이 프로토콜의 개발을위한 자금 조달은 고등 교육의 남동부 펜실베니아 컨소시엄 Cabrini 대학 과학 학부에 의해 제공되었다. 추가 자금 지원은 아카디아 대학과 Immaculata 대학에서왔다. 우리는 특히 친절하게 우리의 고립 된 파지의 전자 현미경 이미지 촬영을 위해 세인트 조셉 대학에서 박사 카렌 Snetselaar 감사합니다. 추가 지원은 유전체학과 진화 과학 (SEA-파지) 프로그램을 진행하는 하워드 휴즈 의학 연구소 과학 교육 얼라이언스 파지 사냥꾼에 의해 제공되었다.

파지 격리를위한 Arthobacter 세포의 1. 준비 <ul><li> 문화 박터 특검팀. KY3901 식민지 2 ~ 3 일 동안 30 ° C에서 배양 루리아 Bertaini (LB) 한천 플레이트 상에 스트리킹. 식민지를 선택하고 30 ° C에서 225 rpm으로 진탕 배양기에서 당황 문화 플라스크에 LB 배지 250 ml의에 추가하고 부화 멸균 루프를 사용합니다. </li><li> 성장의 약 24 시간이 파지 감염 실험 후반 초반 / 지수 정지상 세포를 얻을 수 있습니다. 늦은 성장 정지 상태로 들어가는 중간 세포 않도록 밀접 세균 성장​​의 상태를 모니터한다. 주 : 세포 생장 후술 파지 분리 및 정제 절차 / 단계 0.5 .0.7 사이 광학 밀도 OD (600)로 구성한다. </li></ul> 토양 샘플에서 파지 2. 컬렉션 <ol><li> 원하는 위치에서 토양의 200~400g를 수집합니다. 참고 : Arthroba 세균 이후CTER의 속을 감염 파지가에있는 토양의 대부분의 유형에 만연 될 수있다 그들과 일반적인 토양 박테리아에게 있습니다. </li><li> PB가의 있도록 최소 200 ml의 토양에 파지 버퍼 (PB) (68)의 NaCl, 10mM 황산, 10 mM 트리스 - CL (PH 7.5)] 적어도 400 ML을 추가하고 충분히 큰 플라스크에 혼합 상층 액과 추출 할 수있다. </li><li> 토양이 일시 중단 될 때까지 부드럽게 교반 또는 소용돌이에 의해 혼합 및 토양 침전물, 일반적으로 30 분을 해결 할 수 있습니다. </li><li> 토양 침전물 파편을 제거하기 위해 중력 여과 필터 종이를 통해 &quot;토양 추출물&quot;을 전달합니다. </li><li> LB 분말을 추가하고 2 % 용액 (200 ml의 토양 추출물 4g LB 분말)을 만들기 위해 혼합. </li><li> 멸균 여과 액을 얻기 위해 진공 여과에 의해 0.22 ㎛의 필터를 통해 용액을 통과. 가능하면, 즉시 2.7 단계로 진행합니다. 가능한 파지 입자의 저장 진행하기 전에 주까지 4 ° C에서 샘플을 필터링 할 필요가있는 경우, 그러나, 수감소 될 것이다. </li><li> 나누어지는 다수의 기법을 이용하여 무균 배플 배양 플라스크를 흔들면서 멸균 개별 250 ㎖ 중에 필터 멸균 LB / 토양 추출물 혼합물을 50 ml의 부분. 다른 박터 파지 다른 CaCl2를 조건 하에서 최적의 성장 때문에 원하는 최종 농도 (0-50 mM)을 멸균 2.0 M CaCl2를 솔루션을 추가합니다. 참고 : 우리는 우리가 확인 된 박터 파지의 대부분이 1 MM-2.5 mM의 염화칼슘 2의 범위 내에있는 것을 찾을 수 있습니다. 그러나, 고립 된 파지 중 일부는 매우 높은 염화칼슘이 농도에서 0 mM의 CaCl2를 불러야에서 독점적으로 우리가 성장했다. 3.1 단계로 바로 진행합니다. </li></ol> 3. 파지 격리 <ol><li> 플라스크에 각각 후반 초반 / 지수 정지상 박테리아 문화의 1-2.5 ML을 추가합니다. 0.5-0.7 OD (600)의 경우, 세포를 1 ㎖를 사용합니다. 0.7의 OD보다 높은 OD (600)의 2.5 mL의 사용세포. 참고 : 지수 상 성장 세포는 일반적으로 더 파지과 정지상 성장 세포보다 더 높은 역가를 얻을 수 있습니다. </li><li> 진탕 배양기에서 약 24 시간 동안 30 ° C에서 250 rpm에서 플라스크를 흔들어. </li><li> 24 시간의 잠복기 후 흔들림이 인큐베이터에서 농축 플라스크를 제거하고 농축 샘플을 파지 버퍼에 10 배 희석. </li><li> 후반 초반 / 지수 정지상 박테리아 문화의 0.5 ml의 문화 튜브를 설정하고 다양한 농축 문화의 양 문화 튜브에 (PB의 10 -1 희석 500 μL 5 μL)를 추가합니다. 주 : 각 토양 시료를 농축 한 후에 다른 파지 역가를 생성 이후 농도의 넓은 범위를 테스트하는 것이 바람직하다. </li><li> 원래 농축 플라스크의 농도 현재까지 최종 농도가 동일하게 문화 튜브에 염화칼슘 (2)를 추가합니다. w 많은 파지를 위해 선택하는 다른 염화칼슘 2 농도의 범위를 사용하여i 번째 염화칼슘 2 종속성을 변화. 주 : 대부분의 파지는 1-2.5 mm의 염화칼슘이 범위 내에서 고립되지만 0-50 mM의 염화칼슘이 농도 어디서나 가장 최적의 고립 파지가 있습니다. 이로 인해 그것의 독특한 절연 박터 파지의 수를 최대화하기 위해 CaCl2를 농도의 범위를 확인하는 것이 최상이다. </li><li> 문화 튜브에 LB 위로 한천의 4.5 ml의 혼합 및 LB 한천 접시에 부어 준다. 균일하게 접시에 걸쳐 배포하고 약 15 분 동안 응고 최고 한천 수 있도록 부드럽게 소용돌이. 주 : 한천 큰 일괄 (250 mL) 및 추가 상부 한천 제조 할 수 인기 RT에 저장된 적어도 2 주 동안 후속 실험을 위해 재사용 될 수있다. 상단 한천을 만드는 데 사용 다른 공식이 있지만, 우리는 (LB)와 혼합 한천 7g / L을 사용하여 한천 상단을 60 ° C의 물 중탕에 넣어 상부 한천 실험 과정에서 액체 상태를 유지한다. </li><li> 반전 t그는 판과 원하는 온도 48 시간에 (온도 30 ° C) O / N 품어. 현재 파지를 최적화하려면 다음 조건이 다릅니다. 가장 고립 된 파지는 24 시간의 잠복기 후 1-2.5 mM의 염화칼슘이 농도에서 30 ° C에서 배양 접시에서 왔습니다. </li></ol> 4. 살균 정화 <ol><li> 접시에 플라크 형성을위한 24 시간 배양 검사 후. 더 플라크가 분명하지 않는 경우 최대 72 시간 동안 배양을 계속하거나 추가 플라크가 나타납니다 있는지 확인합니다. 일주일에 최대 4 ° C에서 계속 진행하기 전에에 대한 추정 플라크와 저장 판을 필요로합니다. 참고 : 한 접시에 다른 플라크 형태학의 다양한를 찾는 것은 어려운 일이 아니다. 배양 3 일 후 플라크를 찾는 것은 가능하지만 드문 경우입니다. </li><li> 줄무늬 플라크 방법을 사용하여 명확하게 고립 된 플라크에서 원하는 파지를 정화. 멸균 나무 막대기로 감사패를 터치합니다. 행진, 불꽃을 통해 나무 막대기를 실행 allowin에 의한 플라크g 스틱 간략하게 냉각하고, 그림 1과 같이 다음 부드러운 모션 한천 접시에 스틱을 바르고. 각 패스에 대한 깨끗한 스틱을 사용하여이 작업을 반복합니다. </li><li> 또한, 개별 파지 플라크를 분리하기 위해 기존의 플라크 역가 분석을 사용합니다. 플라크 역가 분석은 파지 플라크 격리를위한 페트리 접시 등의 LB 한천과 LB 위로 한천 및 재료 등 이상의 시약의 사용을 필요로한다. 우리는 행진 플라크 방법을 사용하여 매우 성공적이었다하지만 플라크 역가 분석을 사용하여 개별 플라크를 분리하는 것이 기술적으로 쉽다. </li><li> LB 한천 플레이트를 3 회 이상 스트리킹되면, 추정 된 파지 입자의 낮은 농도를 갖는 영역을 표시하고 부드럽게 상위 판으로서 염화 칼슘의 동일한 농도를 함유하는 (박터 0.5ml의 용융 LB 탑 아가의 4.5 ml의 적용 )과는 접시에 걸쳐 균등하게 할 수 있습니다. </li><li> 반전 및 / N을 판 O를 품어. 줄무늬 플라크를 반복순수한 파지 종을 보장하기 위해 적어도 세 번 반복에 대한 기술은 격리됩니다. 파지 생존 능력의 상당한 손실없이 줄무늬 사이에 필요한 주까지로 4 ° C에서 보관 판. </li><li> 일단 원하는 플라크가 최종 줄무늬 접시에 성장 고립 된, 마이크로 피펫 팁 하나 잘 격리 된 플라크를 터치하고 100 μL의 PB에 다시 일시 중지합니다. 직렬 신선한 PB 180 μL로 샘플의 20 μl를 전달하여 10-8 희석이 10 0 솔루션을 희석. </li><li> 파지를 분리하는데 사용 염화칼슘 (2)의 동일한 농도를 함유 용융 LB 상부 한천 4.5 ml로 감염된 세균을 혼합하여 실온에서 10 분 플레이트 5 배양 관에서 성장 박터 0.5ml를 각 희석액 10 μl를 추가 원래의 토양 샘플에서. 다음 날까지 4 ° C에서 만든 모든 희석을 저장합니다. </li><li> 전통적인 플라크 역가 등을 사용하여 웹 패턴을 확인하는 데 필요한 시리얼 희석 된 파지를 결정말한다. 플라크 역가 분석의 목적은 웹 형판을 얻기 위해 필요한 파지 역가를 결정하는 것이다. 등 대부분의 세균없는 아직 파지에 의해 용해되지 않은 세균의 잔해를 포함하는 웹 패턴 플레이트를 확인합니다. 멸균 50 ㎖ 문화 플라스크에 후반 초반 / 지수 고정 박터 문화의 5 ML을 추가합니다. <ol><li> 문화 플라스크에 희석 (즉 박터 문화에 웹 패턴을 준)의 100 μl를 추가하고 파지는 실온에서 5 ~ 10 분 동안 박테리아 세포를 감염 할 수 있습니다. 처음 파지를 식별하고 10 신선한 LB 플레이트 상에 그 혼합물을 5 mL를 플레이트에 사용 된 염화칼슘 (2)의 동일한 농도를 함유 용융 LB 한천 위쪽으로 섞는다. 응고 및 30 ° C에서 거꾸로 판을 배양 할 수 있습니다. </li></ol></li><li> 다음 날, 4 ° C에서 PB 및 저장 O / N 5 ㎖ 또는 실온에서 4 시간으로 웹 패턴 판을 홍수. </li><li> 0.22 μm의 주사기 필터로 파지 해물 필터4 ℃에서 저장한다. 이러한 파아지 입자의 전자 현미경 화상, 표준 절차를 이용하여 DNA를 제한 효소 분석 및 게놈 시퀀싱 파지 게놈 DNA 분리와 같은 추가적인 실험의 마지막 파지 스톡을 사용. 파지 주식의 장기 저장을 위해, -80 ° C에서 재고 보관을위한 작은 튜브에 멸균 글리세롤로 파지 해물의 동일한 금액을 추가합니다. 참고 :이 파지 기술과 경험하지 않은 사람들을 위해, 훌륭한 자원은 온라인으로 액세스 할 수 www.phagesdb.org 웹 사이트입니다. </li></ol>

<ol>
	<li>Shapir, N., Mongodin, E. F., Sadowsky, M. J., Daugherty, S. C., Nelson, K. E., Wackett, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Catabolic+Pathways:+Genomic+Insights+into+Microbial+s-Triazine+Metabolism.">Evolution of Catabolic Pathways: Genomic Insights into Microbial s-Triazine Metabolism.</a> J Bacteriol. 189 (3), 674-682 (2007).</li><li>Qingyan, L., Ying, L., Xikun, Z., Baoli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Atrazine+Degrading+Arthrobacter+sp.+AD26+and+Use+of+this+Strain+in+Bioremediation+of+Contaminated+Soil.">Isolation and Characterization of Atrazine Degrading Arthrobacter sp. AD26 and Use of this Strain in Bioremediation of Contaminated Soil.</a> J Environ Sci. 20, 1226-1230 (2008).</li><li>Wang, J., Zhu, L., Liu, A., Ma, T., Wang, Q., Xie, H., Wang, J., Jiang, T., Zhao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+an+Arthrobacter+sp.+Strain+HB-5+that+Transforms+Atrazine.">Isolation and characterization of an Arthrobacter sp. Strain HB-5 that Transforms Atrazine.</a> Environ Geochem Hlth. 33, 259-266 (2011).</li><li>Mages, I. S., Frodl, R., Bernard, K. A., Funke, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identities+of+Arthrobacter+spp.+And+Arthrobacter-Like+Bacteria+Encountered+in+Human+Clinical+Specimens.">Identities of Arthrobacter spp. And Arthrobacter-Like Bacteria Encountered in Human Clinical Specimens.</a> J Clin Microbiol. 46 (9), 2980-2986 (2008).</li><li>Brown, D. R., Holt, J. G., Pattee, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Arthrobacter+Bacteriophages+and+Their+Application+to+Phage+Typing+of+Soil+Arthrobacters.">Isolation and Characterization of Arthrobacter Bacteriophages and Their Application to Phage Typing of Soil Arthrobacters.</a> Appl Environ Microbiol. 35 (1), 185-191 (1978).</li><li>Petrovski, S., Seviour, R. J., Tillett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+Gordonia+and+Nocardia+Stabilized+Foam+Formation+by+Using+Bacteriophage+GTE7.">Prevention of Gordonia and Nocardia Stabilized Foam Formation by Using Bacteriophage GTE7.</a> Appl Environ Microbiol. 77 (21), 7864-7867 (2011).</li><li>Le Marrec, C., Moreau, S., Loury, S., Blanco, C., Trautwetter, 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+Characterization+of+Site-specific+Integration+Functions+of+phi+AAU2+infecting+&#8220;Arthrobacter+aureus&#8221;+C70.">Genetic Characterization of Site-specific Integration Functions of phi AAU2 infecting &#8220;Arthrobacter aureus&#8221; C70.</a> J Bacteriology. 178 (7), 1996-2004 (1996).</li><li>Casida, L. E., Liu, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthrobacter+globiformis+and+Its+Bacteriophage+in+Soil.">Arthrobacter globiformis and Its Bacteriophage in Soil.</a> J Appl Microbiol. 28 (6), 951-959 (1974).</li><li>Einck, K. H., Pattee, P. A., Holt, J. G., Hagedorn, C., Miller, J. A., Berryhill, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+a+Bacteriophage+of+Arthrobacter+globiformis.">Isolation and Characterization of a Bacteriophage of Arthrobacter globiformis.</a> J Virol. 12 (5), 1031-1033 (1973).</li><li>Twest, R., Kropinski, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+Enrichment+from+Soil+and+Water.">Bacteriophage Enrichment from Soil and Water.</a> Methods Mol Bio. 501, 15-21 (2009).</li><li>Fullner, K. J., Hatfull, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacteriophage+L5+Infection+of+Mycobacterium+bovis+BCG:+Implications+for+Phage+Genetics+in+the+Slow-growing+Mycobacteria.">Mycobacteriophage L5 Infection of Mycobacterium bovis BCG: Implications for Phage Genetics in the Slow-growing Mycobacteria.</a> Mol Microbiol. 26 (4), 755-766 (1997).</li><li>Robb, S. M., Woods, D. R., Robb, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+Growth+Characteristics+on+Stationary+Phase+Achromobacter+cells.">Phage Growth Characteristics on Stationary Phase Achromobacter cells.</a> J Gen Virol. 41 (2), 265-272 (1978).</li><li>Bolger-Munro, M., Cheung, K., Fang, A., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T4+Bacteriophage+Average+Burst+Size+Varies+with+Escherichia+coli+B23+Cell+Culture+Age.">T4 Bacteriophage Average Burst Size Varies with Escherichia coli B23 Cell Culture Age.</a> Journal of Experimental Microbiology and Immunology. 17, 115-119 (2013).</li></ol>

저자는 더 경쟁 재정적 이익을 선언하지 않습니다.

많은 이전의 시도는 호스트 박터 감염시킬 파아지를 분리에도 불구하고, 우리는 표준 농축 과정을 사용하여 약간의 성공을 거두었 다. 개발 환경 시료로부터 파지를 풍부하게하고 반 Twest Kropinski (10)에 의해 구성된 박테리아의 농축 방법은 일반화 농축 과정의 대부분 기초 남아있다. 이전의 연구에서 증거가 직접 도금의 방법은 분리 (5)의 매우 낮은 성공률에도 불구하고 <em...

토양 환경에서 박터 종의 편재는 호스트 박테리아의이 종으로부터 격리 될 수있는 파지의 광대 한 수와 다양성을 제공합니다. Acintobacteriaceae 가족의 세균 회원 아트라진 및 기타 다양한 살충제와 제초제 1,2,3 등의 난 분해성 화합물을 분해 자신의 이화 작용 경로에 가장 주목할 만하다. 대부분의 연구가 박터의 환경 균주를 사용하여 수행 하였지만,이 속에의 임상 분리는 혈액, 소변, 눈, 모든 계통 발생 학적 이질성 (4)를 표시하는 많은 다른 사람의 소스에서 발견된다. 박터 세균에 대한 연구의 다소 광범위한 몸이 있지만, 단지 소수의 연구는 이러한 다양한 속의 구성원을 감염시킬 파아지에보고한다. 흥미롭게도하지만, 박터에 이전 할 작품은 &lt;토양의 입력으로 몇 가지 주요 별개의 주제에 대한 접촉을 파지EM&gt; 박터 종 활성 오니 처리 설비 (6)에 해로운 거품 감소 및 작업 부위 특이 적 재조합 인테그라 7 유전자를 강조 할 목적으로 5 공업용. 다양한 농축 기술 프로토콜 순수한 파지 박터 종에서 분리 생성하는데 이용되어왔다. 초기 절차는 감염 A. 할 수있는 파지 8 일년 이상주는 상승의 기간 동안 니코틴 염과 같은 추가 독성 제와 토양의 배양을 포함 globiformis. 불안정한 유기물 플라크 분석 기술을 통해 검출 된 파지를 생산하기 위해 등장과 함께 흙을 사용하여 수행 할 연구는 긴 잠복기 (8)을 생략하고, 여과 된. 흥미롭게도하지만, 직접 도금을 닮은 기술은 낮은 성공률과 과거의 연구를 인용, 여전히 연구자 (5)에 의해 현저하게 낮은 성공률을하면서 여러 파지에 이르게 된 과거에 사용되었다 <s&gt; 8입니다. 전반적으로, 과거에 사용 된 분리 기술은 가장 일반적인 호기성 토양을 나타내는 박터 속에도 불구하고 실제로는 약간의 효과를 갖는 떠들썩했었다 자연 4,9에서 분리, 물과 토양에서 파지를 분리하는 반 Twest 및 Kropinski 10 현재 농축 방법 환경 박테리아 균주 만이 우라늄 농축 기술을 풍부하게하는 데 사용 이전 기술에서 적응은 박터 파지를 분리 비효율적 증명했다. 여기에 설명 된 방법의 목적은 이른 농축 방법은 일관되고 효과적으로 박테리아 속에서 파지를 단리와 연관된 이전의 기술적 과제를 극복 박터 파아지를 분리하도록 채택 될 수 있음 &quot;개념 증명&quot;보여주기위한 것이다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>LB Broth powder</td>
			<td>Fisher</td>
			<td>BP9722-2</td>
			<td>It&#39;s best to order these in bulk.</td>
		</tr>
		<tr>
			<td>Granulated Agar</td>
			<td>Fisher</td>
			<td>BP1423-2</td>
			<td>It&#39;s best to order these in bulk.</td>
		</tr>
		<tr>
			<td>0.22 um syringe filters</td>
			<td>Fisher</td>
			<td>09-719A</td>
			<td></td>
		</tr>
		<tr>
			<td>.22 um buchner filters</td>
			<td>Fisher</td>
			<td align="right">430320</td>
			<td>More than 50 mL of liquid can be obtained by carefully swapping the receiving tube.</td>
		</tr>
		<tr>
			<td>Eppendorf Tubes</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL pipets individual</td>
			<td>Fisher</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Fisher</td>
			<td align="right">76002844</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Fisher</td>
			<td align="right">76002845</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL pipets individual</td>
			<td>Fisher</td>
			<td>13-676-10J</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL pipets individual</td>
			<td>Fisher</td>
			<td>13-676-10K</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman qualitative filter paper</td>
			<td>Fisher</td>
			<td>1001-824</td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized enrichment technique for the isolation of arthrobacter bacteriophage species from soil sample isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

박터 파지에 대한 개선 농축 기술의 재현성을 입증하기 위해, 30 개 토양 샘플은 고유 박터 파지이 우라늄 농축을 사용하여 수집 한 토양 샘플의 22에서 얻었다이 30 토양 샘플의 2014 년 봄과 여름 동안 서로 다른 시간과 장소에서 사용되었다 절차. 표준 농축 과정은 동일한 토양 시료로부터 3 고유 파지를 수득 하였다. 농축 샘플 플라크 또는 플라크의 성공적인 검출 용 풍부 박터의<...

Watch this Scientific Journal Video about An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates at JoVE.com

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

의 분리를위한 최적화 된 농축 기법<em&gt; 박터</em&gt; 박테리오파지 생물 종 토양 샘플에서 격리 된 것

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

an-optimized-enrichment-technique-for-isolation-arthrobacter

Research

JoVE Journal

Environment

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

19.3K 조회수.  University of the Sciences. We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

비디오: An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

연속의 설계 및 운영<sup&gt; 13</sup&gt; C 및<sup&gt; 15</sup통일 또는 차등을위한&gt; N 레이블 상공 회의소, 대사 및 구조, 식물 동위 원소 라벨링

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

연구

환경과학

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use due to increased demand for landscape irrigation. Improper irrigation practices and large rainfall events can result in runoff from urban landscapes which has potential to carry nutrients and sediments into local streams and lakes where they may contribute to eutrophication. A 1,000 m2 facility was constructed which consists of 24 individual 33.6 m2 field plots, each equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated urban landscapes. Runoff volumes from the first and second trials had coefficient of variability (CV) values of 38.2 and 28.7%, respectively. CV values for runoff pH, EC, and Na concentration for both trials were all under 10%. Concentrations of DOC, TDN, DON, PO4-P, K+, Mg2+, and Ca2+ had CV values less than 50% in both trials. Overall, the results of testing performed after sod installation at the facility indicated good uniformity between plots for runoff volumes and chemical constituents. The large plot size is sufficient to include much of the natural variability and therefore provides better simulation of urban landscape ecosystems.

This paper describes the design, construction, and function of a 1,000 m2 facility containing 24 individual 33.6 m2 field plots equipped for measuring total runoff volumes with time and collection of runoff subsamples at selected intervals for quantification of chemical constituents in the runoff water from simulated home lawns.

도시 유출의 연구 시설의 설계 및 건설

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to two families: Steinernematidae and Heterorhabditidae. Until now, more than 70 species have been described in the Steinernematidae and there are about 20 species in the Heterorhabditidae. The nematodes have a mutualistic partnership with Enterobacteriaceae bacteria and together they act as a potent insecticidal complex that kills a wide range of insect species.
Herein, we focus on the most common techniques considered for collecting EPN from soil. The second part of this presentation focuses on the insect-baiting technique, a widely used approach for the isolation of EPN from soil samples, and the modified White trap technique which is used for the recovery of these nematodes from infected insects. These methods and techniques are key steps for the successful establishment of EPN cultures in the laboratory and also form the basis for other bioassays that consider these nematodes as model organisms for research in other biological disciplines. The techniques shown in this presentation correspond to those performed and/or designed by members of S. P. Stock laboratory as well as those described by various authors.

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Entomopathogenic nematodes are soil-inhabiting roundworms that parasitize a wide range of insects. We demonstrate sampling methods used for the isolation of these nematodes from the soil using two techniques: the insect baiting and the modified White trap, for the recovery of nematodes from soil samples and infected insect cadavers, respectively.

토양 샘플링과 곤충 병원성 선충의 분리 (Steine​​rnematidae, Heterorhabditidae)

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most experiments examine only two or a few levels of CA concentration and a single soil type, but if CA can be varied as a gradient from subambient to superambient concentrations on multiple soils, we can discern whether past ecosystem responses may continue linearly in the future and whether responses may vary across the landscape. The Lysimeter Carbon Dioxide Gradient Facility applies a 250 to 500 &#181;l L-1 CA gradient to Blackland prairie plant communities established on lysimeters containing clay, silty clay, and sandy soils. The gradient is created as photosynthesis by vegetation enclosed in in temperature-controlled chambers progressively depletes carbon dioxide from air flowing directionally through the chambers. Maintaining proper air flow rate, adequate photosynthetic capacity, and temperature control are critical to overcome the main limitations of the system, which are declining photosynthetic rates and increased water stress during summer. The facility is an economical alternative to other techniques of CA enrichment, successfully discerns the shape of ecosystem responses to subambient to superambient CA enrichment, and can be adapted to test for interactions of carbon dioxide with other greenhouse gases such as methane or ozone.

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 &#181;l L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling.

CO<sub&gt; 2</sub테스트 주식에 대한&gt; 농도 구배 시설<sub&gt; 2</sub초원 생태계 기능에&gt; 농축 및 토양 효과

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

절연 및 토양, Rhizosphere, 및 다년생 잔디 실험에 뿌리에서 미생물 커뮤니티의 분석

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

루트 성장 및 지역화 된 양분에 응답을 시각화 하는 최적화 된 Rhizobox 프로토콜

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

독성물질의 트랜스 및 다세대 효과를 연구하기 위한 예쁜꼬마선충 사용

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

15개의질소 분석을 위한 마이크로플롯 설계 및 식물 및 토양 샘플 준비

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Electrostatic Method to Remove Particulate Organic Matter from Soil

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of roots and plant material from soil can result in highly variable carbon measurements. Methods to remove the plant material are often limited to the largest, most visible plant materials. In this manuscript we describe how electrostatic attraction can be used to remove plant material from a soil sample. An electrostatically charged surface passed close to dry soil naturally attracts both undecomposed and partially decomposed plant particles, along with a small quantity of mineral and aggregated soil. The soil sample is spread in a thin layer on a flat surface or a soil sieve. A plastic or glass Petri dish is electrostatically charged by rubbing with polystyrene foam or nylon or cotton cloth. The charged dish is passed repeatedly over the soil. The dish is then brushed clean and recharged. Re-spreading the soil and repeating the procedure eventually results in a diminishing yield of particulates. The process removes about 1 to 5% of the soil sample, and about 2 to 3 times that proportion in organic carbon. Like other particulate removal methods, the endpoint is arbitrary and not all free particulates are removed. The process takes approximately 5 min and does not require a chemical process as do density flotation methods. Electrostatic attraction consistently removes material with higher than average C concentration and C:N ratio, and much of the material can be visually identified as plant or faunal material under a microscope.

Removing recently deposited and incompletely decomposed plant material from soil samples reduces the influence of temporary seasonal inputs on soil organic carbon measurements. Attraction to an electrostatically charged surface can be used to quickly remove a substantial amount of particulate organic matter.

토양에서 미립자 유기물을 제거하는 정전기 방법

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of ...

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a significant role in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. The cell wall is primarily composed of cellulose, lignin, hemicellulose and pectin. Recently plant cell walls have been targeted for the second-generation biofuel and bioenergy production. Specifically, the cellulose component of the plant cell wall is used for the production of cellulosic ethanol. Estimation of cellulose content of biomass is critical for fundamental and applied cell wall research. The Updegraff method is simple, robust, and the most widely used method for the estimation of crystalline cellulose content of plant biomass. The alcohol insoluble crude cell wall fraction upon treatment with Updegraff reagent eliminates the hemicellulose and lignin fractions. Later, the Updegraff reagent resistant cellulose fraction is subjected to sulfuric acid treatment to hydrolyze the cellulose homopolymer into monomeric glucose units. A regression line is developed using various concentrations of glucose and used to estimate the amount of the glucose released upon cellulose hydrolysis in the experimental samples. Finally, the cellulose content is estimated based on the amount of glucose monomers by colorimetric anthrone assay.

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

Updegraff 방법을 사용하여 식물 바이오매스의 크리스탈 셀룰로오스 함량 추정

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...