미국 국립 과학 재단 (National Science Foundation) 동아시아 및 태평양 서머 연구소; 미국 연구 기금 프로그램의 지질 사회와 대학원 연구 장학금 졸업생 교부금이 작품은 미국 국립 과학 재단 (National Science Foundation) (EAR-0920299 및 EAR-0745808)의 보조금에 의해 지원되었다 오하이오 주립 대학에서. 우리는 자신의 통찰력 덧글에 대한 편집기와 두 개의 익명 리뷰어 감사드립니다.

1. MTB 수집 <ol><li> magnetotactic 박테리아를 수집 할 수있는 담수 사이트 (MTB)에 결정할 때, 그것은 종종 부드러운 진흙 퇴적물 층이있는 연못 또는 느리게 이동 스트림로 시작하는 것이 좋습니다. 이 예제에서 우리는 콜럼버스에있는 오하이오 주립 대학 (OSU), 오하이오 (USA)의 캠퍼스에있는 Olentangy 강의 가장자리에 샘플을 수집했습니다. 이 우리의 데모의 편리한 위치에있을 때, 여기에 설명 된 프로토콜은 수중 위치에 적용됩니다. 이 프로토콜에 사용 된 자료는 표 1에서 찾을 수 있습니다. 물 깊이는 10 100cm 사이의 위치를​​ 찾습니다. 같은 장소에서, 당신은 침전 명확한, 스크류 탑 컨테이너를 사용하는 페이지의 상단에있는 레이어를 수집해야합니다. 이 한 절반 삼분의 침전 물과 남은 볼륨이 가득 될 때까지 컨테이너에 침전물과 물을 퍼내다. 이 물이 가득 될 때까지 잠수 용기를 보관하고 단단히 컨테이너 w를 쏠i 번째의 나사 상단 뚜껑. 이 침전물을 혼합 할 필요가 없습니다. 수건으로 건조 용기의 외부를 닦아 다음 실험실에 표본을 채취하시면됩니다. 당신의 실험실로 샘플을 서두를 필요가 없습니다. 우리는 우리의 실험실로 돌아 오는 전에 몇 일 동안 현장에서 플라스틱 용기에 MTB 샘플을 그만 뒀어요. 이 분야에서 시원하고 그늘진 곳에서 샘플을 저장으로 MTB은 오래 같이 개월 몇 주 동안 가능한 있어야합니다. </li><li> 샘플은 실험실에되면, 뚜껑을 느슨하게하고 증발의 양을 줄일 수있는 용기를 덮고 두십시오. 어두운 방, 서랍에서 실온에서 컨테이너를 저장하거나 완전히 알루미늄 호일로 용기를 다룹니다. 퇴적물과 고급 입자가 완전히 몇 일 몇 시간 동안 방해받지 샘플을 출발하여 컨테이너의 하단에 정착 할 수 있도록 허용합니다. 그것은 퇴적물을 혼합 할 필요가 없습니다, MTB는 그대로 환경을 선호합니다. 플라스틱 용기의 명확한 벽이 입자가 아래로 정착했는지 확인 할 수 있습니다. 샘플에 따라 MTB는 많은 달 동안 샘플에서 살아 남아있을 수 있습니다. </li></ol> 2. MTB 절연 <ol><li> 이 약 1cm 침전물 물 인터페이스 (그림 1A) 위의 플라스틱 용기의 측면에있는 MTB, 장소 자석을 분리 할 준비가되면. 용기의 바닥에있는 퇴적물을 방해하지 않도록주의하십시오. 반대편 (그림 1A)에서 컨테이너와 다른 바 자석의 북쪽의 한쪽면에 바 자석의 남극를 놓습니다. 거의 모든 자석은 이러한 자기 저어 바 또는 대형 냉장고 자석으로 사용하실 수 있습니다. 아무거나는 침전물 물 인터페이스 위에 올바른 높이에서 자석을 지원하는 데 사용할 수 있습니다. 우리는 골판지 또는 플라스틱 상자의 상단에있는 자석을 쉬고 것이 가장 그러나, 자석은 또한 플라스틱 용기의 외부에 붙어 될 수 것으로 나타났습니다. 여러에 30 분을 기다려야박테리아 시간은 자석으로 수영합니다. </li><li> 주의 남쪽 극 바 자석 (북반구에 수집 된 샘플의 경우)의 위치 근처의 컨테이너 (그림 1A) 내부에서 물을 수집하기 위해 멸균 피펫을 사용합니다. 이 물은 남쪽 극 바 자석에 매료 된 MTB를 포함해야합니다. 다음, 모세관 경주가 더욱 MTB을 풍요롭게하는 데 사용되어야합니다. </li></ol> 3. MTB의 경마장 <ol><li> magnetotactic 박테리아와 샘플을 풍요롭게하기 위해, 모세관 경마장이 (인물 1B와 1C)가 필요합니다. 다음은 일반 플라스틱 용기에서 세포를 분리하기 전에 변경해야합니다. </li><li> 경마장을 만들기 위해 5.75 인치 (146mm) 유리 파스퇴르 피펫을 사용합니다. 피펫의 상단을 잘라 다이아몬드 펜이나 파일을 사용 피펫의 길이는 중요하지 않습니다,하지만 물을 약 1-2 ML를 포함 할 수 있습니다. 다음 팁을 녹기 분젠 버너를 사용하는 때문에는 (그림 1C) 밀봉된다. 그 결과 피펫은 오픈 엔드 및 봉인 끝이 있어야합니다. </li><li> 이 racetracks 여러 후 압력솥을 만듭니다. 또한면 여러 긴 금속 바늘을 압력솥해야합니다. </li><li> 필터링 주사기에 부착 된 긴 금속 바늘을 사용하여 autoclaved 경기장으로 그림 1A에 도시 된 침전물 물 인터페이스 근처에서 수집 한 필터링 샘플 물을, 추가 할 수 있습니다. 필터의 기공 크기가 물에서 파편과 오염 물질을 제거하기 위해 0.22 mm해야합니다. 이 모세관 유리에 기포가 없는지 철저하게 확인하는 것이 중요합니다. </li><li> 멸균면 (그림 1B)와 레이스 트랙의 하단을 연결합니다. 거리에 밀봉 된 팁 (그림 1C)에서 1cm -이게 0.5입니다 있도록 경마장의 밀봉 된 끝 부분에 솜을 밀어 금속 바늘을 사용합니다. </li><li> 멸균 피펫 사용하여 샘​​플에 MTB 함유 물을 (섹션 2.2에서) 추가 연구준비된 MTB의 경주 (그림 1B)의 eservoir (오픈 엔드). </li></ol> 4. MTB 농축 <ol><li> 경마장은 샘플 액체로 가득되면, 수평 표면에 옆으로 쏟아 붓는 (예를 들어, benchtop) 및 경마장의 봉인 팁 (인물 1B 옆에 (북반구에서) 바 자석의 남쪽 기둥을 배치 와 1C). </li><li> 면을 통해 마이그레이션 할 수있는 MTB 기준 5 ~ 30 분 정도 기다립니다. 그런 다음 경마장의 끝 근처의 유체를 수집해야합니다. 너무 오래 기다리는 것은 모세관의 끝을하는 다른 운동성 세균 등 오염 물질을, 소개 할 수 있습니다. 선택적으로, 당신은 경주의 끝을 확인하고 MTB는 경마장의 끝에서 수집 볼 가벼운 현미경을 사용할 수 있습니다. 이 당신이면 플러그를 통해 마이그레이션 할 수있는 MTB 소요 시간을 결정 할 수 있습니다. </li><li> 부드럽게면 플러그 근처의 작은 흠집을 만들어 경마장의 끝을 스냅하는 다이아몬드 칼을 사용합니다. </li> &lt;리&gt; 경마장의 끝에서 유체를 제거하는 좁은 바늘 (25 또는 27 게이지)가 1 ML의 주사기를 사용합니다. 이 액체 샘플은 현재 강화 MTB를 포함해야합니다. </li></ol> 5. 가벼운 현미경에 의한 MTB 전망대 <ol><li> coverslip 위에 강화 MTB 샘플의 드롭 (10-20 μl)를 배치합니다. </li><li> 그렇게 빨리 드롭이 현재 줄어들고 직면하고 coverslip에 달려있다 위에 coverslip을 뒤집기. </li><li> 유리 슬라이드에 휴식하는 O-링에 coverslip를 놓습니다. O-링는 약간 작은 직경 coverslip을 (; 그림 2 약 1 cm)이 있어야합니다. </li><li> 가벼운 현미경 단계에이 매달려 드롭을 배치하고 드롭 중 하나를 가장자리에 초점을 맞 춥니 다. 하지만 매달려 드롭 방식 (그림 2)을 사용하기 어려운 오일을 필요로하지 않는다 가장 높은 수치 조리개 값 (예를 들어, 0.93 NA)를 가지고 있기 때문에 60X 건조 목적은 잘 작동합니다. </li><li> 매달려 D에 바 자석 가까이의 남쪽 끝을 배치ROP와 MTB는 자석 (그림 3)에 가장 가까운 드롭의 가장자리으로 마이그레이션하기 시작합니다. 몇 분 이내에 많은 MTB는 드롭의 가장자리 (그림 3)에 있어야합니다. 박테리아가 자석의 극을 반대로하여 자기 것을 이룬 후 박테리아가 반대 방향에서 수영 관찰합니다. </li></ol> 6. 전송 전자 현미경 (TEM)에 의한 MTB 관측 <ol><li> 구리 그리드로 강화 MTB의 드롭 (~ 20 μl)을 놓고 박테리아가 10 분 동안 해결 할 수 있습니다. </li><li> 깨끗한 필터 종이를 초과 물을 위크. </li><li> 선택적으로, 그리드에 부정적인 2 % uranyl 아세테이트, 2% phosphotungstic 산성 산도 7.2, 또는 2.5 %의 나트륨 molybdate 7, 8, 9 물들 할 수 있습니다. 이것은 즉시 강화 MTB와 격자를 잠복기 후 얼룩 한 방울에 구리 그리드를 배치하여 이루어집니다. 부정적인 얼룩으로 격자를 길러, 시간은 세인트에 따라 달라집니다아니야!는 사용 후 깨끗한 필터 종이와 유체를 등심. </li><li> 전송 전자 현미경 (TEM, 그림 4)를 사용하여 MTB를 관찰. 일이 여기에 설명 된 내용은 MTB는 Formvar 안정화와 탄소 코팅 200 메쉬의 구리 격자 (테드 펠라 # 01800)에 adsorbed되었습니다. 격자는 최대 10 분까지에 세포 현탁액의 드롭 탄소 측에 아래로 배치 된 후 즉시 30 초에 물 한 방울에 격자를 배치하여 한 번 씻어. 착색를 들어, 격자는 초는 5 분, 30에 2 % uranyl 아세트산 (테드 펠라 # 19481)의 드롭 위치하고 완전히 필터 종이를 사용하여 건조 하였다. 격자는 TEM은 200kV에서 높은 각도 환형 암시 줄기를 사용하여 80kV 또는 페이 Tecnai F20에 두 페이 Tecnai 정신을 사용하여 분석 하였다. </li></ol>

<ol>
	<li>Blakemore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Science. 190, 377-379 (1975).</li><li>Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Annual Reviews in Microbiology. 36, 217-238 (1982).</li><li>Bazylinski, D. A., Frankel, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Biomineralization+of+Magnetite+(Fe3O4)+and+Greigite+(Fe3S4)+in+a+Magnetotactic+Bacterium.">Controlled Biomineralization of Magnetite (Fe3O4) and Greigite (Fe3S4) in a Magnetotactic Bacterium.</a> Applied and Environmental Microbiology. 61, 3232-3239 (1995).</li><li>Lefevre, C. T., Menguy, N., 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=A+Cultured+Greigite-Producing+Magnetotactic+Bacterium+in+a+Novel+Group+of+Sulfate-Reducing+Bacteria.">A Cultured Greigite-Producing Magnetotactic Bacterium in a Novel Group of Sulfate-Reducing Bacteria.</a> Science. 334, 1720-1723 (2011).</li><li>Simmons, S. L., Bazylinski, D. A., 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=South-seeking+magnetotactic+bacteria+in+the+Northern+Hemisphere.">South-seeking magnetotactic bacteria in the Northern Hemisphere.</a> Science. 311, 371-374 (2006).</li><li>Wolfe, R., Thauer, 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=A+'capillary+racetrack'+method+for+isolation+of+magnetotactic+bacteria.">A 'capillary racetrack' method for isolation of magnetotactic bacteria.</a> FEMS Microbiology Letters. 45, 31-35 (1987).</li><li>Rodgers, F. G., Blakemore, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+structure+in+a+many-celled+magnetotactic+prokaryote.">Intercellular structure in a many-celled magnetotactic prokaryote.</a> Archives of Microbiology. 154, 18-22 (1990).</li><li>Moench, T. T., Konetzka, W., 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=A+novel+method+for+the+isolation+and+study+of+a+magnetotactic+bacterium.">A novel method for the isolation and study of a magnetotactic bacterium.</a> Archives of Microbiology. 119, 203-212 (1978).</li><li>Balkwill, D., Maratea, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+of+a+magnetotactic+spirillum.">Ultrastructure of a magnetotactic spirillum.</a> Journal of Bacteriology. 141, 1399-1408 (1980).</li><li>Lins, U., Freitas, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+homemade+apparatus+for+harvesting+uncultured+magnetotactic+microorganisms.">Simple homemade apparatus for harvesting uncultured magnetotactic microorganisms.</a> Brazilian Journal of Microbiology. 34, 111-116 (2003).</li><li>Jogler, C., Lin, W., 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=Toward+Cloning+of+the+Magnetotactic+Metagenome:+Identification+of+Magnetosome+Island+Gene+Clusters+in+Uncultivated+Magnetotactic+Bacteria+from+Different+Aquatic+Sediments.">Toward Cloning of the Magnetotactic Metagenome: Identification of Magnetosome Island Gene Clusters in Uncultivated Magnetotactic Bacteria from Different Aquatic Sediments.</a> Applied and Environmental Microbiology. 75, 3972-3979 (2009).</li><li>Lin, W., Li, 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=Newly+Isolated+but+Uncultivated+Magnetotactic+Bacterium+of+the+Phylum+Nitrospirae+from+Beijing,+China.">Newly Isolated but Uncultivated Magnetotactic Bacterium of the Phylum Nitrospirae from Beijing, China.</a> Applied and Environmental Microbiology. 78, 668-675 (2012).</li><li>Li, J., Pan, Y., 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=Biomineralization,+crystallography+and+magnetic+properties+of+bullet-shaped+magnetite+magnetosomes+in+giant+rod+magnetotactic+bacteria.">Biomineralization, crystallography and magnetic properties of bullet-shaped magnetite magnetosomes in giant rod magnetotactic bacteria.</a> Earth and Planetary Science Letters. 293, 368-376 (2010).</li><li>Oestreicher, Z., Valerde-Tercedor, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosomes+and+magnetite+crystals+produced+by+magnetotactic+bacteria+as+resolved+by+atomic+force+microscopy+and+transmission+electron+microscopy.">Magnetosomes and magnetite crystals produced by magnetotactic bacteria as resolved by atomic force microscopy and transmission electron microscopy.</a> Micron. 43, 1331-1335 (2012).</li></ol>

관심 없음 충돌이 선언 없습니다.

Magnetotactic 박테리아는 반드시 모든 수생 환경 8 찾을 수 없습니다하지만이 발생 할 때, 100의 순서에서 찾을 수 있습니다 - 밀리리터이 1000 셀. 광학 현미경을 사용하여 MTB를 관찰하기 위해, 당신은 약 50 균 / 샘플 8 ML이 필요합니다. 샘플에 또는 몇 MTB가없는 경우, 당신은 이걸 샘플을 수집하거나 다음 섹션에서 설명하는 기술 중 하나 이상을 시도해야합니다 새로운 환경 사?...

<table border="1"><tbody><tr><td> 항목 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 유리 슬라이드 </td><td> 피셔 과학 </td><td> S95933 </td><td></td></tr><tr><td> 유리 파스퇴르 (Pasteur) pipets </td><td> 피셔 과학 </td><td> 13-678 - 6A </td><td></td></tr><tr><td> O-링 </td><td> 하드웨어 상점 </td><td></td><td></td></tr><tr><td> 커버 용지 </td><td> 피셔 과학 </td><td> 12 542B </td><td></td></tr><tr><td> 바 자석 </td><td> 피셔 과학 </td><td> S95957 </td><td></td></tr><tr><td> 컨테이너 </td><td> 어떤 </td><td></td><td> 모든 plast적어도 0.5 L를 개최 할 수 있으며 밀봉 할 수 IC 또는 유리 용기 </td></tr><tr><td> 면 </td><td> 어떤 </td><td></td><td></td></tr><tr><td> 60X 건조 렌즈 현미경 </td><td> Zeiss </td><td></td><td> 60X 건조 렌즈가 절대적으로 필요하지 않습니다,하지만이 기름을 사용하지 않고 높은 NA를 제공합니다 </td></tr><tr><td> 다이아몬드 펜 </td><td> 피셔 과학 </td><td> 08-675 </td><td></td></tr><tr><td> 0.22 mm 필터 </td><td> 피셔 과학 </td><td> 09-719C </td><td></td></tr><tr><td> 1 ML의 주사기 </td><td> 피셔 과학 </td><td> NC9788564 </td><td></td></tr><tr><td> Microcentrifuge 관 </td><td> 피셔 과학 </td><td> 02-681-320 </td><td></td></tr><tr><td> Formvar / 탄소200 메쉬, 구리 격자 </td><td> 테드 펠라 주식회사 </td><td> 01,800 </td><td></td></tr><tr><td> Uranyl 아세테이트 </td><td> 테드 펠라 주식회사 </td><td> 19,481 </td><td></td></tr><tr><td> Tecnai 정신 TEM </td><td> 페이 </td><td></td><td></td></tr><tr><td> Tecnai F20 S / TEM </td><td> 페이 </td><td></td><td></td></tr></tbody></table>

collection, isolation and enrichment of naturally occurring magnetotactic bacteria from the environment

Magnetotactic 균 (MTB)는 첫째 특히 메사추세츠 (USA)의 염 습지에서 수집 침전 샘플 1975 1에서 설명 된 수중 미생물 있습니다. 이후 MTB는 모두 세계 2 이상에서 층상 물과 침전물-열에서 발견되었습니다. 모든 MTB에 일반적인 기능들은 세포, 막 바인딩 된 자성 자철광의 nanocrystals (철 3 O 4) 및 / 또는 greigite (철 3 S 4) 또는 두 가지 모두 3 4아르 magnetosomes를 포함한다는 것입니다. 남반구에서 사람들은 보통 자석 3,5의 북쪽 끝 부분에 매력을하는 동안 북반구에서는 MTB는 일반적으로, 바 자석의 남쪽 끝 부분에 매력을 수 있습니다. 환경 샘플에서 MTB를 분리 할 때이 속성을 악용 할 수 있습니다. MTB를 풍성하게하는 가장 일반적인 방법 중 하나는, 천연 소스에서 침전물과 물을 수집하기 위해 투명한 플라스틱 용기를 사용하는 것입니다담수 연못 등. 북반구에서는, 바 자석의 남쪽 끝 단지 침전물 물 인터페이스에서 침전물 위의 컨테이너의 외부에 대한 배치됩니다. 약간의 시간 후, 박테리아는 피펫으로 자석 근처에있는 컨테이너의 내부에서 제거 할 수 있습니다 다음 모세관 경주 6 자석을 사용하여 더 값진 경험을했습니다. 일단, 풍부한 박테리아는 매달려 드롭 방법을 사용하여 현미경 슬라이드에 배치 할 수 있으며 가벼운 현미경에서 관찰 또는 구리 그리드에 입금 및 전송 전자 현미경 (TEM)을 사용하여 관찰했다. 이 방법 사용 절연 MTB는 미세 또한 수영과 같은 특성을 결정하기 위해 연구 될 수 세포, 자기 결정의 모양, magnetosomes 수, 각 셀에 magnetosome 체인의 수의 구성의 flagella, 세포 형태학의 행동 유형과 번호 nanomineral 결정하고, 세포 내 액포의 존재.

자석은 환경 샘플 (그림 1A)에 포함 된 magnetotactic 세균 (MTB)를 분리하는 데 사용할 수있는 효과적인 도구입니다. 모세관 경마장 (그림 1B)은 그들이 또한 환경 시료 내에 포함 된 비 magnetotactic 미생물에서 분리 될 수있는면 플러그인을 통해 유치를 MTB의 자기 속성을 사용합니다. <img alt="그림 1" src="/files/ftp_upload/50123/50123fig1.jpg...

Watch this Scientific Journal Video about Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment at JoVE.com

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

우리는 자연 물에 적용 할 수있는 magnetotactic 박테리아 (MTB)를 수집하는 방법을 보여줍니다. MTB는 고립과 박테리아의 자연 자기의 활용 비교적 간단한 설정을 사용하여 침전 샘플 풍부하게 할 수 있습니다. 절연 MTB 그런 다음 빛과 전자 현미경를 사용하여 자세하게 검토 할 수 있습니다.

당연히 환경에서 Magnetotactic 박테리아를 발생의 수집, 절연 및 강화

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

collection-isolation-enrichment-naturally-occurring-magnetotactic

Research

JoVE Journal

Biology

22.9K 조회수.  The Ohio State University. 우리는 자연 물에 적용 할 수있는 magnetotactic 박테리아 (MTB)를 수집하는 방법을 보여줍니다. MTB는 고립과 박테리아의 자연 자기의 활용 비교적 간단한 설정을 사용하여 침전 샘플 풍부하게 할 수 있습니다. 절연 MTB 그런 다음 빛과 전자 현미경를 사용하여 자세하게 검토 할 수 있습니다.

비디오: Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

StemCell 기술에서 RosetteSep 키트와 함께 인간의 피를에서 NK 세포의 농축

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

연구

생물학

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

미국 호르세쇼 게, Limulus Polyphemus의 혈액 수집

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

인간 혈청에서 작은 비 암호화 RNA를의 분리

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

조류 정액 컬렉션 Cloacal 마사지와 정액에서 DNA의 분리

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ability to biomineralize magnetosomes, which are magnetic nanometer-sized crystals of magnetite (Fe3O4) or greigite (Fe3S4) surrounded by a lipid bilayer membrane, within their cytoplasm. For most known magnetotactic bacteria, magnetosomes are assembled in chains inside the cytoplasm, thereby conferring a permanent magnetic dipole moment to the cells and causing them to align passively with external magnetic fields. Because of these specific features, magnetotactic bacteria have a great potential for commercial and medical applications. However, most species are microaerophilic and have specific O2 concentration requirements, making them more difficult to grow routinely than many other bacteria such as Escherichia coli. Here we present detailed protocols for growing three of the most widely studied strains of magnetotactic bacteria, all belonging to the genus Magnetospirillum. These methods allow for precise control of the O2 concentration made available to the bacteria, in order to ensure that they grow normally and synthesize magnetosomes. Growing magnetotactic bacteria for further studies using these procedures does not require the experimentalist to be an expert in microbiology. The general methods presented in this article may also be used to isolate and culture other magnetotactic bacteria, although it is likely that growth media chemical composition will need to be modified.

Growing Magnetotactic Bacteria of the Genus Magnetospirillum: Strains MSR-1, AMB-1 and MS-1

We present a procedure for growing several strains of Magnetospirillum in two different types of growth media. Magnetospirillum gryphiswaldense strain MSR-1 is grown in both liquid and O2 concentration gradient semi-solid media while M. magneticum strain AMB-1 and M. magnetotacticum strain MS-1 are grown in liquid medium.

속 Magnetospirillum의 Magnetotactic 박테리아 성장: 변종 MSR-1, 자녀-1 및 MS-1

Magnetotactic bacteria are Gram-negative, motile, mainly aquatic prokaryotes ubiquitous in freshwater and marine habitats. They are characterized by their ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

성인 뮤 린 눈물과 턱 밑 동맥에서 Myoepithelial 세포의 고립

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

오가노이드 배양을 위한 인간 폐 상피 전구 세포의 분리 및 농축

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Collection and Identification of Pollen from Honey Bee Colonies

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate pesticide exposure of bees via pollen. Described herein is an effective pollen-trapping method for collecting corbicular pollen from honey bees returning to their hives. This collection method results in large quantities of corbicular pollen that can be used for research purposes. Honey bees collect pollen from many plant species, but typically visit one species during each collection trip. Therefore, each corbicular pollen pellet predominantly represents one plant species, and each pollen pellet can be described by color. This allows the sorting of samples of corbicular pollen by color to segregate plant sources. Researchers can further classify corbicular pollen by analyzing the morphology of acetolyzed pollen grains for taxonomic identification. These methods are commonly used in studies related to pollinators such as pollination efficiency, pollinator foraging dynamics, diet quality, and diversity. Detailed methodologies are presented for collecting corbicular pollen using pollen traps, sorting pollen by color, and acetolyzing pollen grains. Also presented are results pertaining to the frequency of pellet colors and taxa of corbicular pollen collected from honey bees in five different cropping systems.

We describe methods for collecting corbicular pollen from honey bees as well as protocols for color sorting, acetolysis, and microscope slide preparation of pollen for taxonomic identification. In addition, we present pellet color and taxonomic diversity of corbicular pollen collected from five cropping systems using pollen traps.

꿀벌 식민지에서 꽃가루의 수집 및 식별

Researchers often collect and analyze corbicular pollen from honey bees to identify the plant sources on which they forage for pollen or to estimate ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

도시 환경 면봉 - 환경 저수지에서 SARS-CoV-2의 샘플링 및 검출을 위한 파이프라인

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

진핵 세포에서 번역 단지의 신속한 생체 내 고정 및 격리

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...