We thank Heather Maughan and Linda M. Kalikin for careful editing of this manuscript. This work was supported by NIH NHLBI (grant 5R01HL136647-04).

1. Headspace Sorbent Pen (HSP) and sample analysis considerations
NOTE: The HSP containing the sorbent Tenax TA was selected to capture a broad range of volatiles. Tenax has a lower affinity for water compared to other sorbents, which enables it to trap more VOCs from higher-moisture samples. Tenax also has a low level of impurities and can be conditioned for re-use. Sorbent selection was also made in consideration with the column installed in the GC-MS (see the Table of Materials).
<ol>
	<li>Generate negative controls by extracting media and/or sample blanks with the same conditions used for sample extraction.</li>
	<li>Analyze a blank HSP (previously confirmed to be clean and free of significant background) on the GC-MS before analyzing extracted samples. Run blanks between sample types (e.g., three replicates of bacteria mono-culture, blank, three replicates of bacteria co-culture, blank, etc.).</li>
	<li>Limit use of fragrant personal care items or consumption of smelly foods prior to sample extraction and analysis. Ideally, prepare samples in a biosafety hood that has not been cleansed by alcohol or other volatile cleaners for at least 30 min. Turn on airflow in the biosafety hood for 30-60 min prior to sample preparation.</li>
	<li>Keep samples on ice to limit volatile release during sample preparation.</li>
</ol>
2. Mono- and co-culture preparation
<ol>
	<li>In the biosafety hood, inoculate cultures of A. baumannii, S. aureus, and P. aeruginosa in Todd Hewitt growth media. Incubate overnight at 37 &#176;C with 200 rpm agitation.</li>
	<li>After the overnight incubation, perform culture handling in the biosafety hood. Dilute each culture to optical density 0.05 at 500 nm.</li>
	<li>Mix co-cultures in equal parts, and pipette 200 &#181;L of control media, mono-, or co-culture into each well of a 96-well plate, and place in 37 &#176;C incubator for 24 h. Prepare a second plate for a 48-h incubation.</li>
	<li>At the end of the incubation period, prepare samples for extraction in section 4. Pipette liquid cultures into microcentrifuge tubes and store at -80 &#176;C. 
	NOTE: At this point, samples can be stored at -80 &#176;C to extract later if needed.</li>
</ol>
3. Stable isotope probing in biological samples preparation
NOTE: The feces and saliva samples were donated from anonymous donors with approval from the University of California Irvine Institutional Review Board (HS# 2017-3867). The sewage came from San Diego, CA. The sputum samples were collected from subjects with cystic fibrosis as part of a larger study approved by the University of Michigan Medical School Institutional Review Board (HUM00037056).
<ol>
	<li>Perform all biological sample preparations in the biosafety hood.
	<ol>
		<li>To prepare fecal samples, add 1 mL of deionized water to 100 mg of feces in a 1.5 mL microcentrifuge tube and vortex for 3 min. Place on ice when not in use.
		<ol>
			<li>To 15 &#181;L of fecal and water mixture, add 485 &#181;L of Brain Heart Infusion (BHI) medium with 20 mM 13C glucose, or BHI with 30% deuterium (D2O). Ensure that the final volume of the sample is 500 &#181;L. Prepare samples in technical triplicates.</li>
		</ol>
		</li>
		<li>To prepare sewage samples, add 500 &#181;L of sewage to 500 &#181;L of BHI with 20 mM 13C glucose or BHI with 30% D2O for a total volume of 1 mL. Prepare samples in triplicate. Place on ice when not in use.</li>
		<li>To prepare saliva samples, add 50 &#181;L of saliva to 500 &#181;L of BHI with 20 mM 13C glucose or BHI with 30% D2O for a total volume of 550 &#181;L. Prepare samples in triplicate. Place on ice when not in use.</li>
		<li>To prepare sputum samples to compare the volatiles present in the sample prior to and after culturing, perform a first extraction with 15 &#181;L of sputum. Prepare samples in triplicate. Place on ice when not in use. Proceed to section 4 for sample extraction, and extract for 18 h at 37 &#176;C with 200 rpm agitation.</li>
		<li>After the completion of the first extraction of the uncultured sputum samples, save the vials with sputum. Add 500 &#181;L of BHI with 20 mM 13C glucose to the vials with sputum from 3.5.1. Place on ice when not in use.</li>
	</ol>
	</li>
	<li>Proceed to section 4 for sample extraction.</li>
</ol>
4. Sample extraction
<ol>
	<li>Place empty volatile organic analysis (VOA) vials (20 mL) on the cold plate, and place the cold plate on ice in the biosafety hood.</li>
	<li>Turn on the 5600 sorbent pen extraction unit (SPEU), and adjust to the desired temperature as required for each method. 
	NOTE: For stable isotope probing experiments at 37 &#176;C, reaching the setpoint can take up to 15 min. For mono- and co-culture experiments at 70 &#176;C, reaching the setpoint can take up to 60 min.</li>
	<li>Collect clean HSPs that are equal to the number of samples prepared, including HSPs for media or sample controls.</li>
	<li>Label 20 mL VOA vials according to samples, replicates, and HSP IDs as needed. Use a marker that resists water in case condensation forms on the outside of the vial while on ice.</li>
	<li>Inside the biosafety hood, unscrew the white cap on the vial, quickly pipet sample into the vial, and assemble the black cap, lid liner, and HSP. 
	NOTE: Samples should not come into contact with the HSP, and sample volume will depend on sample type.</li>
	<li>Place the vial containing the sample and HSP back on the cold plate.</li>
	<li>Repeat steps 4.5 and 4.6 for each sample. Perform these steps per sample instead of all at once to prevent sample warming and thus, premature volatile release.</li>
	<li>Once all samples have been prepared in the glass vials, perform the following steps outside the biosafety hood on the bench. Turn on the vacuum pump, place the vials under vacuum to 30 mmHg, and remove the vacuum source. 
	NOTE: The vials do not need to be on the cold tray after vacuum application has been completed.</li>
	<li>Double-check the pressure after placing all samples under vacuum using the pressure gauge. If a vial has a leak, ensure that the cap is screwed on tightly, and that the white O-rings of the HSP and lid liners are properly in place. 
	NOTE: A compromised seal can result in decreased volatile detection compared to a vial under vacuum.</li>
	<li>Place vials in the SPEU for the optimized time and temperature with agitation at 200 rpm. Extract cultures for 1 h at 70 &#176;C. Extract stable isotope probing experiments with fecal, sewage, saliva, and sputum samples for 18 h at 37 &#176;C.</li>
	<li>Place the cold plate at -80 &#176;C for use after the extraction period is complete.</li>
	<li>When extraction is complete, place samples on the cold plate for 15 min to draw out water vapor from the HSP and vial headspace.</li>
	<li>Transfer the HSPs to their sleeves. 
	&#8203;NOTE: The experiment can be paused here for up to ~1 week at room temperature before losing the more highly volatile compounds from the HSPs.</li>
</ol>
5. Analyze samples on the gas chromatography - mass spectrometer (GC-MS)
<ol>
	<li>Use the following GC-MS (see the Table of Materials) settings: 35 &#176;C with a 5 min hold, 10 &#176;C/min ramp to 170 &#176;C, and a 15 &#176;C/min ramp to 230 &#176;C with a 20:1 split ratio and a total runtime of 38 min.</li>
	<li>Set the desorption method as follows: 2 min, 70 &#176;C preheat; 2 min 260 &#176;C desorption; 34 min, 260 &#176;C bakeout; and 2 min, 70 &#176;C post bake.</li>
	<li>Set up the sequence of samples, and start the run according to instrumentation.
	<ol>
		<li>To set up a sequence on the Entech Software, open the program. In the options to the right of the instrument dropdown menu, select 5800 | Sequence.</li>
		<li>Observe the sequence table in the Entech software similar to that in the GC-MS software. Name the Sample ID column according to Current date_vial number. Keep in mind that Name is analogous to Name in the GC-MS sequence table, and 5800 Method determines the rate of temperature ramp, holding times, etc. (opens a menu to select the method generated in step 5.2).</li>
		<li>Keep in mind that the Tray and Position columns determine where the Sample Preparation Rail (SPR) will go to pick up the HSPs.
		<ol>
			<li>Observe the two trays with 30 spots each to the immediate left, laid out as six columns with five spots each.&#160;The tray position that is left most and closest to the user (front) is position 1, while the rightmost, furthest away is position 30.</li>
			<li>Note that these trays are HSP A or B, where HSP B is the tray closer to the SPR (innermost tray), and directly behind HSP B is HSP Blank. Place the extracted samples into the trays, and select the spot on the sequence accordingly.</li>
		</ol>
		</li>
		<li>Save the sequence table, select Run on the left-hand side, then Start with blank in desorber if the blank HSP is in the desorber (denoted by a HSP marked by yellow label).</li>
	</ol>
	</li>
	<li>Note that HSPs will be handled by the SPR for each sample in the sequence. Let the SPR warm up, then a message will appear at the top of the screen to confirm if the blank is in the desorber. Click on Skip to confirm that the pen is there. Allow the SPR to run all samples automatically, and the sequence on the GC-MS side will automatically record the data in separate files.</li>
</ol>
6. Data analysis
<ol>
	<li>Quality-filter data on GC-MS software (Table of Materials).
	<ol>
		<li>Review each peak on the chromatogram, and annotate peaks that match the National Institute of Standards &#38; Technology (NIST) library (or with another available library).</li>
		<li>Add annotated chromatogram peaks to the processing method. Set the criteria for selecting peaks to include compounds with a greater than 75% probability, and ensure that the alignment of each identifying ion of the compound lies within the center of the peak.
		<ol>
			<li>To add a peak to the processing method, select Calibrate | Edit Compound | Name | insert compound under External Standard Compound. Add the name of the compound, retention time, Quant Signal Target Ion. Add the three largest peaks. To save, select ok | Method | Save.</li>
		</ol>
		</li>
		<li>Once the process method is set up, proceed to Quantitate | Calculate, and View | QEdit Quant Result.</li>
		<li>Inspect each compound to ensure that the peaks align with their expected retention times and are above background noise.</li>
		<li>Once QEdit has been completed, select Exit | Yes to save the QEdits and return to the main chromatogram. Export the area integrations by opening the file on the left-hand side. Select Quantitate | Generate Report.</li>
		<li>To export files for use in DExSI, select File | Export Data to AIA format | Create New Directory, and select a location for the file or Use Existing Directory.</li>
		<li>Observe a new window opening up to select files for export. Move the files to the right side of the window and click on Process. Wait for a few seconds to a few minutes depending on the number of files being converted.</li>
	</ol>
	</li>
	<li>Correct for isotope abundance in DExSI according to instructions for the DExSI software (https://github.com/DExSI/DExSI), and perform analysis with a favorite software or program (e.g., R).&#160;Scripts used to generate the figures are located at&#160;<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/joannlp/VOC_SIP&amp;source=gmail&amp;ust=1646398895210000&amp;usg=AOvVaw18Jx-lKhdf3eNanCnF4OhQ" href="https://github.com/joannlp/VOC_SIP" target="_blank">https://github.com/joannlp/VOC_SIP</a>.</li>
</ol>

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V. L. V and S. J. B. D. were former employees of Entech Instruments Inc., and K. W. is a member of Entech&#39;s University Program. J. P., J. K., and C. I. R. have no conflicts of interest to declare.

To identify volatile production in in vitro cultures and human-associated samples, volatile analysis of mono- and co-cultures of P. aeruginosa, S. aureus, and A. baumanii and stable isotope probing of different biological samples were performed. In the analysis for the mono- and co-cultures, volatiles were detected by performing a short extraction for 1 h at 70 &#176;C. The volatile analysis of mono- and co-cultures allowed the survey of the compounds produced both by individual species and dur...

Volatile organic compounds (VOCs) have great promise for bacterial detection and identification because they are emitted from all organisms, and different microbes have unique VOC signatures. Volatile molecules have been utilized as a non-invasive measurement for detecting various respiratory infections including chronic obstructive pulmonary disease1, tuberculosis2 in urine3, and ventilator-associated pneumonia4, in addition to distinguishing subjects with cystic fibrosis (CF) from healthy control subjects5,6. Volatile signatures have even been used to distinguish specific pathogen infections in CF (Staphylococcus aureus7, Pseudomonas aeruginosa8,9, and S. aureus vs. P. aeruginosa10). However, with the complexity of such biological samples, it is often difficult to pinpoint the source of specific VOCs.
One strategy for disentangling the volatile profiles from multiple infecting microbes is to perform headspace analysis of microorganisms in both mono- and co-culture11. Headspace analysis examines the analytes emitted into the &#34;headspace&#34; above a sample rather than those embedded in the sample itself. Microbial metabolites have often been characterized in mono-cultures because of the difficulty in determining the origin of microbial metabolites in complex clinical samples. By profiling volatiles from bacterial mono-cultures, the types of volatiles a microbe produces in vitro may represent a baseline of its volatile repertoire. Combining bacterial cultures, e.g., creating co-cultures, and profiling the volatile molecules produced may reveal the interactions or cross-feeding between the bacteria12.
Another strategy for identifying the microbial origin of volatile molecules is to provide a nutrient source that is labeled with a stable isotope. Stable isotopes are naturally occurring, non-radioactive forms of atoms with a different number of neutrons. In a strategy that has been utilized since the early 1930s to trace active metabolism in animals13, the microorganism feeds off of the labeled nutrient source and incorporates the stable isotope into its metabolic pathways. More recently, a stable isotope in the form of heavy water (D2O) has been used to identify metabolically active S. aureus in a clinical CF sputum sample14. In another example, 13C-labeled glucose has been used to demonstrate the cross-feeding of metabolites between CF clinical isolates of P. aeruginosa and Rothia mucilaginosa12 .
With the advancement of mass spectrometry techniques, methods of detecting volatile cues have moved from qualitative observations to more quantitative measurements. By using gas chromatography mass spectrometry (GC-MS), processing of biological samples has become within reach for most laboratory or clinical settings. Many methods to survey volatile molecules have been used to profile samples such as food, bacterial cultures, and other biological samples, and air and water to detect contamination. However, several common methods of volatile sampling with high-throughput require solvent and are not performed with the advantages provided by vacuum extraction. In addition, larger volumes or quantities (greater than 0.5 mL) of sampled materials are often required for analysis15,16,17,18,19, although this is substrate-specific and requires optimization for each sample type and method.
Here, vacuum-assisted sorbent extraction (VASE) followed by thermal desorption on a GC-MS was employed to survey the volatile profiles of bacterial mono- and co-cultures and identify actively produced volatiles with stable isotope probing from human feces, saliva, sewage, and sputum samples (Figure 1). With limited sample quantities, VOCs were extracted from as little as 15 &#181;L of sputum. Isotope probing experiments with human samples required adding a stable isotope source, such as 13C glucose, and media to cultivate the growth of the microbial community. The active production of volatiles was identified as a heavier molecule by GC-MS. Extraction of volatile molecules under a static vacuum enabled the detection of volatile molecules with increased sensitivity20,21,22.

<table><tbody><tr><td>&lt;sup&gt;13&lt;/sup&gt;C glucose</td><td>Sigma-Aldrich</td><td>389374-1G</td><td></td></tr><tr><td>2-Stg Diaph Pump</td><td>Entech Instruments</td><td>01-10-20030</td><td></td></tr><tr><td>20 mL VOA vials</td><td>Fisher Scientific</td><td>5719110</td><td></td></tr><tr><td>24 mm Black Caps with hole, no septum</td><td>Entech Instruments</td><td>01-39-76044B</td><td>holds lid liner in place on vial</td></tr><tr><td>24 mm vial liner for sorbent pens</td><td>Entech Instruments</td><td>SP-L024S</td><td>allows pens to make a vacuum seal at top of vial</td></tr><tr><td>5600 Sorbent pen extraction unit (SPEU)</td><td>Entech Instruments</td><td>5600-SPES</td><td>5600 Sorbent Pen Extraction Unit -120 VAC</td></tr><tr><td>96-well assay plates</td><td>Genesee</td><td>25-224</td><td></td></tr><tr><td>Brain Heart Infusion (BHI) media</td><td>Sigma-Aldrich</td><td>53286-500G</td><td></td></tr><tr><td>ChemStation Stofware</td><td>Agilent</td><td></td><td></td></tr><tr><td>DB-624 column</td><td>Agilent</td><td>122-1364E</td><td>60 m, 0.25 mm ID, 1.40 micron film thickness, in GC-MS</td></tr><tr><td>Deuterium oxide</td><td>Sigma-Aldrich</td><td>151882-1L</td><td></td></tr><tr><td>Dexsi sofware</td><td>Dexsi (open source)</td><td></td><td></td></tr><tr><td>GC-MS (7890A GC and 5975C inert XL MSD with Triple-Axis Detector)</td><td>Agilent</td><td></td><td>7890A GC and 5975C inert XL MSD with triple-axis detector</td></tr><tr><td>Headspace Bundle HS-B01, 120VA</td><td>Entech Instruments</td><td>SP-HS-B01</td><td>Items for running headspace extraction included in bundle</td></tr><tr><td>Headspace sorbent pen (HSP) - blank</td><td>Entech Instruments</td><td>SP-HS-0</td><td></td></tr><tr><td>Headspace sorbent pen (HSP) Tenax TA (35/60 Mesh)</td><td>Entech Instruments</td><td>SP-HS-T3560</td><td></td></tr><tr><td>Microcentrifuge tubes (2 mL)</td><td>VWR</td><td>53550-792</td><td></td></tr><tr><td>O-rings</td><td>Entech Instruments</td><td>SP-OR-L024</td><td></td></tr><tr><td>Sample Preparation Rail</td><td>Entech Instruments</td><td></td><td></td></tr><tr><td>Sorbent pen thermal conditioner</td><td>Entech Instruments</td><td>3801-SPTC</td><td></td></tr><tr><td>Todd Hewitt (TH) media</td><td>Sigma</td><td>T1438-500G</td><td></td></tr></tbody></table>

capturing actively produced microbial volatile organic compounds from human-associated samples with vacuum-assisted sorbent extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

Mono- and co-cultures of S. aureus, P. aeruginosa, and A. baumannii 
The mono- and co-cultures consisted of the bacterial species S. aureus, P. aeruginosa, and A. baumannii. These are common opportunistic pathogens found in human wounds and chronic infections. To identify the volatile molecules present in the mono- and co-cultures, a short 1-h extraction was performed at ...

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Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

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Research

JoVE Journal

Biochemistry

4.0K Views.  University of California Irvine. Dit protocol stelt ons in staat om vluchtige metabolieten en actief geproduceerde vluchtige stoffen uit microbiële organismen in verschillende biologische monsters gemakkelijk te concentreren en te identificeren. VASE is een gebruiksvriendelijkere methode om vluchtige stoffen met een lage abundantie te concentreren. Het enige wat je nodig hebt is een monster onder bijna-vacuüm, en laat de natuurkunde de rest doen.Toegang tot vluchtige analyse van klinische monsters heeft belangrijke ...

Video: Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

Bioengineering

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

Environment

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 ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

Chemistry

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Rapid Collection of Floral Fragrance Volatiles using a Headspace Volatile Collection Technique for GC-MS Thermal Desorption Sampling

Fragrances of many flower families have been sampled and the volatiles analyzed. Knowing the compounds that make up the fragrances can be an important step to conservation of flowers that are threatened or endangered. Because floral fragrance is critical for attracting pollinators, this method could be used to better understand or even enhance pollination. We present a protocol using a portable charcoal air filter and vacuum to collect floral fragrance volatiles, which are then analyzed by a GC-MS. By using this method, fragrance volatiles can be sampled using a non-destructive method with a machine that is easily transported. This methodology uses a rapid sampling procedure, cutting sampling time down from 2-3 hours to approximately 10 minutes. Using GC-MS, the fragrance compounds can be identified individually, based on authentic standards. The steps used for collecting fragrance and control data are presented, from material setup to collecting the data output.

Here, we present a protocol for collecting the floral fragrance volatiles from blooming flowers, using a non-destructive sampling procedure.

Fragrances of many flower families have been sampled and the volatiles analyzed. Knowing the compounds that make up the fragrances can be an important ...

Sampling and Analysis of Animal Scent Signals

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry, to understand how they may be used in animal communication. This technique allows the semi-quantitative analysis of the volatile components of odor secretions by enabling the separation and tentative identification of the components in the sample, followed by the analysis of peak area ratios to look for trends that could signify compounds that may be involved in signaling. The key strengths of this current approach are the range of sample types that can be analyzed; the lack of need for any complex sample preparation or extractions; the ability to separate and analyze the components of a mixture; the identification of the components detected; and the capability to provide semi-quantitative and potentially quantitative information on the components detected. The main limitation to the methodology relates to the samples themselves. Since the components of specific interest are volatile, and these could easily be lost, or their concentrations altered, it is important that the samples are stored and transported appropriately after their collection. This also means that sample storage and transport conditions are relatively costly. This method can be applied to a variety of samples (including urine, feces, hair and scent-gland odor secretions). These odors consist of complex mixtures, occurring in a range of matrices, and thus necessitate the use of techniques to separate the individual components and extract the compounds of biological interest.

We have developed an effective methodology for sampling and analysis of odor signals in order to understand how they may be used in animal communication. Particularly, we use headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry to analyze the volatile components of animal odors and scent-markings.

We have developed an effective methodology for sampling and analysis of odor signals, by using headspace solid-phase microextraction coupled with gas ...

Behavior

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars with enhanced organoleptic characteristics and thus, to increase consumer acceptance. High-throughput metabolomic platforms have been recently developed to quantify a wide range of metabolites in different plant tissues, including key compounds responsible for fruit taste and aroma quality (volatilomics). A method using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is described here for the identification and quantification of VOCs emitted by ripe blackcurrant fruits, a berry highly appreciated for its flavor and health benefits.
Ripe fruits of blackcurrant plants (Ribes nigrum) were harvested and directly frozen in liquid nitrogen. After tissue homogenization to produce a fine powder, samples were thawed and immediately mixed with sodium chloride solution. Following centrifugation, the supernatant was transferred into a headspace glass vial containing sodium chloride. VOCs were then extracted using a solid-phase microextraction (SPME) fiber and a gas chromatograph coupled to an ion trap mass spectrometer. Volatile quantification was performed on the resulting ion chromatograms by integrating peak area, using a specific m/z ion for each VOC. Correct VOC annotation was confirmed by comparing retention times and mass spectra of pure commercial standards run under the same conditions as the samples. More than 60 VOCs were identified in ripe blackcurrant fruits grown in contrasting European locations. Among the identified VOCs, key aroma compounds, such as terpenoids and C6 volatiles, can be used as biomarkers for blackcurrant fruit quality. In addition, advantages and disadvantages of the method are discussed, including prospective improvements. Furthermore, the use of controls for batch correction and minimization of drift intensity have been emphasized.

A headspace solid-phase microextraction-gas-chromatography platform is described here for fast, reliable, and semi-automated volatile identification and quantification in ripe blackcurrant fruits. This technique can be used to increase knowledge about fruit aroma and to select cultivars with enhanced flavor for the purpose of breeding.

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

Gas Chromatography-Mass Spectrometry Paired with Total Vaporization Solid-Phase Microextraction as a Forensic Tool

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including controlled substances, ignitable liquids, and explosives. GC-MS can be coupled with Solid-Phase Microextraction (SPME), in which a fiber with a sorptive coating is placed into the headspace above a sample or immersed in a liquid sample. Analytes are sorbed onto the fiber which is then placed inside the heated GC inlet for desorption. Total Vaporization Solid-Phase Microextraction (TV-SPME) utilizes the same technique as immersion SPME but immerses the fiber into a completely vaporized sample extract. This complete vaporization results in a partition between only the vapor phase and the SPME fiber without interference from a liquid phase or any insoluble materials. Depending upon the boiling point of the solvent used, TV-SPME allows for large sample volumes (e.g., up to hundreds of microliters). On-fiber derivatization may also be performed using TV-SPME. TV-SPME has been used to analyze drugs and their metabolites in hair, urine, and saliva. This simple technique has also been applied to street drugs, lipids, fuel samples, post-blast explosive residues, and pollutants in water. This paper highlights the use of TV-SPME to identify illegal adulterants in very small samples (microliter quantities) of alcoholic beverages. Both gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) were identified at levels that would be found in spiked drinks. Derivatization by a trimethylsilyl agent allowed for conversion of the aqueous matrix and GHB into their TMS derivatives. Overall, TV-SPME is quick, easy, and requires no sample preparation aside from placing the sample into a headspace vial.

Total Vaporization Solid Phase Microextraction (TV-SPME) completely vaporizes a liquid sample whilst analytes are sorbed onto a SPME fiber. This allows for partitioning of the analyte between only the solvent vapor and the SPME fiber coating.

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including ...

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

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