作者承认瑞士国家科学基金会的批准号:31003A-一分之一十三万二千三百五十八，31003A_152972和No. 151948，和基金会皮埃尔·名士倒拉科学。

1.制备化学品和设备<ol><li>制成500毫升1％的六偏磷酸钠（SHMP）（磷酸钠3）N溶液和通过高压灭菌消毒。 </li><li>消毒硝化纤维素（NC）的过滤器（孔径0.22μm，直径47个毫米）通过高压灭菌在密闭的玻璃培养皿。 </li><li>通过高压在密闭玻璃培养皿消毒的NC滤波器（孔径0.22μm，直径25毫米）。 </li><li>称量并注意无菌50ml试管的空机重量（含上限）（每个样品一管）。 </li><li>制备的Tris-EDTA缓冲液（TE缓冲液）1×:使10毫摩尔Tris（ 三（羟甲基 ）氨基甲烷）和1mM EDTA缓冲溶液。调节pH至8，并通过高压灭菌消毒。 </li><li>通过在1ml的TE缓冲液中溶解0.02克溶菌酶制备溶菌酶溶液（20毫克/毫升）。理想的情况下，使新鲜每次。存放在4℃下不超过1周。 </li><li>通过将8克/升的氯化钠制备生理溶液（氯化钠）在蒸馏水。高压灭菌。 </li></ol>从沉积物2.分离生物质<ol><li>添加3克沉积物样品，以预先称重无菌使用乙醇火烧金属勺50ml试管。执行此在一个干净，UV消毒生物安全柜，以避免污染。 </li><li>加入15mL 1％的无菌（高压灭菌）SHMP溶液使用无菌刻度滴管样品。该SHMP溶液还可以进行筛选，以避免污染。 </li><li>均化沉渣和SHMP溶液与液体分散器/均化器（例如，超TURRAX均化）。 70％的乙醇 - 消毒或高压灭菌，使用前分散转子。运行同质化1分钟17,500转。让样品休息2分钟，在相同的转子速度重复均质化1分钟。 </li><li>让样品静置10分钟。在该步骤中，最重的粒子（矿物）会沉淀。将细胞和样品然而瓦特任何有机成分病留在溶液中。之后，转移上清液溶液（含有细胞生物量）到一个干净的50ml管中，同时小心不要扰乱沉积的颗粒。 </li><li>向沉淀添加粒料再次将15ml 1％的无菌（高压灭菌）SHMP使用无菌刻度滴管溶液。然后重复步骤2.3和2.4。这种重复可以确保电池和有机粒子的最大量，从沉淀物的矿物组分分离。此第二分离的上清液可以合并与来自第一分离上清液。 注:以下步骤都做对上清（含细胞生物量）。矿物组分（沉淀沉淀）可以被丢弃。 </li><li>离心样品以20×g离心1分钟。这一步增加了G力足以解决小矿粒，而生物细胞物质仍然在溶液中。离心后，转移上清液溶液（含有细胞的生物质）转化为干净的50ml管中。弃矿物颗粒。 </li><li>确定包含生物量通过称重样品溶液的最终体积。权重确定避免了必须将样品转移至量筒并降低污染的风险。 </li></ol> 3.生物质收集在滤膜<ol><li>制备过滤单元（47毫米直径的膜）和真空泵。或者通过高压灭菌或（如果派热克斯玻璃）用70％的乙醇喷洒和用本生灯燃烧它消毒过滤单元。让与协议，然后再继续它冷却下来。 </li><li>用乙醇 - 熄火灭菌镊子添加无菌NC膜的过滤单元。 </li><li>加入一半的上清液样品（来自步骤2.6）的到膜过滤单元和收集细胞上使用真空泵膜上。 </li><li>当液体已充分通过过滤器，停止真空泵，小心地用乙除去膜OL火焰消毒镊子。放置膜进入无菌培养皿。 <ol><li>使用乙醇火烧消毒剪刀剪膜的一半。该膜的每一半添加到单独的2毫升管。膜的一个半部分将被用于DNA提取和整个细菌群落分析。滤波器的另一半将被保存在-80℃，并作为一个备份。 </li></ol></li><li>放置新的NC膜到过滤单元，并使用真空泵收集从样品体积（来自步骤2.6）的第二半的生物质。 </li><li>当液体已充分通过过滤器，停止真空泵，并仔细用乙醇 - 熄火灭菌镊子除去该膜。将整个膜到一个单独的2毫升管。该样品将被用于对生孢子从营养细胞分离的治疗。可将样品储存在-20℃直至使用。 </li></ol> 4.裂解营养细胞<ol><li>执行处理分离先前在NC膜（步骤3.6）收集的生物量营养细胞内生孢子。 <ol><li>如果膜被冻结，离开它在室温10分钟以解冻。然后放置在无菌培养皿的膜和使用乙醇火烧灭菌剪刀剪（约4倍）成更小的碎片。然后将所有的膜过滤片放入一个无菌的ml管。 </li></ol></li><li>加入900微升1×TE（的Tris-EDTA）缓冲液（见1.5），以含有样品膜管涡旋调匀。在此步骤中，所述生物质从膜进TE-缓冲液的溶液中除去。 </li><li>将管在培养箱在65℃下进行10分钟，80转。然后从孵化器中取出管，让它冷却15分钟。 </li><li>加入100微升新鲜制备的溶菌酶（见1.5），以达到2毫克/毫升的最终浓度。不要添加溶菌酶前将样品冷却到37℃，因为这可能DEG拉德的酶。 </li><li>孵育样品在37℃下60分钟，80转，对于溶菌酶的最佳条件以溶解营养细胞。 </li><li>裂解完成后，加入250微升3当量的氢氧化钠（氢氧化钠）和250微升6％十二烷基硫酸钠（SDS）溶液到样品中。通过添加此，样品体积达到1.5 ml和有0.5N的氢氧化钠的最终浓度和1％SDS的终浓度。 </li><li>孵育这种混合在室温进行60分钟和80转。加入碱和洗涤剂将有助于最终的细胞溶解。这些洗涤剂的浓度进行了优化，以不损害孢子，而裂解营养细胞。 </li><li>准备保存25mm直径的膜通过高压灭菌，或者（如果派热克斯玻璃）喷洒用70％乙醇和用本生灯燃烧它的无菌过滤单元。让它冷却下来。 </li><li>用乙醇 - 火焰灭菌镊子将过滤单元上的0.2微米的NC膜（25毫米直径）。&lt;/ LI&gt; <li>从步骤4.7添加示例到膜上，并使用真空泵过滤液体。当液体已通过，关闭真空泵。在该步骤中，裂解营养细胞材料被去除，因为它不保留在膜。只有内生孢子将留在该膜上。 </li><li>加入2毫升无菌生理溶液洗掉残留的清洁剂和用真空泵过滤液体。 </li><li>当液体完全过滤，关闭真空泵。离开过滤单元的膜。 </li></ol> 5. DNA酶处理注意:直接在过滤膜执行DNase处理。重要的是，过滤部不泄漏是重要的，所述真空泵是关闭的。 <ol><li>加入450微升无菌水，50微升DNA酶反应直接缓冲液（1x）和0.5微升DNA酶的酶附着在过滤器膜上，静置15分钟。如果可能的话，做到这一点消化，房间里稍微战争聚体比平均室温，由于酶的工作温度为25℃以上更好。 注意:保留本生灯一旁过滤单元也增加的温度和具有保持周围环境的无菌的附加好处，因此降低了样品的污染的风险。 </li><li>当DNA酶消化完成后，开启真空泵以从样品除去酶。 </li><li>通过添加和筛选1 ml的生理溶液洗掉残留的酶。 </li><li>如果液体已全面通过，关闭真空泵，并删除含有使用无菌镊子，并放入无菌培养皿内生孢子的过滤膜。 注意:分离内生孢子的样品现在准备用于下游分析。如果使用储存在-20℃下进行DNA提取或替代地，在4℃下，如果用于发芽和栽培。 </li></ol>

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内生孢子外部攻击性理化因素 （如温度或清洁剂）的电阻来设计一种方法，从营养细胞环境样品中分离的细菌芽孢。这是第一个全面的方法来隔离环境样品的内生孢子在非破坏性的方式。以前的方法来定量，检测或分析样品中内生孢子是根据特定的代理内生孢子诸如吡啶二羧酸或特定的标记基因的测定。与此相反，与协议这里介绍，比生孢子其他样品组分被去除，所以内生孢子最终仍作为...

这项工作的目的是提供一种协议，用于细菌内生孢子的从营养细菌细胞环境样品中分离。细菌内生孢子的形成是一个生存策略，通常由饥饿触发，在多个属于脊索动物门厚壁菌门1细菌群体的发现。形成内生孢子的细菌被很好的研究，主要是因为一些菌株是病原体，因此医学重要（例如， 炭疽芽孢杆菌或艰难梭菌 ）。形成内生孢子菌的环境菌株已分离出几乎所有的环境（土壤，水，沉淀物，空气，冰，人体肠道，内脏动物，和更多）1-3。因此，厚壁菌在培养物保藏4第二个最丰富的门。 由于其耐寒外皮层和保护核心蛋白，内生孢子能够生存极端的环境条件范围FROM干燥高辐射，极端温度和有害化学物质5。这一显着性使它成为一个挑战提取芽孢6-8 DNA。这可能解释了为什么他们被忽视了环境测序研究9,10。其他方法，如环境样品中内生孢子的荧光抗体11靶向，吡啶二羧酸的定量（DPA）中土壤12和沉积物13，流式细胞仪14或巴氏灭菌和随后的培养15,16已被用于在检索或量化生孢子环境样品。在最近几年，优化的DNA提取方法以及具体的分子的引物靶向生孢子特异性基因序列已经被开发10,17-20。这有助于揭示这组细菌21之间更多的生物多样性，并且也导致了内生孢子的检测应用在工业和医药中，例如在奶粉19。 这里介绍的协议是基于到有害的物理化学条件（例如热和洗涤剂）相对于营养细胞细菌内生孢子的电阻的差别。以破坏营养细胞的样品中，我们连续施加热，溶菌酶和低浓度的洗涤剂。这些治疗的时间和强度都得到了优化，以便不破坏孢子，但以裂解所有营养细胞。一些细胞在一个环境室池比其它更耐磨，所以为了提高破坏所有营养细胞的概率，我们应用了三种不同的治疗方法。这种方法的优点和新颖性在于仍保持原样，在处理后的内生孢子可用于进一步下游分析。这些包括DNA提取，定量PCR（qPCR的）和扩增子或宏基因组测序（专门针对组内生孢子的，从而减少ðiversity，同时增加覆盖）。的内生孢子也可以用于下行种植或定量由荧光显微镜，流式细胞仪，或检测DPA的。这种方法的一个重要特征是，通过未处理样品与处理过的样品进行比较，可以推断出内生孢子的数量和多样性环境样品中除了对应于营养细胞的组件。

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			<td height="63" style="height:63px;width:216px;">Whatman nitrocellulose membrane filters, 0,2 um pore size, 47 mm diameter</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td colspan="2" style="width:285px;">WHA7182004 Aldrich&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris(hydroxymethyl)aminomethane</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td>252859 Sigma-Aldrich</td>
			<td>CAS 77-86-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884 Sigma-Aldrich</td>
			<td>CAS&#160; 60-00-4&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysozyme from chicken egg white</td>
			<td>Sigma-Aldrich</td>
			<td>62971 Fluka</td>
			<td>powder, CAS&#160; 12650-88-3&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-Turrax homogenizer T18 basic</td>
			<td style="width:95px;">IKA</td>
			<td>3720000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass filter holders for 47 mm membranes, pyrex glass</td>
			<td style="width:95px;">EMD Millipore</td>
			<td>XX10 047 00</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Chemical duty vacuum pump</td>
			<td style="width:95px;">Millipore</td>
			<td style="width:119px;">WP6122050</td>
			<td>220 V/50 Hz</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Manifold sampling filtration for 25 mm membranes</td>
			<td style="width:95px;">Millipore</td>
			<td>1225 Sampling Manifold</td>
			<td>polypropylene</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dnase</td>
			<td style="width:95px;">New England Biolabs</td>
			<td>M0303S</td>
			<td>Rnase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S5881 Sigma-Aldrich</td>
			<td>CAS&#160; 1310-73-2&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium dodecyl sulfphate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771 Sigma&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Whatman nitrocellulose membrane filters, 0,2 um pore size, 25 mm diameter</td>
			<td>Sigma-Aldrich</td>
			<td colspan="2">WHA7182002 Aldrich</td>
		</tr>
	</tbody>
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physical isolation of endospores from environmental samples by targeted lysis of vegetative cells

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

这里介绍的结果前面已经10,21公布。请参阅那些条数据的环境解释和讨论。 整体过程总结在图1中，对应于三个主要步骤:首先，从沉淀物或任何其他环境矩阵生物质的分离;第二，营养细胞的破坏;和第三，分离的内生孢子的下游分析。下游分析可以由，例如，DNA提取和扩增子测序以确定多样性。的DNA提取需要优化以保证内生孢子的裂解。在沉积物样品，我?...

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

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

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

从环境样品中的内生孢子通过无性细胞定向裂解物理隔离

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

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

Research

JoVE Journal

Environment

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

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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

科研

环境科学

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.

Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

物理，化学和六Biochars生产的生物表征污染场地修复的

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

去除微量元素的氧化铜纳米粉体制备铀<em&gt;原位</em&gt;恢复泌水及其对细胞活力的影响

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

准备和测试阻抗为基础与RTgill-W1细胞饮用水进行快速评估水样进行毒性射流生物芯片

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

A Bending Test for Determining the Atterberg Plastic Limit in Soils

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a considerable subjective judgment from the operator that carries out the test is involved during its performance, which may affect the final result significantly. Different alternative methods have been put forward, but they cannot compete with the standard rolling test in speed, simplicity and cost.
In an earlier study by the authors, a simple method with a simple device to determine the PL was presented (the &#34;thread bending test&#34; or simply &#34;bending test&#34;); this method allowed the PL to be obtained with minimal operator interference. In the present paper a version of the original bending test is shown. The experimental basis is the same as the original bending test: soil threads which are 3 mm in diameter and 52 mm long are bent until they start to crack, so that both the bending produced and its related moisture content are determined. However, this new version enables the calculation of PL from an equation, so it is not necessary to plot any curve or straight line to obtain this parameter and, in fact, the PL can be achieved with only one experimental point (but two experimental points are recommended).
The PL results obtained with this new version are very similar to those obtained through the original bending test and the standard rolling test by a highly experienced operator. Only in particular cases of high plasticity cohesive soils, there is a greater difference in the result. Despite this, the bending test works very well for all types of soil, both cohesive and very low plasticity soils, where the latter are the most difficult to test via the standard thread rolling method.

The traditional standardized test for determining the plastic limit in soils is performed by hand, and the result varies depending on the operator. An alternative method based on bending measurements is presented in this study. This allows the plastic limit to be obtained with a clear and objective criterion.

弯曲试验在土壤确定阿太堡限塑

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a ...

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 ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

纳米材料在水介质中的分散: 对协议优化的研究

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (Formicidae) for Volatilome Analysis by GC-MS and MetaboliteDetector

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean &#39;exploding ants&#39; belonging to the Colobopsis cylindrica (COCY) group. For this purpose, the model species C. explodens is used. Ants belonging to the minor worker caste possess distinctive hypertrophied mandibular glands (MGs). In territorial combat, they use the viscous contents of their enlarged mandibular gland reservoirs (MGRs) to kill rival arthropods in characteristic suicidal &#39;explosions&#39; by voluntary rupture of the gastral integument (autothysis). We show the dissection of worker ants of this species for the isolation of the gastral portion of the wax-like MGR contents as well as listing the necessary steps required for solvent-extraction of the therein contained volatile compounds with subsequent gas chromatography-mass spectrometry (GC-MS) analysis and putative identification of metabolites contained in the extract. The dissection procedure is performed under cooled conditions and without the use of any dissection buffer solution to minimize the changes in the chemical composition of the MGR contents. After solvent-based extraction of volatile metabolites contained therein, the necessary steps for analyzing the samples via liquid-injection-GC-MS are presented. Lastly, data processing and putative metabolite identification with the use of the open-source software MetaboliteDetector is shown. With this approach, the profiling and identification of volatile metabolites in MGRs of ants belonging to the COCY group via GC-MS and the MetaboliteDetector software become possible.

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' Formicidae for Volatilome Analysis by GC-MS and MetaboliteDetector

Minor workers of the ant species Colobopsis explodens are dissected to isolate the wax-like content stored in their hypertrophied mandibular gland reservoirs for subsequent solvent-extraction and analysis by gas chromatography-mass spectrometry. The&#160;annotation and identification of volatile constituents using the open-source software MetaboliteDetector is also described.

婆罗洲 "爆炸蚂蚁" (蚁科昆虫) 对下颌腺储层含量的分离--gc-ms 和 MetaboliteDetector Volatilome 分析

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean &#39;exploding ants&#39; belonging to the Colobopsis ...