저자는 그랜트 번호 31003A-1분의 132,358, 31003A_152972 및 번호 151948에 대한 스위스 국립 과학 재단 (National Science Foundation)을 인정하고 Fundation 피에르 메르 라 과학을 붓는다.

화학 물질 및 장비의 1. 준비 <ol><li> 1 %의 나트륨 헥사 (SHMP) (나포 3) N 용액 500 mL로하고 고압 증기 멸균에 의해 소독. </li><li> 폐 유리 페트리 접시에서 고압 증기 멸균에 의해 멸균 니트로 셀룰로오스 (NC) 필터 (기공 크기 0.22 μm의 직경 47mm). </li><li> 폐 유리 페트리 접시에서 고압 증기 멸균에 의해 NC 필터 (기공 크기 0.22 μm의 직경 25mm)를 소독. </li><li> 무게 (에 캡) 멸균 50 ㎖ 튜브의 빈 무게 (샘플 당 하나의 튜브를) 참고. </li><li> EDTA 트리스 완충액 (TE 버퍼) (1X)를 준비 : 10 mM 트리스 (트리스 (히드 록시 메틸) 아미노 메탄) 및 1 mM의 EDTA 완충액 용액을 만든다. 8 산도를 조정하고 고압 증기 멸균에 의해 소독. </li><li> 1 ㎖의 TE 완충액에 리소자임 0.02 g을 용해하여 리소자임 용액 (20 ㎎ / ㎖)을 제조. 이상적으로, 때마다 신선한합니다. 더 이상 1 이상 주 4 ℃에서 보관하십시오. </li><li> 8g / L 염화 나트륨을 용해시켜 용액을 제조 생리증류수 (염화나트륨). 고압 증기 멸균에 의해 소독. </li></ol> 퇴적물에서 바이오 매스 2. 분리 <ol><li> 퇴적물 시료 3 g을 에탄올 골조 금속 국자를 사용하여 50 ㎖ 튜브를 멸균 무게를 사전에 추가합니다. 오염을 방지하기 위해 깨끗하고 자외선 살균 바이오 안전성 캐비닛에이를 수행합니다. </li><li> 멸균이 뷰렛을 졸업 사용하여 샘​​플에 1 % 살균 (멸균) SHMP 솔루션의 15 ML을 추가합니다. SHMP 용액은 오염을 방지하기 위해 필터링 될 수있다. </li><li> 액체 분산기 / 균질 (예를 들어, 울트라 타락 균질)와 침전물과 SHMP 솔루션을 균질화. 70 % 에탄올 소독 또는 사용하기 전에 분산 로터를 압력솥. 17,500 rpm에서 1 분간 균질화를 실행합니다. 2 분 동안 샘플 나머지를하자 같은 회 전자 속도에서 1 분 동안 균질화를 반복합니다. </li><li> 샘플은 10 분 동안 서 보자. 이 단계에서 가장 무거운 입자 (미네랄) 해결됩니다. 그러나 세포와 w 샘플 중 유기 성분병이 용액에 남아 있습니다. 퇴적물 펠렛을 방해하지 않도록주의하면서 그 후, 깨끗한 50 ML 튜브에 뜨는 용액 (포함하는 세포 바이오 매스)을 전송합니다. </li><li> 침전물 펠릿에 멸균이 뷰렛을 졸업하여 1 % 살균 (멸균) SHMP 솔루션 다시 15 ml를 추가합니다. 그런 다음 반복 2.3 및 2.4 단계를 반복합니다. 이 반복은 퇴적물의 미네랄 성분으로부터 세포 및 유기 입자의 최대 양의 분리를 보장한다. 이 제 2 분리의 상등액을 제 1 분리에서 상등액과 병합 될 수있다. 주 : 다음 단계는 모든 (셀 바이오 매스를 포함) 상층 액에서 수행된다. 미네랄 성분 (퇴적물 펠렛) 폐기 될 수있다. </li><li> 1 분 20 XG에서 샘플을 원심 분리기. 생물 세포 물질은 여전히​​ 용액에 남아있는 동안이 단계는 작은 광물 입자를 해결하기에 충분 G-힘을 증가시킨다. 원심 분리 후, 상청에 용액 (세포 함유 매스)를 전송할깨끗한 50 ML 튜브. 미네랄 펠렛을 폐기하십시오. </li><li> 샘플을 계량하여 바이오 매스를 함유하는 용액의 최종 부피를 결정한다. 가중치 결정 눈금 실린더에 시료를 전송하는 데 및 오염의 위험을 감소 피한다. </li></ol> 필터 멤브레인에 바이오 매스의 3 컬렉션 <ol><li> 여과 장치 (47mm 직경 멤브레인) 및 진공 펌프를 준비합니다. 고온 가압 멸균하여 70 % 에탄올을 분무 분젠 버너로 타오르는 그래피 (파이렉스 유리의 경우) 중 여과 부 소독. 이 프로토콜을 계속하기 전에 식혀 보자. </li><li> 에탄올 골조 멸균 집게를 사용하여 여과 장치에 멸균 NC 멤브레인을 추가합니다. </li><li> 진공 펌프를 사용하여 멤브레인에 세포 막 여과 장치에 (단계 2.6)에서 시료의 상층 액의 절반을 첨가하고 수집한다. </li><li> 액체가 완전히 필터를 통과 한 경우, 진공 펌프를 정지하고 이단을 사용하여 조심스럽게 제거 막OL-불꽃 집게를 소독. 멸균 페트리 접시에 막 놓습니다. <ol><li> 에탄올 골조 멸균 가위를 사용하여 반으로 막을 잘라. 별도의 2 ML 튜브에 막의 각각의 절반을 추가합니다. 멤브레인의 절반은 DNA 추출 및 사회 전체 박테리아의 분석을 위해 사용된다. 필터의 다른 절반은 -80 ° C에서 저장 및 백업의 역할을한다. </li></ol></li><li> 여과 유닛 상에 새로운 NC 멤브레인을두고 진공 펌프를 사용하여 (단계 260)에서 샘플 볼륨의 후반에서 미생물을 모은다. </li><li> 액체가 완전히 필터를 통과 한 경우, 진공 펌프를 정지하고 신중 에탄올 골조 멸균 집게를 사용하여 멤브레인을 제거한다. 별도의 2 ML 튜브로 전체 막 놓습니다. 이 샘플은 식물 세포로부터 내생 포자를 분리하는 처리를 위해 사용될 것이다. 시료를 사용할 때까지 -20 ℃에서 저장 될 수있다. </li></ol> 식물 세포의 용해 (4) <ol><li> 수행치료 이전에 NC 막 (단계 3.6)에 수집 된 바이오 매스에 식물 세포에서 내생 포자를 분리합니다. <ol><li> 멤브레인이 동결 된 경우 해동 실온에서 10 분 동안두고. 그런 다음 멸균 페트리 접시에서 막을 배치하고 에탄올 골조 멸균 가위를 사용하여 작은 조각으로 (약 4 배)를 잘라. 그런 다음 멸균 2 ML 튜브에 모든 멤브레인 필터 조각을 배치합니다. </li></ol></li><li> 샘플 멤브레인을 포함하는 튜브에 1X TE (트리스 - EDTA) 버퍼의 900 μl를 (1.5 참조) 추가 소용돌이에 의해 철저하게 섞는다. 이 단계에서, 바이오 매스-TE 완충액으로 막으로부터 제거된다. </li><li> 10 분 80 rpm으로 65 ° C에서 인큐베이터에서 튜브를 놓습니다. 그 후 인큐베이터에서 튜브를 제거하고 15 분 동안 냉각 수 있습니다. </li><li> 2 mg / ml의 최종 농도에 도달하기 위해 새로 제조 라이소자임 (1.5 참조) 100 ㎕를 추가한다. 샘플을 37 ℃로 냉각되기 전에이 내지 수 있기 때문에, 라이소자임를 추가하지 마십시오효소 rade. </li><li> 리소자임에 대한 최적 조건은 식물 세포를 용해시키기 위하여, 60 분, 80 rpm으로 37 ℃에서 샘플을 배양한다. </li><li> 용해가 완료되면, 3 N 수산화 나트륨 (NaOH) 샘플 6 % 도데 실 황산나트륨 (SDS) 용액을 250 μL의 250 μL를 추가한다. 이 첨가에 의해, 시료 부피는 1.5 ㎖에 도달하여, 0.5 N NaOH를 최종 농도 1 % SDS의 최종 농도가있다. </li><li> 60 분 80 rpm으로 실온에서이 믹스를 품어. 베이스와 세제를 추가하면 최종 세포 용해에 도움이 될 것입니다. 식물 세포를 용균 동안 이러한 세제의 농도는, 내생 포자를 해치지하도록 최적화되어있다. </li><li> (파이렉스 유리의 경우) 70 % 에탄올로 분무하고 분젠 버너 화염을 오토 클레이브에 의해 직경 25mm 막을 보유, 또는 멸균 여과 유닛을 준비한다. 이 식히십시오. </li><li> 에탄올 화염 멸균 집게를 사용하여 여과 유닛에 0.2 μm의 NC 막 (25mm 직경)을 배치했다. &lt;/ 리&gt; <li> 멤브레인 상 단계 4.7에서 샘플을 추가하고, 진공 펌프를 사용하는 액체 필터. 액체가 통과되면, 진공 펌프의 전원을 끄십시오. 이 막 상에 유지되지 않기 때문에이 단계에서 분해 된 식물 세포의 재료가 제거된다. 만 내생 포자는 막에 남아있을 것이다. </li><li> 잔류 세제를 씻어 진공 펌프를 사용하여 액체를 필터링하는 멸균 생리 용액 2 ㎖를 추가한다. </li><li> 액체가 완전히 여과되면, 진공 펌프의 전원을 끄십시오. 여과 장치의 멤브레인을 남겨주세요. </li></ol> 5. DNase의 치료 주 : 여과막에 직접 D​​Nase의 처리를 수행한다. 이 여과 장치가 누출되지 않는 것이 중요하고, 진공 펌프는 턴 오프된다. <ol><li> 필터 막에 멸균 물 450 μL, 직접 D​​Nase의 반응 버퍼 (1X) 0.5 μL DNase의 효소의 50 μl를 추가하고 15 분 동안 서 보자. 가능하면, 약간 전쟁 방에서이 소화 할평균 RT보다 르, 효소는 위의 25 ° C에서의 온도에서 잘 작동하기 때문이다. 주 : 분젠 버너를 늘리지 따로 여과 장치는 또한 온도가 증가하고 무균 환경을 유지하는 추가적인 장점을 가지며, 따라서 샘플의 오염의 위험을 감소시켰다. </li><li> DNase의 분해가 완료되면, 샘플로부터 효소를 제거하기 위해 진공 펌프를 켜. </li><li> 추가 및 생리 액 1ml를 필터링하여 잔여 효소를 씻어 내십시오. </li><li> 액체가 완전히 통과 한 경우, 진공 펌프를 끄고 멸균 집게를 사용하여 멸균 페트리 접시에 배치 내생 포자가 들어있는 필터 멤브레인을 제거합니다. 주 : 분리 내생 포자의 샘플을 지금 다운 스트림 분석을위한 준비가되어 있습니다. 발아와 재배에 사용되는 경우 -20 ° C에서 보관은 DNA 추출을 위해 또는 다른 4 ° C에서 사용합니다. </li></ol>

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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=Dispersal+of+thermophilic+Desulfotomaculum+endospores+into+Baltic+Sea+sediments+over+thousands+of+years.">Dispersal of thermophilic Desulfotomaculum endospores into Baltic Sea sediments over thousands of years.</a> ISME J. 7 (1), 72-84 (2013).</li><li>Hubert, C., 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=Thermophilic+anaerobes+in+Arctic+marine+sediments+induced+to+mineralize+complex+organic+matter+at+high+temperature.">Thermophilic anaerobes in Arctic marine sediments induced to mineralize complex organic matter at high temperature.</a> Environ Microbiol. 12 (4), 1089-1104 (2010).</li><li>Garbeva, P., van Veen, J. A., van Elsas, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+Bacillus+spp.+in+agricultural+soil+under+different+management+regimes+detected+via+PCR-DGGE.">Predominant Bacillus spp. in agricultural soil under different management regimes detected via PCR-DGGE.</a> Microb Ecol. 45 (3), 302-316 (2003).</li><li>Brill, J. A., Wiegel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+between+spore-forming+and+asporogenic+bacteria+using+a+PCR+and+southern+hybridization+based+method.">Differentiation between spore-forming and asporogenic bacteria using a PCR and southern hybridization based method.</a> J Microbiol Methods. 31 (1-2), 29-36 (1997).</li><li>Rueckert, A., Ronimus, R. S., Morgan, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+real-time+PCR+assay+targeting+the+sporulation+gene,+spo0A.,+for+the+enumeration+of+thermophilic+bacilli+in+milk+powder.">Development of a real-time PCR assay targeting the sporulation gene, spo0A., for the enumeration of thermophilic bacilli in milk powder.</a> Food Microbiol. 23 (3), 220-230 (2006).</li><li>Bueche, 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=Quantification+of+endospore-forming+firmicutes+by+quantitative+PCR+with+the+functional+gene+spo0A.">Quantification of endospore-forming firmicutes by quantitative PCR with the functional gene spo0A.</a> Appl Environ Microbiol. 79 (17), 5302-5312 (2013).</li><li>Wunderlin, T., Junier, T., Roussel-Delif, L., Jeanneret, N., Junier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endospore-enriched+sequencing+approach+reveals+unprecedented+diversity+of+Firmicutes+in+sediments.">Endospore-enriched sequencing approach reveals unprecedented diversity of Firmicutes in sediments.</a> Environ Microbiol Rep. 6 (6), 631-639 (2014).</li><li>Nicholson, W. L., Law, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+purification+of+bacterial+endospores+from+soils:+UV+resistance+of+natural+Sonoran+desert+soil+populations+of+Bacillus+spp.+with+reference+to+B.+subtilis+strain+168.">Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp. with reference to B. subtilis strain 168.</a> J Microbiol Methods. 35 (1), 13-21 (1999).</li></ol>

저자가 공개하는 게 없다.

외부 공격적인 물리 화학적 요인 (예를 들면, 온도 또는 세제)에 내생 포자의 저항은 환경 시료에서 식물 세포에서 세균 내생 포자를 분리하는 방법을 고안 하였다. 이것은 비파괴 방식으로 환경 시료로부터 내생 포자를 분리하는 제 광범위한 방법이다. , 양을 감지하거나 샘플 내생 포자를 분석하는 이전 방법 등 dipicolinic 산 또는 특정 마커 유전자와 같은 내생 포자에 대한 특정 프록시의 ?...

이 연구의 목적은 환경 시료에서 식물 세균 세포에서 세균 내생 포자의 분리를위한 프로토콜을 제공하는 것이다. 박테리아 내생 포자의 형성은 문 후벽 균 1 그룹에 속하는 박테리아의 수에서 발견 생존 전략, 보통 기아에 의해 유발된다. 내생 포자 형성 세균이 잘 균주의 수는 병원균 따라서 의료의 중요성 (예를 들어, 탄저균이나 클로스 트리 디움 디피)의 주로하기 때문에, 연구한다. 내생 포자 형성 박테리아의 환경 균주는 거의 모든 환경 (토양, 물, 퇴적물, 공기, 얼음, 인간의 직감, 동물의 창자 등) 1-3에서 고립되었다. 따라서, 후벽 균 배양 컬렉션 (4)에서 두 번째로 가장 풍부한 문이다. 때문에 튼튼한 외부 피질 및 보호 핵심 단백질, 내생 포자는 FR에 이르기까지 극단적 인 환경 조건을 견딜 수높은 방사선, 극한의 온도 및 유해 화학 물질 5 옴 탈수. 이 놀라운 저항은 내생 포자 6-8에서 DNA를 추출하는 도전한다. 그들은 환경 시퀀싱 연구 9,10에서 간과 된 이유는 가능성을 설명합니다. 형광 항체 (11)에 의해 환경 시료의 내생 포자의 대상과 같은 다른 방법, 토양 12, 퇴적물 13 dipicolinic 산의 정량 (DPA)은 14 유동 세포 계측법 또는 저온 살균 이후 재배 (15, 16)는에 내생 포자를 검색하거나 정량화하는데 사용되어왔다 환경 시료. 최근, 최적화 된 DNA 추출 방법뿐만 아니라, 특정 분자 프라이머 내생 포자 특정 유전자 서열은 10,17-20 개발 된 타겟팅한다. 이 박테리아 (21)이 그룹들 사이에서 더 많은 생물 다양성을 나타 내기 위해 도움이 또한 내생 포자의 검출을위한 산업 및 의학에 응용하게되었다, 우유 분말 (19)의 예를 들어. 여기에 제시된 프로토콜은 식물 세포에 대하여 내생 포자의 세균 (예 : 열 및 세제 등) 유해한 물리 화학적 조건에 저항의 차이에 기초한다. 샘플에서 식물 세포를 파괴하기 위해, 우리는 연속적으로 열, 리소자임과 세제의 낮은 농도를 적용합니다. 포자를 파괴하지만, 모든 식물 세포를 용균하지 않도록 이러한 치료의 시간과 강도를 최적화되었다. 환경 세포 풀의 일부 셀들은 다른 것보다 더 내성이므로, 모든 식물 세포 파괴의 확률을 증가시키기 위해, 우리는 세 가지 다른 치료를 적용한다. 이 방법의 장점 및 신규성은 치료 후 내생 포자는 그대로이며, 하류의 분석에 이용 될 수 있다는 것이다. 이들은 D를 감소 따라서 DNA 추출, 정량 PCR (qPCR에) 및 앰플 리콘 또는 특히 내생 포자의 그룹을 대상으로 metagenomic 시퀀싱 (및 포함iversity) 범위 증가한다. 또한 하류 내생 포자 배양 또는 형광 현미경으로 정량에 사용될 수있는, 유동 세포 계측법, 또는 DPA의 검출. 이 방법의 중요한 특징은 처리 된 샘플과 미처리 샘플과 비교하여, 하나의 셀에 대응하는 식물 성분 외에 환경 샘플에서 내생 포자의 양과 다양성을 추론 할 수 있다는 점이다.

<table><tbody><tr><td>Whatman nitrocellulose membrane filters, 0.2 &amp;#956;m pore size, 47 mm diameter</td><td>Sigma-Aldrich</td><td>WHA7182004 Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma-Aldrich</td><td>252859 Sigma-Aldrich</td><td>CAS 77-86-1</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884 Sigma-Aldrich</td><td>CAS&amp;nbsp; 60-00-4&amp;nbsp;</td></tr><tr><td>Lysozyme from chicken egg white</td><td>Sigma-Aldrich</td><td>62971 Fluka</td><td>powder, CAS&amp;nbsp; 12650-88-3&amp;nbsp;</td></tr><tr><td>Ultra-Turrax homogenizer T18 basic</td><td>IKA</td><td>3720000</td><td></td></tr><tr><td>Glass filter holders for 47&amp;nbsp;mm membranes, Pyrex glass</td><td>EMD Millipore</td><td>XX10 047 00</td><td></td></tr><tr><td>Chemical duty vacuum pump</td><td>Millipore</td><td>WP6122050</td><td>220 V/50 Hz</td></tr><tr><td>Manifold sampling filtration for 25&amp;nbsp;mm membranes</td><td>Millipore</td><td>1225 Sampling Manifold</td><td>polypropylene</td></tr><tr><td>Dnase</td><td>New England Biolabs</td><td>M0303S</td><td>Rnase free</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S5881 Sigma-Aldrich</td><td>CAS&amp;nbsp; 1310-73-2&amp;nbsp;</td></tr><tr><td>Sodium dodecyl sulfphate (SDS)</td><td>Sigma-Aldrich</td><td>L3771 Sigma&amp;nbsp;</td><td></td></tr><tr><td>Whatman nitrocellulose membrane filters, 0.2 &amp;#956;m pore size, 25 mm diameter</td><td>Sigma-Aldrich</td><td>WHA7182002 Aldrich</td><td></td></tr></tbody></table>

physical isolation of endospores from environmental samples by targeted lysis of vegetative cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

여기에 제시된 결과는 이전 10,21 발표되었다. 데이터의 환경 적 해석과 설명은 해당 문서를 참조하십시오. 전체 절차를도 1에 요약 및 세 개의 주요 단계에 해당된다 : 첫째, 침전물 또는 다른 환경적인 행렬에서의 바이오 매스의 분리; 둘째, 식물 세포의 파괴; 그리고 세 번째, 분리 된 내생 포자의 하류 분석. 하류 분석 다양성을 결정하는 DNA 추출 및 앰플...

Watch this Scientific Journal Video about Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells at JoVE.com

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

식물 세포의 표적으로 용해하여 환경 시료에서 내생 포자의 물리적 분리

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

physical-isolation-endospores-from-environmental-samples-targeted

Research

JoVE Journal

Environment

14.1K 조회수.  Macquarie University. A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

비디오: Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

배양 및 유지<em&gt; 클로 스트 리듐 디피</em&gt; 혐기성 환경에서

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

연구

면역학 및 감염병학

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats(수생 서식지에서 탄화수소 대사 특성을 가진 박테리아 종의 분리, 번식 및 식별)

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

환경과학

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

세균 포자 발아 동안 코어 텍스 조각 검출

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

Germinosomes과 새 균의 subtilis 포자에 내부 막의 시각화

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

생체공학

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

해로운 효과의 낮은 압력 플라즈마 살 균 새 균의 subtilis 포자를 사용 하 여 라이브 셀 현미경의 생존에 조사

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

생물학

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Microrespiration 분석 및 기정을 사용 하 여 Teliospore 발 아 조사

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

토양 샘플에서 내토모 병원성 곰팡이의 격리 및 선택 및 곤충 해충에 대한 곰팡이 독성의 평가

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

토양 샘플에서 엔토모병원성 곰팡이를 분리하는 방법의 비교

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

곰팡이 개체군 구조를 조사하기 위해 토양에서 배양 가능한 효모와 곰팡이를 분리합니다.

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

Mycoheterotrophic 식물에서 내생균 균류를 식별하기 위한 분리, 특성화 및 전체 DNA 추출

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...