edinilmiş Anaerobik Mikroorganizmaların Kültürlenmesi için Bir Dizi Yerinde Bilgilendirilmiş Simüle Edilmiş Ortam Formatı

ve Cefoperazone-tedavi edilmiş fare modeli klinik olarak ilgili

Morfolojik Değişiklikleri Karakterize Eden Bir Protokol

Yapay Olarak Yetiştirilen Bakteriler Tarafından Murin Vajinal Kolonizasyon Modelleri

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

Anaerobik büyüme ve bakım memeli hücre hatları

Ağız Boşluğundan Simbiyotik Sakkarakterinin Kararlı İkili Kültürlerinin Kurulması