The authors thank Dr. Yoav Barak from the Chemistry Faculty, Weizmann Institute, for support and advice. This study was supported by the Israel Science Foundation (grant # 913/12), and by the Minerva Foundation with funding from the Federal German Ministry for Education and Research.

<ol>
	<li>Lodge, J. P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods of Air Sampling and Analysis. , (1988).</li><li>Costa, V., 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=Characteristics+of+carbonaceous+aerosols+in+Emilia-Romagna+(Northern+Italy)+based+on+two+fall/winter+field+campaigns.">Characteristics of carbonaceous aerosols in Emilia-Romagna (Northern Italy) based on two fall/winter field campaigns.</a> Atmos. Res. , (2015).</li><li>Brent, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development, enhancement, and evaluation of aircraft measurement techniques for national ambient air quality standard criteria pollutants [dissertation]. , (2014).</li><li>Okuda, T., Schauer, J. J., Shafer, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+elemental+analysis+of+atmospheric+aerosols+for+evaluating+human+health+impacts+of+aerosols+in+East+Asia.">Improved methods for elemental analysis of atmospheric aerosols for evaluating human health impacts of aerosols in East Asia.</a> Atmos. Environ. 97, 552-555 (2014).</li><li>Hospodsky, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+airborne+fungal+and+bacterial+concentrations+and+emission+rates+in+six+occupied+children's+classrooms.">Characterizing airborne fungal and bacterial concentrations and emission rates in six occupied children's classrooms.</a> Indoor air. , (2014).</li><li>Bottos, E., Woo, A., Zawar-Reza, P., Pointing, S., Cary, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+Bacterial+Populations+Above+Desert+Soils+of+the+McMurdo+Dry+Valleys,+Antarctica.">Airborne Bacterial Populations Above Desert Soils of the McMurdo Dry Valleys, Antarctica.</a> Microb. Ecol. 67, 120-128 (2014).</li><li>Ovadnevaite, 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=Submicron+NE+Atlantic+marine+aerosol+chemical+composition+and+abundance:+Seasonal+trends+and+air+mass+categorization.">Submicron NE Atlantic marine aerosol chemical composition and abundance: Seasonal trends and air mass categorization.</a> J. Geophys. Res. Atmos. 119, (2014).</li><li>Lodge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ES&amp;T+Books:+Methods+of+Air+Sampling+and+Analysis,+3rd+ed.">ES&amp;T Books: Methods of Air Sampling and Analysis, 3rd ed.</a> Environ. Sci. Technol. 23, 938 (1989).</li><li>Pramod, K., Baron, P. A., Willeke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerosol Measurement: Principles, Techniques, and Applications. , (2011).</li><li>Vincent, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerosol Sampling: Science, Standards, Instrumentation and Applications. , (2007).</li><li>Duquenne, P., Marchand, G., Duchaine, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Endotoxins+in+Bioaerosols+at+Workplace:+A+Critical+Review+of+Literature+and+a+Standardization+Issue.">Measurement of Endotoxins in Bioaerosols at Workplace: A Critical Review of Literature and a Standardization Issue.</a> Ann. Occup. Hyg. 57, 137-172 (2013).</li><li>Okuda, T., Schauer, J. J., Shafer, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+elemental+analysis+of+atmospheric+aerosols+for+evaluating+human+health+impacts+of+aerosols+in+East+Asia.">Improved methods for elemental analysis of atmospheric aerosols for evaluating human health impacts of aerosols in East Asia.</a> Atmos. Environ. 97, 552-555 (2014).</li><li>Lewtas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Air+pollution+combustion+emissions:+Characterization+of+causative+agents+and+mechanisms+associated+with+cancer,+reproductive,+and+cardiovascular+effects.">Air pollution combustion emissions: Characterization of causative agents and mechanisms associated with cancer, reproductive, and cardiovascular effects.</a> Mutat. Res.-Rev. Mutat. 636, 95-133 (2007).</li><li>Lang-Yona, N., Lehahn, Y., Herut, B., Burshtein, N., Rudich, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+aerosol+as+a+possible+source+for+endotoxins+in+coastal+areas.">Marine aerosol as a possible source for endotoxins in coastal areas.</a> Sci. Total. Environ. 499, 311-318 (2014).</li><li>Levin, J., Bang, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clottable+protein+in+Limulus:+its+localization+and+kinetics+of+its+coagulation+by+endotoxin.">Clottable protein in Limulus: its localization and kinetics of its coagulation by endotoxin.</a> Thromb. Diath. Haemorrh. 19, 186-197 (1968).</li><li>Heid, C. A., Stevens, J., Livak, K. J., Williams, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+quantitative+PCR.">Real time quantitative PCR.</a> Genome Res. 6, 986-994 (1996).</li><li>O'Dowd, C. D., de Leeuw, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+aerosol+production:+a+review+of+the+current+knowledge.">Marine aerosol production: a review of the current knowledge.</a> Phil. Trans. R. Soc. A. 365, 1753-1774 (2007).</li><li>O'Dowd, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenically+driven+organic+contribution+to+marine+aerosol.">Biogenically driven organic contribution to marine aerosol.</a> Nature. 431, 676-680 (2004).</li><li>Yang, R., Paparini, A., Monis, P., Ryan, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+next-generation+droplet+digital+PCR+(ddPCR)+with+quantitative+PCR+(qPCR)+for+enumeration+of+Cryptosporidium+oocysts+in+faecal+samples.">Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples.</a> Int. J. Parasitol. 44, 1105-1113 (2014).</li><li>Stetzenbach, L. D., Buttner, M. P., Cruz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+enumeration+of+airborne+biocontaminants.">Detection and enumeration of airborne biocontaminants.</a> Curr. Opin. Biotechnol. 15, 170-174 (2004).</li><li>Dannemiller, K. C., Gent, J. F., Leaderer, B. P., Peccia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+housing+characteristics+on+bacterial+and+fungal+communities+in+homes+of+asthmatic+children.">Influence of housing characteristics on bacterial and fungal communities in homes of asthmatic children.</a> Indoor air. , (2015).</li><li>Yamamoto, N., Hospodsky, D., Dannemiller, K. C., Nazaroff, W. W., Peccia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indoor+emissions+as+a+primary+source+of+airborne+allergenic+fungal+particles+in+classrooms.">Indoor emissions as a primary source of airborne allergenic fungal particles in classrooms.</a> Environ. Sci. Technol. 49, 5098-5106 (2015).</li><li>Yamamoto, 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=Particle-size+distributions+and+seasonal+diversity+of+allergenic+and+pathogenic+fungi+in+outdoor+air.">Particle-size distributions and seasonal diversity of allergenic and pathogenic fungi in outdoor air.</a> ISME J. 6, 1801-1811 (2012).</li><li>Karottki, D. G., 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=Cardiovascular+and+lung+function+in+relation+to+outdoor+and+indoor+exposure+to+fine+and+ultrafine+particulate+matter+in+middle-aged+subjects.">Cardiovascular and lung function in relation to outdoor and indoor exposure to fine and ultrafine particulate matter in middle-aged subjects.</a> Environ Int. 73, 372-381 (2014).</li><li>Guy, D., Hodges, N., Hanlon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endotoxins+and+Depyrogenation.">Endotoxins and Depyrogenation.</a> Industrial Pharmaceutical Microbiology: Standards and Controls. , 12.1-12.15 (2003).</li><li>Thorne, P. S., Bartlett, K. H., Phipps, J., Kulhankova, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Five+Extraction+Protocols+for+Quantification+of+Endotoxin+in+Metalworking+Fluid+Aerosol.">Evaluation of Five Extraction Protocols for Quantification of Endotoxin in Metalworking Fluid Aerosol.</a> Ann. Occup. Hyg. 47, 31-36 (2003).</li><li>Thornton, B., Basu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+(qPCR)+primer+design+using+free+online+software.">Real-time PCR (qPCR) primer design using free online software.</a> Biochem. Mol. Biol. Educ. 39, 145-154 (2011).</li><li>Madigan, M. T., Clark, D. P., Stahl, D., Martinko, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brock Biology of Microorganisms. , (2011).</li><li>Watson, J. G., Chow, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerosol Measurement: Principles and Techniques. , 591-613 (2011).</li><li>Mueller-Anneling, L., Avol, E., Peters, J. M., Thorne, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ambient+endotoxin+concentrations+in+PM10+from+Southern+California.">Ambient endotoxin concentrations in PM10 from Southern California.</a> Environ. Health Perspect. 112, 583-588 (2004).</li><li>Hospodsky, D., Yamamoto, N., Peccia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy,+Precision,+and+Method+Detection+Limits+of+Quantitative+PCR+for+Airborne+Bacteria+and+Fungi.">Accuracy, Precision, and Method Detection Limits of Quantitative PCR for Airborne Bacteria and Fungi.</a> Appl. Environ. Microbiol. 76, 7004-7012 (2010).</li><li>Kutyavin, I. V., 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=3&#8242;-Minor+groove+binder-DNA+probes+increase+sequence+specificity+at+PCR+extension+temperatures.">3&#8242;-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.</a> Nucleic Acids Res. 28, 655-661 (2000).</li><li>Brankatschk, R., Bodenhausen, N., Zeyer, J., B&#252;rgmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+absolute+quantification+method+correcting+for+quantitative+PCR+efficiency+variations+for+microbial+community+samples.">Simple absolute quantification method correcting for quantitative PCR efficiency variations for microbial community samples.</a> Appl. Environ. Microbiol. 78, 4481-4489 (2012).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appendix+3B,+Trypan+Blue+Exclusion+Test+of+Cell+Viability.">Appendix 3B, Trypan Blue Exclusion Test of Cell Viability.</a> Current Protocols in Immunology. , (2001).</li><li>Hollander, A., Heederik, D., Versloot, P., Douwes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+and+enhancement+in+the+analysis+of+airborne+endotoxin+levels+in+various+occupational+environments.">Inhibition and enhancement in the analysis of airborne endotoxin levels in various occupational environments.</a> Am Ind Hyg Assoc J. 54, 647-653 (1993).</li><li>Kennedy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PCR troubleshooting and optimization : the essential guide. , (2011).</li><li>Joiner, T. J., Kraus, P. F., Kupiec, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Endotoxin+Testing+Methods+for+Pharmaceutical+Products.">Comparison of Endotoxin Testing Methods for Pharmaceutical Products.</a> Int J Pharm Compd. 6, 408-409 (2002).</li><li>Ebentier, D. L., 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=Evaluation+of+the+repeatability+and+reproducibility+of+a+suite+of+qPCR-based+microbial+source+tracking+methods.">Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods.</a> Water Res. 47, 6839-6848 (2013).</li></ol>

The authors have nothing to disclose.

这部作品描述了收集器的气溶胶样品中既定量的内毒素和DNA提取存在和检测方法。该方法要求精确的例程和只要实验者附着在这里讨论了一些基本的和重要的点可以容易地进行。 用于内毒素检测步骤，请注意，裂解物溶液是相当粘稠，趋于在移液以产生气泡。它是难以除去你气泡，并且它们导致在微孔板阅读器值的变化。因此，要首先放置在样品中的板和仅确保所有的气泡消失后，继续加入裂解物溶液是很重要的。一个有用的技术，以防止在这一步骤中气泡的形成是用吸管慢慢地工作，而不是排水裂解液入井时按它所有的方式。作为反应立即开始，一旦裂解物被添加到endoto辛提取物，有必要尽早开始读取。为了缩短溶解产物添加步骤，可以使用多道移液器。注意，在一些试剂盒和协议有样品在37℃下进行10分钟的额外预孵育步骤中，它被放置在微偷笑板后29仅此步骤之后，可以加入裂解物。 对于内毒素的环境样品提取的重要的一点是在样品中的抑制剂的存在下（ 例如高污染空气样品中）。在这种情况下，稀释变得至关重要，并能防止假增强，通常为未稀释的样品观察到的。38 同样，也有重要的几个点，以确保所述Q-PCR检测的成功和可靠性。 1）作为溶液的体积是非常小的，如果它们不放置在井的底部，在该DNA滴的水含量可以通过蒸发控制而减少Ë安排在板样品。为了防止此问题，将DNA样品在井的底部，通过将它放置在冰上或其它冷却平台冷却该板，并准备提前一切加快该步骤尽可能。 2）当制备Q-PCR反应混合物（ 表4），用铝箔覆盖的管，以防止光降解。 3）在本实验中描述了一个10微升反应的准备。然而，在某些Q-PCR的仪器，将反应体积为20微升，然后各试剂的体积相应地相乘。 4）最佳的热循环条件可以为不同的引物和聚合酶混合物改变。39例如，退火温度应在约引物的熔化温度被设置，并且根据经验确定为在该扩增最有效地发生的温度。由于这个原因，同样重要的是，以制备校准曲线为每个分析Q-PCR板，以确保该量化是准确的。校正曲线以及标准掺入滤波器也用作阳性对照，以确保没有抑制发生。为了避免抑制，建议使用设计用于从环境样品中移除抑制剂提取试剂盒。 5）在本实验中的热循环数设定为最大数，45中，为了提高灵敏度作为来自滤波器提取的DNA的浓度是非常低的。 6）确保循环至阈值（对于所有DNA样品Cτ）值在标准曲线的动态范围内。还确保阴性对照样品的任何扩增比DNA样品大至少八个周期。 7）如果用的SYBR Green试剂工作，确保有每个反应没有多重熔化温度峰值。 对于浊度测试中，两款内毒素定量方法:动力学方法和endpoi新台币显色法。在动力学的方法，因为在这个协议进行的，该样品所需要的时间，以达到最大吸收进行测量。这里，更短的反应时间间隔指示样品中更高的内毒素浓度。在端点发色的方法，光吸收所定义的孵育时间，以确定内毒素浓度后测定的。这两种方法都需要定量的标准曲线。40 虽然上述的内毒素提取的精度和准确度是非常高的，14中的提取效率可以改善。这可能是不同类型的过滤器或额外的洗涤剂（如聚山梨醇酯20），以在萃取步骤可以提高从滤波器提取的内毒素的产量。内毒素提取效率还可以通过在并行采样过滤器上的不同的颗粒的影响。33对于我们的实验装置，该方法的检测限估计为0.001欧米-3 14 用于量化的DNA提取物，可以作为替代Q-PCR技术包括克隆方法和数字液滴的PCR（DD-PCR）。 Q-PCR的以上物种鉴定的克隆方法的优点是它的更大的敏感性，而且它要求初始扩增步骤低级DNA浓度。此外，与Q-PCR，克隆方法，该方法是劳动密集且耗时的，是不是定量的。为将DNA提取物的定量的另一种方法为DD-PCR 19这个技术的优点在于，它不要求一个校准曲线，该检测是基于在每个液滴单一DNA菌株。然而，对于环境样品，没有可靠的方法迄今用于从过滤器中提取的DNA的液滴检测存在。 与DNA被提取fr处的效率嗡滤波器，以及该DNA的测量的准确度和精确度，可以由诸如过滤器类型，靶标微生物，和仪表。34,41因此应在试样分析之前定义的影响。在我们的实验装置，检测限可以达到降低到3.5基因组米-3 14 最后，此处所描述的方法适用于从两个人为和自然来源空气采样生物物种的鉴定和定量。精确使用适当的测定使得进行复杂的环境问题有价值的调查。

在过滤器上空气采样是在大气气溶胶研究的基本工具。1所述的取样过滤器可用于各种化学，物理和所收集的环境粒子的生物表征的起点。2-11这种方法的优点是，各种分析可离线对同一样品执行的。编译来自所有不同分析的数据使研究者获得的收集的颗粒和助剂的特性很好地理解在解决在大气科学的复杂问题。12,13例如，在相同的拍摄海洋和内陆空气采样期间可相对于所采样的颗粒的毒性和生物组合物。14本研究进行比较，脂多糖（LPS），对革兰氏阴性细菌细胞壁，也被称为内毒素组分，从过滤器中提取的采样在岸和在内陆部位，并使用该评价鲎变形细胞溶解物（LAL）测试。并行地，细菌含量的基因组的评估（总菌，革兰氏阴性，和蓝藻）是使用定量聚合酶链反应（Q-PCR）对同一样品执行的。在LAL试验是基于形成的加成从鲎， 美洲鲎阿米巴细胞的含水提取物之后，向含有内毒素的水溶液浊度的测量。样品在较高的内毒素浓度，更快浊度开发15的Q-PCR分析是基于发射作为特定的DNA片段被扩增荧光信号16由信号的实时监测在PCR指数期反应和校准用标准曲线，初始的DNA量进行量化。这两种的组合与他人一起分析，如别处详述，14可提供的内毒素和水平的良好估计样品中的源的细菌量。 这里提出的方法的目的是获得从过滤器中提取的生物气溶胶的内毒素和DNA含量的高度准确和可靠的分析。而用于采样气溶胶的物理和无机化学特性的方法是，最近，已开发了探讨其有机质成分众所周知，17一直存在很少研究气溶胶的生物成分上18为当前的理方法是通过在详细介绍，用于提取，分析和确定空气气溶胶的生物分数一个可靠的方法来解决这个间隙14中这里详述的方法可望找到在涉及过滤分析生物的室内和室外的气溶胶研究项目广泛使用。20-24

<table border="0" cellpadding="0" cellspacing="0" width="631">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Filter sampling</td>
			<td style="width:123px;"></td>
			<td style="width:103px;"></td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">HiVol 3000 - High Air Volume Sampler</td>
			<td style="width:123px;">Ecotech</td>
			<td style="width:103px;"></td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Quartz Microfiber Filters</td>
			<td style="width:123px;">Whatman</td>
			<td style="width:103px;">1851-865</td>
			<td style="width:220px;">203mm X 254mm</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">ELF - Laboratory Chamber Furnaces</td>
			<td style="width:123px;">Carbolite</td>
			<td style="width:103px;">ELF 11series</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Aluminum foil</td>
			<td style="width:123px;">Opal</td>
			<td style="width:103px;"></td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:187px;">Name</td>
			<td style="width:123px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td style="width:220px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Endotoxin</td>
			<td style="width:123px;"></td>
			<td style="width:103px;"></td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Ethanol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;">16368&#160;</td>
			<td style="width:220px;">Laboratory Reagent, 96%</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Airstream Class II Biological Safety Cabinet AC-4E1</td>
			<td style="width:123px;">ESCO</td>
			<td style="width:103px;">10011712</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Pyrotell -T</td>
			<td style="width:123px;">Associates of Cape Cod, Inc.</td>
			<td style="width:103px;">T0051</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Control Standard Endotoxin</td>
			<td style="width:123px;">Associates of Cape Cod, Inc.</td>
			<td style="width:103px;">E0005-1</td>
			<td style="width:220px;">Escherichia coli&#160;O113:H10, 0.5 &#181;g/vial 1 Pack</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">LAL Reagent Water</td>
			<td style="width:123px;">Associates of Cape Cod, Inc.</td>
			<td style="width:103px;">W0051</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">10 mL sterile syringe with Luer-Lok Tip</td>
			<td style="width:123px;">Becton-Dickinson &#38; Co.</td>
			<td style="width:103px;">309605</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">BD Precisionglide syringe needle</td>
			<td style="width:123px;">Becton-Dickinson &#38; Co.</td>
			<td style="width:103px;">305129</td>
			<td style="width:220px;">Sterile&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Parafilm-M sealing tape</td>
			<td style="width:123px;">Parafilm</td>
			<td style="width:103px;">P7543</td>
			<td style="width:220px;">Sigma catalog number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Microtubes</td>
			<td style="width:123px;">Axigen</td>
			<td style="width:103px;">MCT-200-C</td>
			<td style="width:220px;">2ml, pyrogen free</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">1.12 cm diameter Cork Borer</td>
			<td style="width:123px;">Boekel Scientific</td>
			<td style="width:103px;">1601 BD Series - Steel</td>
			<td style="width:220px;">Part of a cork borer set containing borers with various diameters.&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">50 mm Petri Dish</td>
			<td style="width:123px;">Miniplast Ein-Shemer</td>
			<td style="width:103px;">72050-01</td>
			<td style="width:220px;">Aseptic</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Vortex Genie 2&#160;</td>
			<td style="width:123px;">Scientific Industries, inc.</td>
			<td style="width:103px;">SI-0297</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Microcentrifuge 5415 D</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:103px;">22621408</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:187px;">TC MicroWell 96 F SI w/lid</td>
			<td style="width:123px;">Nunc</td>
			<td style="width:103px;">167008</td>
			<td style="width:220px;">Flat bottom wells (with lid (individually wrapped)), sterile, pyrogen free</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:187px;">Synergy HT Multi-Detection Microplate Reader</td>
			<td style="width:123px;">Biotek</td>
			<td style="width:103px;">7091000</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:187px;">Name</td>
			<td style="width:123px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td style="width:220px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">DNA</td>
			<td style="width:123px;"></td>
			<td style="width:103px;"></td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">DNA away</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;">7010</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:187px;">Standard DNA of the microbial species of interest</td>
			<td style="width:123px;">ATCC or other culture collection</td>
			<td style="width:103px;"></td>
			<td style="width:220px;">Either the appropriate microbial strain for DNA extraction or the extracted DNA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Neubauer-improved</td>
			<td style="width:123px;">Marienfeld</td>
			<td style="width:103px;">640030</td>
			<td style="width:220px;">hemocytometer</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">TE buffer, Low EDTA</td>
			<td style="width:123px;">Life Technologies</td>
			<td style="width:103px;">12090-015</td>
			<td style="width:220px;">10 mM Tris-HCl (pH 8.0)&#160;0.1 mM EDTA&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Nuclease-free PCR-grade water&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;">3315959001</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">PCR primers</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;"></td>
			<td style="width:220px;">Targets the microbial species of interest</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Dual-Labeled Probes</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;"></td>
			<td style="width:220px;">Targets the microbial species of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Screw cap tubes</td>
			<td style="width:123px;">Axigen</td>
			<td style="width:103px;">ST-200-SS</td>
			<td>2 mL&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">PowerSoil DNA extraction kit&#160;</td>
			<td style="width:123px;">Mo Bio Laboratories</td>
			<td style="width:103px;">12888-100</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Glass beads, acid-washed 425-600 Microns</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;">G8772-100G</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Glass beads, acid-washed &#60;106 microns</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:103px;">G4649-100G</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">PowerSoil Solution C1</td>
			<td style="width:123px;">Mo Bio Laboratories</td>
			<td style="width:103px;">12888-100-1</td>
			<td style="width:220px;">Cell lysis buffer , Power soil Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Magic Touch ice bucket</td>
			<td style="width:123px;">Bel-Art</td>
			<td style="width:103px;">18848-4001</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:187px;">Mini-Beadbeater-16</td>
			<td style="width:123px;">BioSpec</td>
			<td style="width:103px;">607EUR</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">StepOnePlus Real-Time PCR System</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4376600</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">Fast SYBR Green Master Mix</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4385612</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4370048</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:187px;">MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4346906</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">MicroAmp Splash-Free 96-Well Base</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4312063</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:187px;">MicroAmp Optical Adhesive Film</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:103px;">4311971</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:187px;">Centrifuge 5810 R</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:103px;">5811 000.010</td>
			<td style="width:220px;">Rotor A-4-62 with MTP buckets&#160;</td>
		</tr>
	</tbody>
</table>

air-sampled filter analysis for endotoxins and dna content

室外气溶胶的研究通常使用的过滤器采样的颗粒物。此过程使得能够并行执行所收集的颗粒的各种特征。这里提出的方法的目的是获得从过滤器中提取的生物气溶胶的内毒素和DNA含量的高度准确和可靠的分析。高的分子量的有机分子，如脂多糖，从采样滤波器的提取包括摇动在一个无热原的水性介质中的样品。随后的分析是基于可使用浊度测量来检测酶反应。作为上采样滤波器的高有机含量的结果是，使用该最初设计用于土壤和修改，以提高DNA产量市售的DNA提取试剂盒进行DNA的样​​品的提取。的检测和使用定量聚合酶链reacti特定微生物物种的量化上（Q-PCR）分析中描述并与其他可用的方法进行对比。

它是常见使用"离线"采样滤波器的分析来研究大气气溶胶（参见图2）。取样物质包括有机（ 例如蛋白质，烃类分子，糖类）和无机（ 例如金属，盐）含量的32化学分析。生物分析包括存活和非存活的微生物含量，使用DNA的方法或显微镜物种鉴定，以及基于DNA的定量。 <img alt="图2" src="/files/ftp_upload/53444/53444fig2.jpg" /> 图2: 文件管理器试样分析 流程图空气采样（A）的后过滤，制备的过滤器下游分析（B）和样品组分（C）的分析。 E ="1"&gt;对于内毒素的提取，滤波子样本（1.12 平方厘米）的1ml PFW摇动，在室温下60分钟。然后将样品离心在375×g下10分钟。不同离心速度可导致不同的萃取效率，如在图3A中表示。 对提取效率的初步试验应进行选择的特定的过滤器和协议进行。在图3B中 ，较高的萃取效率从与采样石英滤波器相比扣球干净获得，并且这两个效率都比通过直接检测标准溶液的实现低。如其他地方所讨论的，在过滤器上采样的环境气溶胶的性质也可以影响内毒素提取效率。14,33 g3.jpg"/&gt; 图 3: 内毒素提取效率离心速度（A）和过滤器的负载（B）的内毒素提取效率的影响。误差线表示标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53444/53444fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 用于内毒素检测的反应包括一个1:LAL试剂的到内毒素标准，样本提取内毒素，或坯件1的比例。它建议使用一式三份试样分析。然而，对于标准曲线，重复就足够了。当的吸收读数（在405nm处）完成后，将所得的光密度（OD）是出口它到用于数据的进一步分析的数据分析的电子表格程序后进行分析。 <p class="jove_content" fo:keep-together.within页="1"&gt;从空气取样滤波器DNA的提取是直接从子采样滤波器部执行。 DNA的提取是用市售的土壤样本DNA分离试剂盒，有一些修改，以增加DNA的产率进行。因此，玻璃珠加入到搅拌的珠打浆机的过滤器，以提高DNA的释放到该溶液中。为了防止样品加热（这可能会导致该DNA的变性），这一步分为五个1分钟的间隔，与该管上冷却间隔之间的冰。然后提取可根据制造商的过程继续进行。 如内毒素提取，它的具体的实验条件下进行的DNA的提取效率的初步分析是很重要的。这是通过掺加标准生物体细胞溶液已知量上从过滤器切下一块，按照以上描述的DNA提取协议完成。 <suP&gt; 34 从采样的气溶胶提取靶微生物的浓度是用Q-PCR确定的。这里水解探针技术被使用，其具有因为结合于模板DNA的额外探针的高特异性的优势，35的SYBR绿方法也可以适用于该目的。 校准曲线从从感兴趣的选择的生物体中提取DNA来源，使用上述的提取方法。反应的化合物的混合池，不包括DNA样品，标准或对照，制备和保持在黑暗中，并在冰上直至需要。然后，将混合池的等分试样在96微孔光学板加入到每个孔中。该DNA标准样品，或控制是根据片阵列的反应混合物后加入。之后所有的试剂已经被插入到板，它是海导致用光学粘接膜并旋转以收集所有的液滴在孔的底部。该板比插入的q PCR仪进行热循环扩增和信号检测。 输出数据由PCR的Ct值，其被定义为在该扩增的DNA量到达阈值电平的周期数目。使用校准曲线值，能够得到目标DNA的浓度（参见图4）。36的微生物的细胞浓度可以从与标准生物体溶液的血球计数相比，提取效率的Ct值来计算。37 <img alt="图4" src="/files/ftp_upload/53444/53444fig4.jpg" /> 图 4: 定量PCR扩增曲线 （A）和校准。离子曲线范围为代表的革兰阴性细菌的DNA进行2-4之间×10 -2×10 2（B）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53444/53444fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Air-sampled Filter Analysis for Endotoxins and DNA Content at JoVE.com

Air-sampled Filter Analysis for Endotoxins and DNA Content

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

内毒素和DNA含量的空气采样滤波器分析

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

air-sampled-filter-analysis-for-endotoxins-and-dna-content

Nanyang Technological University

Research

JoVE Journal

Environment

9.6K 次观看.  Weizmann Institute of Science. Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

视频: Air-sampled Filter Analysis for Endotoxins and DNA Content

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

脂肪酸组分和含量微藻分析

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

科研

环境科学

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

Modification and Application of a Leaf Blower-vac for Field Sampling of Arthropods

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropod populations in rice. When used in combination with an enclosure, application of this sampling device provides absolute estimates of the populations of arthropods as numbers per standardized sampling area. The sampling efficiency depends critically on the sampling duration. In a mature rice crop, a two-minute sampling in an enclosure of 0.13 m2 yields more than 90% of the arthropod population. The device also allows sampling of arthropods dwelling on the water surface or the soil in rice paddies, but it is not suitable for sampling fast flying insects, such as predatory Odonata or larger hymenopterous parasitoids. The modified blower-vac is simple to construct, and cheaper and easier to handle than traditional suction sampling devices, such as D-vac. The low cost makes the modified blower-vac also accessible to researchers in developing countries.

Assessment of arthropod abundance in crops is critical for investigating population dynamics and species interactions. Here we describe the modification and application of a leaf blower-vac for suction sampling of arthropods in rice.

修改和叶鼓风机-VAC的节肢动物领域采样中的应用

Rice fields host a large diversity of arthropods, but investigating their population dynamics and interactions is challenging. Here we describe the ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

滤芯水过滤样品和环境DNA的提取使用

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

基因土壤分析的纳米 DNA 测序

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

真菌 DNA 分析用职业空气标本的收集与提取

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

以玉米 (玉蜀黍) 为例量化植物可溶性蛋白和可消化碳水化合物含量

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

Combining Eye-tracking Data with an Analysis of Video Content from Free-viewing a Video of a Walk in an Urban Park Environment

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. Eye-tracking informatics are known to connect to a range of feelings, health conditions, mental states and actions. But because vision is the result of constant eye-movements, teasing out what is important from what is noise is complex and data intensive. Furthermore, a significant challenge is controlling for what people look at compared to what is presented to them.
The following presents a methodology for combining and analyzing eye-tracking on a video of a natural and complex scene with a machine learning technique for analyzing the content of the video. In the protocol we focus on analyzing data from filmed videos, how a video can be best used to record participants&#39; eye-tracking data, and importantly how the content of the video can be analyzed and combined with the eye-tracking data. We present a brief summary of the results and a discussion of the potential of the method for further studies in complex environments.

The objective of the protocol is to detail how to collect video data for use in the laboratory; how to record eye-tracking data of participants looking at the data and how to efficiently analyze the content of the videos that they were looking at using a machine learning technique.

将眼动追踪数据与从自由观看城市公园环境中的步行视频中获得的视频内容进行分析

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. ...