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. 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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. 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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. 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The authors have nothing to disclose.

This work describes extraction and detection methods for quantifying both endotoxins and DNA present in aerosol samples collected on filters. The methods require accurate routines and can be performed easily as long as the experimentalist adheres to a few essential and important points discussed here.
For the endotoxin detection step, note that the lysate solution is quite viscous and tends to produce bubbles upon pipetting. It is difficult to remove thee bubbles, and they lead to changes in t...

Air sampling on filters is a basic tool in atmospheric aerosols research.1 The sampled filters are the starting point for various chemical, physical, and biological characterizations of the collected ambient particles.2-11 The advantage of this approach is that various analyses can be performed off-line on the same sample. Compiling the data from all the different analyses enables the researcher to obtain a good understanding of the characteristics of the collected particles and aids in solving complex questions in the atmospheric sciences.12,13 For example, marine and inland air-samples taken during the same period can be compared with respect to the sampled particle toxicity and biological composition.14 For this study, lipopolysaccharides (LPS), components on gram-negative bacterial cell-walls, also known as endotoxins, were extracted from filters sampled on-shore and at an inland site, and were evaluated using the limulus amebocyte lysate (LAL) test. In parallel, a genomic evaluation of the bacterial content (total bacteria, gram negative, and cyanobacteria) was performed on the same sample using the quantitative polymerase chain reaction (q-PCR). The LAL test is based on measurements of turbidity formed following the addition of an aqueous extract of amebocytes from the horseshoe crab, Limulus polyphemus, to an aqueous solution containing the endotoxins. The higher the endotoxin concentration in the sample, the faster turbidity develops.15 The q-PCR analysis is based on a fluorescence signal emitted as a specific DNA fragment is amplified.16 By real time monitoring of the signal during the exponential phase of the PCR reaction and calibrating with a standard curve, the initial DNA amount can be quantified. The combination of these two analyses together with others, as detailed elsewhere,14 can provide a good estimation of the levels of endotoxin and the amount of the source bacteria in the samples.
The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. While methods for sampling the physical and inorganic chemical characteristics of aerosols are well known and, more recently, methods have been developed to investigate its organic matter component,17 there has been scant research on the biological component of aerosols.18 The rationale for the current method is to address this gap by presenting in detail a robust method for extracting, analyzing, and identifying the biological fraction of airborne aerosols.14
The method detailed here is expected to find wide-spread use in biological indoor and outdoor aerosol research projects involving filter analysis.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

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

It is common to study atmospheric aerosols using &#34;off-line&#34; analyses of sampled filters (see Figure 2).32 Chemical analyses of the sampled matter include organic (e.g. protein, hydrocarbon molecules, saccharides) and inorganic (e.g. metals, salts) content. Biological analyses include viable and non-viable microorganism content, species identification using a DNA approach or microscopy, as well as DNA-based quantification.
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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.

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 Views.  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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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