Name: collection and extraction of occupational air samples for analysis of fungal dna
Uploaded: 2018-05-02T13:00:00
Description: Watch this Scientific Journal Video about Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA at JoVE.com

This work was supported in part by an interagency agreement between NIOSH and NIEHS (AES12007001-1-0-6).

1. Preparing the NIOSH aerosol sampler
NOTE: The NIOSH aerosol sampler is a two-stage cyclone aerosol sampler that collects bioaerosols using two sampling tubes and a polytetrafluoroethylene (PTFE) filter.
<ol>
	<li>Before assembly, carefully inspect the sampler. Check the sampler to ensure it is not damaged, all screws are in place and snug, and the sealing tape around the seam formed by the two halves is intact. Inspect the sampler O-ring to ensure it does not have any nicks, cracks or tea...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-stage+cyclone+using+microcentrifuge+tubes+for+personal+bioaerosol+sampling.">A two-stage cyclone using microcentrifuge tubes for personal bioaerosol sampling.</a> J Environ Monit. 8 (11), 1136-1142 (2006).</li><li>Rittenour, W. R., Park, J. H., Cox-Ganser, J. M., Beezhold, D. H., Green, B. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+bias+associated+with+incomplete+extraction+of+microbial+DNA+from+soil.">Assessment of bias associated with incomplete extraction of microbial DNA from soil.</a> Appl Environ Microbiol. 75 (16), 5428-5433 (2009).</li><li>Keswani, J., Kashon, M. L., Chen, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+interference+to+conventional+and+real-time+PCR+for+detection+and+quantification+of+fungi+in+dust.">Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust.</a> J Environ Monit. 7 (4), 311-318 (2005).</li><li>Bellemain, E., Carlsen, T., Brochmann, C., Coissac, E., Taberlet, P., Kauserud, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITS+as+an+environmental+DNA+barcode+for+fungi:+an+in+silico+approach+reveals+potential+PCR+biases.">ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases.</a> BMC Microbiol. 10, 189 (2010).</li><li>Farrelly, V., Rainey, F. A., Stackebrandt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+genome+size+and+rrn+gene+copy+number+on+PCR+amplification+of+16S+rRNA+genes+from+a+mixture+of+bacterial+species.">Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species.</a> Appl Environ Microbiol. 61 (7), 2798-2801 (1995).</li><li>Vesper, S., 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=Development+of+an+Environmental+Relative+Moldiness+index+for+US+homes.">Development of an Environmental Relative Moldiness index for US homes.</a> J Occup Environ Med. 49 (8), 829-833 (2007).</li></ol>

Determining the fungal diversity within an occupational environment using sequencing-based approaches has improved fungal hazard identification and exposure assessment. Using this approach has allowed for the detection of many additional fungal species that are often not detected using culture or microscopy-based methods of assessment. A method for sampling bioaerosols from occupational and indoor environments and the extraction of genomic DNA from air samples for ITS amplification and sequencing is presented here. Deter...

Fungal exposures in indoor and occupational environments can result in respiratory morbidities, including allergic sensitization and asthma1. Identification of fungal hazards is important for assessing risk and preventing worker exposure. These fungal hazards may be a result of indoor contamination, outdoor air intrusion, or environmental disturbances that result in the transport of fungal materials into areas where workers are present2. Methods to assess fungal exposure have included viable culture sampling as well as microscopic identification of fungal spores. These approaches have several limitations and often overlook many fungal species that could be contributing to the overall fungal burden3. Culture-based approaches can only differentiate those viable fungal organisms that can be cultivated on nutrient media. Identifying fungal spores to species level via microscopy can be confounded by spores sharing similar morphologies. Both methods are highly dependent on mycologists to analyze and identify the fungal species, with many remaining unidentified.
To improve upon existing methodologies used in occupational hazard identification and exposure assessments, many researchers have turned to molecular-based technologies. Sequencing-based approaches for assessing microbial diversity within indoor and occupational environments have revealed a broader spectrum of fungal species encountered compared to methods such as microscopy and viable culture3,4,5. The method presented here describes the air sampling of occupational environments and extraction of genomic DNA for the identification of potential fungal hazards. Hazard identification is accomplished by sequencing the nuclear ribosomal internal transcribed spacer, or ITS, regions that are highly variable among fungi and have been commonly used to differentiate fungal species6,7,8,9. Many species found in occupational settings, such as some species belonging to the phylum Basidiomycota, are not identifiable in viable culture and are difficult to differentiate microscopically. These fungi have been observed in high relative abundance within indoor and occupational environments assessed by sequencing fungal ITS regions3,4,10. ITS sequencing has provided greater knowledge into the diversity of fungi encountered within indoor and occupational environments.
The protocol described here details the methods used to collect, extract, and amplify fungal ITS regions from bioaerosols for sequence analysis. This approach utilizes the National Institute for Occupational Safety and Health (NIOSH) two-stage cyclone aerosol sampler to collect particulates in the air. This sampler was developed to collect bioaerosols and separate respirable (&#8804;4 &#181;m aerodynamic diameter) and non-respirable (&#62;4 &#181;m aerodynamic diameter) particles, which allows for identification of fungal organisms within indoor environments that are most likely to be inhaled by a worker11. Other air samplers, including cyclone samplers, are available on the market that have the ability to collect particles within the respirable range (&#60;4 &#181;m) using filters12,13. In contrast, the NIOSH two-stage cyclone aerosol sampler separates fungal species based on their aerodynamic diameter into disposable, polypropylene tubes that can be immediately processed for downstream applications14.
The processes of extracting genomic DNA and amplifying the fungal ITS regions are detailed in this protocol. The extraction methodologies presented have been developed specifically for the extraction of genomic DNA from fungi and bacteria, as many commercial kits target mammalian cells, bacteria, or specifically yeasts15. The primers used in this study are selected based on their overall coverage of both the fungal ITS 1 and ITS 2 regions4,5. Sequencing of these regions allows for the comparison of many banked ITS sequences, including those that sequence the ITS 1 region, the ITS 2 region, or both the ITS 1 and ITS 2 regions. The fungal diversity of air samples collected in an indoor setting using these methods are shown, revealing a substantial number of sequences placed in the phyla Ascomycota and Basidiomycota as well as other sequences belonging to less dominant fungal phyla, such as Zygomycota. The broad diversity of fungal sequences identified using this approach would not be captured using traditional hazard identification methodologies like cultivation or microscopy. Sequencing of fungal ITS regions provides an enhanced method to identify fungal hazards and allow for a better understanding of indoor and occupational fungal exposures.

<table>
	<tbody>
		<tr>
			<td>NIOSH BC251 bioaerosol cyclone sampler</td>
			<td>NIOSH</td>
			<td>BC251</td>
			<td>The NIOSH sampler is not yet commercially available. Please contact William Lindsley, PhD (wlindsley@cdc.gov) for information on obtaining the NIOSH sampler</td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Microcentrifuge Tubes with Screw Caps</td>
			<td>Fisher Scientific</td>
			<td>02-681-373</td>
			<td>1.5 mL polypropylene microcentrifuge tubes for air sampling; screw top threading must match the threading of the NIOSH sampler</td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge Tubes</td>
			<td>Corning</td>
			<td>352096</td>
			<td>15 mL polypropylene tubes for air sampling</td>
		</tr>
		<tr>
			<td>Clean Room Vinyl Tape, Easy-Remove, 1/4&#34; Width</td>
			<td>McMaster-Carr</td>
			<td>76505A1</td>
			<td>sealing tape</td>
		</tr>
		<tr>
			<td>Filter Cassette, Clear Styrene, 37 mm</td>
			<td>SKC Inc.</td>
			<td>225-3LF</td>
			<td>3-piece sampling cassette (no filter). Contains: cassette base, extension cowl, cassette cap and inlet/outlet plugs</td>
		</tr>
		<tr>
			<td>PTFE hydrophobic fluoropore membrane filters, 3.0 &#181;m, 37 mm</td>
			<td>EMD Millipore</td>
			<td>FSLW03700</td>
			<td>Contains: 37 mm, 3.0 &#181;m PTFE filters and support pads</td>
		</tr>
		<tr>
			<td>Fisherbrand filter forceps</td>
			<td>Fisher Scientific</td>
			<td>09-753-50</td>
			<td>filter forceps</td>
		</tr>
		<tr>
			<td>Model 502 Precision PanaPress</td>
			<td>PanaVise</td>
			<td>502</td>
			<td>pneumatic cassette press is constructed from this precision arbor press</td>
		</tr>
		<tr>
			<td>Scotch Super 33+ vinyl electrical tape</td>
			<td>McMaster-Carr</td>
			<td>76455A21</td>
			<td>19 mm tape</td>
		</tr>
		<tr>
			<td>Multi-purpose Calibration Jar, Large</td>
			<td>SKC Inc.</td>
			<td>225-112</td>
			<td>calibration jar</td>
		</tr>
		<tr>
			<td>Universal PCXR4 Sample Pump</td>
			<td>SKC Inc.</td>
			<td>224-PCXR4</td>
			<td>sampling pump</td>
		</tr>
		<tr>
			<td>Mass Flowmeter 4140</td>
			<td>TSI Inc.</td>
			<td>4140</td>
			<td>flow meter</td>
		</tr>
		<tr>
			<td>Roche High Pure PCR Template Kit</td>
			<td>Roche Diagnostics</td>
			<td>11796828001</td>
			<td>Kit used for genomic DNA extraction. Contains: Lysis buffer, Binding buffer, Proteinase K, Inhibitor removal buffer, Wash buffer, Elution buffer, Glass fiber filter tubes and 2 ml collection tubes</td>
		</tr>
		<tr>
			<td>Fisherbrand 2 mL Reinforced Polypropylene Screw Cap Tubes with Caps</td>
			<td>Fisher Scientific</td>
			<td>15340162</td>
			<td>2 mL reinforced tubes for bead homogenization</td>
		</tr>
		<tr>
			<td>Glass beads, acid washed, 212-300 &#181;m</td>
			<td>Sigma-Aldrich</td>
			<td>G1277</td>
			<td>glass beads</td>
		</tr>
		<tr>
			<td>Fisher Scientific Bead Mill 24 Homogenizer</td>
			<td>Fisher Scientific</td>
			<td>15-340-163</td>
			<td>bead homogenizer</td>
		</tr>
		<tr>
			<td>CelLytic B Cell Lysis Reagent, 10X</td>
			<td>Sigma-Aldrich</td>
			<td>C8740</td>
			<td>lysis reagent</td>
		</tr>
		<tr>
			<td>Platinum Taq polymerase</td>
			<td>Invitrogen</td>
			<td>10966-018</td>
			<td>Contains: Platinum Taq polymerase, 10X PCR buffer (no MgCl2), 50 mM MgCl2, KB Extender</td>
		</tr>
		<tr>
			<td>dNTP Mix</td>
			<td>Invitrogen</td>
			<td>18427-088</td>
			<td>10 mM dNTP mix</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td>Kit used to purify fungal amplicons. Contains: Buffer PB (binding buffer), Buffer PE (washing buffer), Buffer EB (elution buffer), pH Indicator dye (optional), and GelPilot loading dye</td>
		</tr>
		<tr>
			<td>Owl EasyCast Mini Gel Electrophoresis System</td>
			<td>Thermo Fisher</td>
			<td>B1 or B2</td>
			<td></td>
		</tr>
		<tr>
			<td>TrackIt 1 KB Plus DNA Ladder</td>
			<td>Thermo Fisher</td>
			<td>10488-085</td>
			<td>DNA ladder</td>
		</tr>
	</tbody>
</table>

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.

The species distribution within an environment can be assessed using relative abundance by determining the number of clones of each OTU identified in the air samples. Figure 6 is a Krona chart representing the taxonomically placed species within an indoor environment following 60 min of air sampling. It can be observed that the environment contains a variety of species within two major fungal phyla, Ascomycota and Basidiomycota, as well as species belonging t...

Watch this Scientific Journal Video about Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA at JoVE.com

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

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 ...

The overall goal of this air sampling and DNA extraction methodology is to obtain fungal genomic DNA that can amplified, sequenced, and taxonomically placed to identify potential fungal hazards within occupational settings. This method can help answer key questions in the occupational exposure assessment field, such as what fungal hazards are workers exposed to that may result in negative health effects. The main advantage of this technique is that fungal species that remain undetected using culture or microscopy-based methods can be elucidated using sequencing-based approaches.To assemble the air-sampling filter cassette, place a cellulose filter support pad on the gridded surface of the base piece, then, with the help of filter forceps, place a filter on top of the filter support pad with the collection side facing upwards. After assembling, seal the filter cassette by inserting the extension cowl and pressing it down tightly and evenly with a manual or pneumatic press. Then, fit the assembled cassette on top of the NIOSH aerosol sampler and push down as much as possible.Use a piece of 19-millimeter tape to wrap around the outside of the filter cassette and sampler holding the filter cassette in place and acting as a back-up seal to prevent leaks. Screw the collection tubes tightly and fully into the sampler until they bottom out and wrap the sealing tape around the tubes acting as secondary seal against leakage. To conduct personal aerosol sampling, place the sampler within the person's breathing zone.Attach the sampler to the lapel, shoulder, or chest and then keep the sampling pump at the waist level or in a backpack. Turn the pumps on and check after a minute if the pump is functioning. Upon completing air sampling, collect the air sampler from the subject.Remove the sealing tape, unscrew the tubes, and then put a cap on them. Place the third piece of filter cassette over the filter. First, wipe the sampling cassette with 70%ethanol, then use a cassette opening tool to open the sampling cassette while working in a Class II biological safety cabinet.Next, use a filter lifter to lift the support pad and filter upwards. Then, use filter forceps to remove the filter and place it in a sterile Petri dish. Cut the filter into six equal pieces with a steril scalpel and then place them in a two-milliliter reinforced tube containing 300 milligrams of 200 to 500 micrometer glass beads.Immerse the filter-containing tube in liquid nitrogen for 30 seconds and then quickly place it in a bead mill homogenizer at a speed of 4.5 meters per second for 30 seconds. Add 0.5 milliliters of lysis buffer to tubes containing the remaining small pieces of intact filter, then add 0.3 milliliter of lysis buffer to the 15 and 1.5 milliliter air sampler collection tubes and vortex the tubes in an upright and inverted position for 10 to 15 seconds respectively. Transfer the lysis buffer from each collection tube to two-milliliter reinforced tubes containing 300 milligrams of glass beads.Process the tubes in the bead mill homogenizer for 30 seconds, then centrifuge the samples at 20, 000 times gravity for one minute at 22 degrees Celsius. After centrifugation, transfer the supernatant in a sterile 1.5-milliliter microcentrifuge tube and again centrifuge the supernatant at 20, 000 times gravity for one minute at 22 degrees Celsius. After adding 30 microliters of lysis reagent, incubate the tubes at 37 degrees Celsius for 15 minutes.Then, add 0.2 milliliter of binding buffer and 100 micrograms per milliliter of Proteinase K to the tube, then leave the tubes for incubation in a block heater at 70 degrees Celsius for 10 minutes. Finally, add 100 microliters of isopropanol to each tube. Then, place glass fiber filter tubes with a capacity of 700 microliters in two-milliliter collection tubes and transfer the extract solutions into it.Following transfer, centrifuge the collection tubes at 20, 000 times gravity for 30 seconds at 22 degrees Celsius. After centrifugation, insert the glass filter fiber in fresh collection tubes and discard the collection tube. To each tube add 0.5 milliliter of inhibitor removal buffer and conduct centrifugation at 20, 000 times gravity for 30 seconds at 22 degrees Celsius.Discard the collection tube after centrifugation and place the filter tubes into new collection tubes. Then, add 0.5 milliliter of wash buffer to each tube and again subject to centrifugation at 20, 000 times gravity for 30 seconds at 22 degrees Celsius. Discard the collection tube and replace with fresh ones after the centrifugation.Again, add 0.5 milliliter of wash buffer to each tube and centrifuge at 20, 000 times gravity for 30 seconds at 22 degrees Celsius. To remove any residual wash buffer, finally centrifuge the collection tubes at 20, 000 times gravity for one minute at 22 degrees Celsius and discard the collection tubes and replace them with fresh ones. After adding 100 microliters of warm elution buffer, incubate the tubes on the lab bench at room temperature for one to two minutes and centrifuge the collection tubes at 20, 000 times gravity for 30 seconds at 22 degrees Celsius.Place the filter tubes in a sterile 1.5-milliliter microcentrifuge tube and reapply the eluates for another round centrifugation. Use 0.5 milliliter PCR tubes to set up 50-microliter reactions in triplicate with five microliters of already-extracted DNA for each reaction, then program the thermocycler and start the cycle. Mix all three PCR reactions together in sterile 1.5 milliliter microcentrifuge tubes.Add 750 microliters of binding buffer and mix thoroughly by pipetting up and down 10 times. Add 450 microliters of the total mixture to spin columns inserted in collection tubes and start centrifuging at 17, 900 times gravity for 30 seconds, then discard the filtrate and add the remaining 450 microliters of the mixture for a second round of centrifugation. Again, discard the filtrate and add 750 microliters of wash buffer to the spin columns and centrifuge at 17, 900 times gravity for 30 seconds at 22 degrees Celsius.Finally, discard the filtrate and re-spin the empty columns at 17, 900 times gravity for one minute at 22 degrees Celsius. After transferring the spin columns to 1.5-milliliter sterile microcentrifuge tubes, pipette 45 microliters of elution buffer and incubate the tubes on the lab bench at room temperature for five minutes. Then, centrifuge the columns at 17, 900 times gravity for one minute at 22 degrees Celsius.Immerse a 1%agarose gel in one-fold TAE buffer in an electrophoresis chamber, then mix two microliters of five-fold loading buffer with eight microliters of amplified DNA. Finally, load the entire 10 microliters of the sample on the gel along with the DNA ladder. Run the gel at 75 volts for approximately 90 minutes.A representative agarose gel demonstrates amplification of internal transcribed spacer regions of fungal genomic DNA derived from occupational air samples. The lane on the extreme left side represents the DNA marker lane. The bands on the right represent the internal transcribed spacer regions amplified from different occupational air samples with sizes ranging from 750 to 1, 000 base pairs.A representative Krona chart shows the presence of diverse taxonomic fungal species in an indoor environment. The NIOSH bio-aerosol cyclone sampler was used in homes to collect bio-aerosols for one hour to determine the fungal species present. Tabular data represents the diversity and richness indices of fungal species in indoor environments.Data shows higher Chao-1 Richness indices, Shannon Diversity indices, and Bray-Curtis dissimilarity coefficients in air-conditioned and evaporative cooler environments. After watching this video, you should have a good understanding of how to collect indoor and occupational air samples and extract genomic DNA for the analysis of microbial populations. While attempting this procedure, it is important to remember to utilize the best aseptic methods to prevent microbiological contamination of the samples.

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

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

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.

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 ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

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 ...

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence the oxidizing capacity of the atmosphere through interactions with ozone and hydroxyl radicals. The main sink of NOx is the formation and deposition of nitric acid, a component of acid rain and a bioavailable nutrient. NOx is emitted from a mixture of natural and anthropogenic sources, which vary in space and time. The collocation of multiple sources and the short lifetime of NOx make it challenging to quantitatively constrain the influence of different emission sources and their impacts on the environment. Nitrogen isotopes of NOx have been suggested to vary amongst different sources, representing a potentially powerful tool to understand the sources and transport of NOx. However, previous methods of collecting atmospheric NOx integrate over long (week to month) time spans and are not validated for the efficient collection of NOx in relevant, diverse field conditions. We report on a new, highly efficient field-based system that collects atmospheric NOx for isotope analysis at a time resolution between 30 min and 2 hr. This method collects gaseous NOx in solution as nitrate with 100% efficiency under a variety of conditions. Protocols are presented for collecting air in urban settings under both stationary and mobile conditions. We detail the advantages and limitations of the method and demonstrate its application in the field. Data from several deployments are shown to 1) evaluate field-based collection efficiency by comparisons with in situ NOx concentration measurements, 2) test the stability of stored solutions before processing, 3) quantify in situ reproducibility in a variety of urban settings, and 4) demonstrate the range of N isotopes of NOx detected in ambient urban air and on heavily traveled roadways.

Automated, High-resolution Mobile Collection System for the Nitrogen Isotopic Analysis of NOx

Previous work suggests that the nitrogen isotopic composition of atmospheric nitrogen oxides might distinguish the influence of different sources in the environment. We report on an automated, mobile, field-based method for the high collection efficiency of atmospheric NOx for N isotopic analysis at an hourly time resolution.

Nitrogen oxides (NOx = NO + NO2) are a family of atmospheric trace gases that have great impact on the environment. NOx concentrations directly influence ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

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 ...