私たちは親切嫌気室の写真を提供するためのコイ研究所に感謝したいと思います。この作品は、DK087763（SMM）とSTEP /ハワードヒューズカリキュラム開発フェローシップ（ANE）を付与国立衛生研究所によってサポートされていました。

<ol>
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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. 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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>

利害の対立が宣言されていません。

ここに記載された方法は、Cの簡単かつ迅速なリカバリを可能にヒト、マウスおよびハムスター、ならびにC.の長期保存を含む糞便サンプル、種々からディフィシルグリセロールまたは胞子ストックとしてディフィシル 。 でディフィシルは、育成することは困難生物であってもよいが、慎重な嫌気性環境の維持と無菌技術の適用は、堅調な成長と汚染の低減?...

クロストリジウム·ディフィシルは、絶対嫌気性菌と人間と動物の致命的な胃腸病原体であるグラム陽性、芽胞形成細菌である。最初は新生児1、Cから糞便中に見られる共生生物として1935年に記載されてディフィシルは後に抗生物質治療2に関連した偽膜性大腸炎の原因物質であることが実証されました。Cをディフィシル感染症（CDI）は、典型的にはCのニッチを作成して、正常な結腸細菌叢の破壊をもたらし、抗生物質による治療が先行している2℃に発育ディフィシルディフィシルは、糞口経路を介して休眠胞子として送信され、その後、いくつかの毒素を生成し、重篤な疾患や大腸炎3を引き起こすことができる栄養細胞を生産する、消化管の中に発芽している。 CDIは、多くの場合、従来の治療と、これらの中に抵抗性fectionsは、頻繁に再発4です。その結果、CDIは米国5-7の医療費で最大48億ドルを担当している。 クロストリジウム·ディフィシルは、環境中の酸素でさえ低レベルに非常に敏感である。 Cのディフィシルが環境中に残留し、効率的にホストにホストから送信され、代謝的に不活性胞子の形成が重要で8です。なぜならCの実験室でのメンテナンスと操作ディフィシルは、これらの技術は、嫌気性チャンバーの使用を必要とし、制御され、嫌気性環境を必要とする。嫌気性チャンバーの使用は、偏性嫌気性菌の増加9-11回収及び単離をもたらした、分子の多くの技術が嫌気雰囲気下で行うことを可能にした。 C.に加えてディフィシルは 、ここで説明する嫌気性チャンバーの使用と保守が適用されますこのような他のクロストリジウム種（ 例えばウェルシュ菌 ）、他の胃腸の種（ 例えばバクテロイデス種12）と歯周病原体（ 例えばペプトストレプトコッカス属 13）のような他の偏性嫌気性菌に。

<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

クロストリジウム·ディフィシルは、抗生物質関連下痢症（AAD）の主な原因であり、重要な院内感染病原体であるグラム陽性嫌気性、胞子形成細菌である。C.ディフィシルは隔離し、育成することで悪名高いことは困難であり、環境中の酸素でさえ低いレベルに非常に敏感である。ここでは、Cを単離する方法糞便サンプルからディフィシル 、その後培養することで長期保存用グリセロールストックの調製のためディフィシルが提示される。顕微鏡や動物実験を含む下流の様々なアプリケーションのために、実験室での胞子株を作製し、列挙するための手法についても説明します。これらの技術は、最適C.ための適切な条件を確保するための一貫性嫌気性環境を維持する嫌気性チャンバーを必要とするディフィシル成長。我々は、約なくチャンバの内外に物質を移送するためのプロトコルを提供する効率的かつ一貫したC.ための適切な嫌気性環境を維持するのに必要な定期的なメンテナンスのための提案と共に有意な酸素汚染を用いディフィシル栽培。

Cの例BHISコロンビア嫌気ヒツジ血液寒天培地上で増殖させディフィシルは、 図2に見ることができる。C.ディフィシルは平坦であり、両方のメディアに明らかであるすりガラスの外観を持っている不規則なコロニーを形成している。ここでは、Cのエリスロマイシンに敏感な臨床分離株ディフィシル 、630E 30は 、37°C（ 図2A）で...

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

クロストリジウム·ディフィシルは、厳密な嫌気性菌で、抗生物質関連下痢（AAD）を引き起こす病原性細菌である。ここで、単離培養し、Cを維持するための方法ディフィシル栄養細胞と芽胞が記載されている。これらの技術は、最適C.ための適切な条件を確実にするために定期的なメンテナンスを必要とする、嫌気性チャンバーを必要とディフィシル栽培。

培養およびメンテナンス<em&gt;クロストリジウム·ディフィシル</em&gt;嫌気性環境での

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

38.4K ビュー.  Emory University School of Medicine.  クロストリジウム·ディフィシルは、厳密な嫌気性菌で、抗生物質関連下痢（AAD）を引き起こす病原性細菌である。ここで、単離培養し、Cを維持するための方法ディフィシル栄養細胞と芽胞が記載されている。これらの技術は、最適C.ための適切な条件を確実にするために定期的なメンテナンスを必要とする、嫌気性チャンバ?...

ビデオ: 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.

厳密嫌気性/無酸素条件下での微生物の培養のための培地製剤

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

生化学

環境微生物培養のためのIn Situ模擬嫌気性法

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.

著者スポットライト:In Situ Culturingによる未知の環境における陸生嫌気性微生物の謎の解明

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."

環境的に獲得した嫌気性微生物を培養するための In Situ 情報に基づく模擬培地フォーマットのセット

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

環境学

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.

臨床的に関連のセフォペラゾン処理されたマウスモデル<em&gt;クロストリジウム・ディフィシル</em&gt;ひずみR20291

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

免疫・感染学

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.

形態変化を特徴付けるプロトコル<em&gt;クロストリジウム・ディフィシル</em&gt;抗生物質治療に対する反応

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.

嫌気性増殖細菌によるマウス膣コロニー形成モデル

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.

哺乳類セルラインの嫌気性成長と維持

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 ...

生物学

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.

口腔から共生サッカリバクテリアの安定したバイナリ培養を確立する

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

培養およびメンテナンス

要約

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厳密嫌気性/無酸素条件下での微生物の培養のための培地製剤

環境的に獲得した嫌気性微生物を培養するための In Situ 情報に基づく模擬培地フォーマットのセット

環境的に獲得した嫌気性微生物を培養するための In Situ 情報に基づく模擬培地フォーマットのセット

環境的に獲得した嫌気性微生物を培養するための In Situ 情報に基づく模擬培地フォーマットのセット

臨床的に関連のセフォペラゾン処理されたマウスモデル

形態変化を特徴付けるプロトコル

嫌気性増殖細菌によるマウス膣コロニー形成モデル

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

哺乳類セルラインの嫌気性成長と維持

口腔から共生サッカリバクテリアの安定したバイナリ培養を確立する