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>
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The authors have nothing to disclose.

Este trabajo describe los métodos de extracción y detección para cuantificar tanto las endotoxinas y ADN presente en las muestras recogidas en los filtros de aerosol. Los métodos requieren rutinas precisos y se pueden realizar fácilmente, siempre que el experimentador se adhiere a algunos puntos esenciales e importantes discutidos aquí. Para la etapa de detección de endotoxinas, tenga en cuenta que la solución de lisado es bastante viscoso y tiende a producir burbujas en la pipeta. E...

El muestreo del aire en los filtros es una herramienta básica en la investigación de los aerosoles atmosféricos. 1 Los filtros de la muestra son el punto de partida para diversos productos químicos, físicos y caracterizaciones biológicas de las partículas ambientales recogidos. 2-11 La ventaja de este enfoque es que varios análisis pueden ser realizado fuera de línea en la misma muestra. La compilación de los datos de todos los diferentes análisis permite al investigador para obtener una buena comprensión de las características de las partículas y las ayudas recogidas en la resolución de cuestiones complejas en las ciencias de la atmósfera 12,13. Por ejemplo, los acondicionadores de muestras marinas y continentales tomadas durante el mismo período puede compararse con respecto a la toxicidad de las partículas en la muestra y la composición biológica. 14 para este estudio, los lipopolisacáridos (LPS), componentes de paredes celulares bacterianas gram-negativas, también conocidos como endotoxinas, se extrajeron de los filtros en la muestra en tierra y en un sitio en el interior, y se evaluaron utilizando elamebocitos de Limulus lisado (LAL). En paralelo, una evaluación genómica del contenido bacteriano (total de bacterias, gram negativo, y cianobacterias) se realizó en la misma muestra usando la reacción en cadena de la polimerasa cuantitativa (Q-PCR). La prueba de LAL se basa en mediciones de turbidez formados después de la adición de un extracto acuoso de amebocitos del cangrejo de herradura, Limulus polyphemus, a una solución acuosa que contiene las endotoxinas. Cuanto mayor sea la concentración de endotoxina en la muestra, la turbidez más rápido se desarrolla. 15 El análisis q-PCR se basa en una señal de fluorescencia emitida como un fragmento de ADN específico se amplifica. 16 Por la monitorización en tiempo real de la señal durante la fase exponencial de la PCR reacción y de calibración con una curva estándar, la cantidad inicial de ADN se pueden cuantificar. La combinación de estos dos análisis junto con otros, como se detalla en otro lugar, 14 puede proporcionar una buena estimación de los niveles de endotoxina y de lacantidad de las bacterias de origen en las muestras. El propósito del método presentado aquí es obtener un análisis muy preciso y fiable del contenido de endotoxina y ADN de bio-aerosoles extraídos de filtros. Si bien los métodos de muestreo de las características físicas y químicas inorgánicas de aerosoles son bien conocidos y, más recientemente, se han desarrollado métodos para investigar su componente de materia orgánica, 17 no ha sido escasa la investigación sobre el componente biológico de aerosoles. 18 La razón de la corriente método consiste en llenar este vacío mediante la presentación en detalle un método robusto para la extracción, el análisis y la identificación de la fracción biológica de los aerosoles en el aire. 14 Se espera que el método detallado aquí para encontrar amplio uso en proyectos de investigación en aerosol interiores y exteriores biológicos que implican análisis de filtro. 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

la investigación de aerosol al aire libre comúnmente utiliza partículas muestreada en los filtros. Este procedimiento permite diversas caracterizaciones de las partículas recogidas que se realizan en paralelo. El propósito del método presentado aquí es obtener un análisis muy preciso y fiable del contenido de endotoxina y ADN de bio-aerosoles extraídos de filtros. La extracción de las moléculas de alto peso molecular orgánicos, tales como lipopolisacáridos, de los filtros de la muestra implica agitando la muestra en un medio a base de agua libre de pirógenos. El posterior análisis se basa en una reacción enzimática que puede detectarse utilizando una medición turbidimétrico. Como resultado de la alto contenido orgánico en los filtros en la muestra, la extracción de ADN de las muestras se realiza usando un kit de extracción de ADN comercial que fue diseñado originalmente para suelos y modificado para mejorar el rendimiento de ADN. La detección y cuantificación de especies microbianas específicas utilizando Reacti cadena de la polimerasa cuantitativaen el análisis (q-PCR) se describen y se comparan con otros métodos disponibles.

Es común para estudiar los aerosoles atmosféricos utilizando &quot;off-line&quot; los análisis de los filtros incluidos en la muestra (véase la Figura 2). Analiza 32 Química de la incluirá orgánica (por ejemplo, proteínas, moléculas de hidrocarburos, sacáridos) e inorgánicos (por ejemplo, metales, sales) contenido en la muestra cuestión . análisis biológicos incluyen contenido viable y no viable de microorganismos, la identifica...

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.

Análisis del filtro de aire en la muestra de endotoxinas y contenido de ADN

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

Research

JoVE Journal

Environment

9.6K vistas.  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.

Video: 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.

Análisis de contenido de ácido graso y Composición en microalgas

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

Investigación

Medio ambiente

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.

El uso de Chironomidae (Diptera) en superficie flotante pupal Exuviae como Protocolo Bioassessment rápida para las Masas de Agua

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.

Modificación y aplicación de un soplador de hojas-vac de campo Muestreo de artrópodos

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.

El uso de un cartucho de filtro para la filtración de agua Las muestras y extracción de ADN del Medio Ambiente

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.

Protocolo para microplásticos muestreo en la superficie del mar y Análisis de las muestras

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.

Extracción de plaguicidas organoclorados de pellets de plástico y análisis de tipos de plástico

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.

Secuencia de la DNA Nanopore para análisis de metagenómica suelo

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.

Colección y extracción de muestras de aire profesional para análisis de ADN fúngico

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.

Cuantificación de proteínas solubles de la planta y Digestible contenido de hidratos de carbono, utilizando el maíz (Zea mays) como un ejemplar

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.

Combinando los datos de seguimiento ocular con un análisis de contenido de vídeo de la visualización gratuita de un vídeo de un paseo en un entorno de parque urbano

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