Name: tracking microbial contamination in retail environments using fluorescent powder - a retail delicatessen environment example
Uploaded: 2014-03-05T17:00:00
Description: Watch this Scientific Journal Video about Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example at JoVE.com

The authors would like to thank all of the food safety lab students and volunteers who helped with this study. This research has been funded by a USDA National Integrated Food Safety Initiative (NIFSI) grant (Award #10507316).

1. Standard Curve
<ol>
	<li>Weigh 37.5 mg fluorescent powder and dissolve in 6 ml 200 proof ethanol. Make 2-fold serial dilutions of this solution in 200 proof ethanol.</li>
	<li>Vortex all the samples for 10 sec and measure the absorbance at 370 nm.</li>
	<li>Calculate the amount of fluorescent powder per 1 ml 200 proof ethanol.</li>
	<li>Prepare the standard curve (concentration in mg/ml&#160;on the y-axis and absorbance at 370 nm on the x-axis) to demonstrate the absorbance based on fluorescent powder concentration....

<ol>
	<li>Sheen, S., Hwang, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+modeling+the+cross-contamination+of+Escherichia+coli+O157:+H7+on+the+surface+of+ready-to-eat+meat+product+while+slicing.">Mathematical modeling the cross-contamination of Escherichia coli O157: H7 on the surface of ready-to-eat meat product while slicing.</a> Food Microbiol. 27, 37-43 (2010).</li><li>Crandall, P., Neal, 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=Minimizing+the+risk+of+Listeria+monocytogenes+in+retail+delis+by+developing+employee+focused,+cost+effective+training.">Minimizing the risk of Listeria monocytogenes in retail delis by developing employee focused, cost effective training.</a> Agric. Food Anal. Bacteriol. 1, 159-174 (2011).</li><li>Koo, O. K., Sirsat, S. A., 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=Physical+and+chemical+control+of+Salmonella+in+ready-to-eat+products.">Physical and chemical control of Salmonella in ready-to-eat products.</a> Agric. Food Anal. Bacteriol. 2, 56-68 (2012).</li><li>Hanning, I. B., Johnson, M. G., Ricke, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precut+prepackaged+lettuce:+a+risk+for+listeriosis?.">Precut prepackaged lettuce: a risk for listeriosis?.</a> Foodborne Pathog. Dis. 5, 731-746 (2008).</li><li>Hanning, I. B., Nutt, J. D., Ricke, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonellosis+outbreaks+in+the+United+States+due+to+fresh+produce:+sources+and+potential+intervention+measures.">Salmonellosis outbreaks in the United States due to fresh produce: sources and potential intervention measures.</a> Foodborne Pathog. Dis. 6, 635-648 (2008).</li><li>Zhu, M., Du, M., 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=Control+of+Listeria+monocytogenes+contamination+in+ready&#8208;to&#8208;eat+meat+products.">Control of Listeria monocytogenes contamination in ready&#8208;to&#8208;eat meat products.</a> Compr. Rev. Food Sci. Food Safety. 4, 34-42 (2005).</li><li>Lianou, A., Sofos, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+incidence+and+transmission+of+Listeria+monocytogenes+in+ready-to-eat+products+in+retail+and+food+service+environments.">A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments.</a> J. Food Protect. 70, 2172-2198 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+risk+assessment+of+relative+risk+to+public+health+from+foodborne+Listeria+monocytogenes+among+selected+categories+of+ready-to-eat+foods.">Quantitative risk assessment of relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods.</a> , .</li><li>Endrikat, S., Gallagher, 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=A+comparative+risk+assessment+for+Listeria+monocytogenes+in+prepackaged+versus+retail-sliced+deli+meat.">A comparative risk assessment for Listeria monocytogenes in prepackaged versus retail-sliced deli meat.</a> J. Food Protect. 73, 612-619 (2010).</li><li>Pradhan, A. K., Ivanek, R., 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=Comparison+of+public+health+impact+of+Listeria+monocytogenes+product-to-product+and+environment-to-product+contamination+of+deli+meats+at+retail.">Comparison of public health impact of Listeria monocytogenes product-to-product and environment-to-product contamination of deli meats at retail.</a> J. Food Protect. 74, 1860-1868 (2011).</li><li>Jones, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handwashing+Technique+Analysis.">Handwashing Technique Analysis.</a> Patent number 5900067. , (1999).</li><li>Gibson, K. E., Koo, O. K., 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=Observation+and+relative+quantification+of+cross-contamination+within+a+mock+retail+delicatessen+environment.">Observation and relative quantification of cross-contamination within a mock retail delicatessen environment.</a> Food Control. 31, 116-124 (2013).</li><li>Xiao, M., Zheng, G., 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=Method+and+System+for+Fecal+Detection.">Method and System for Fecal Detection.</a> Patent number 5821546. , (1998).</li><li>Waldroup, A., Kirby, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+and+System+for+Fecal+Detection.">Method and System for Fecal Detection.</a> Patent number 5621215. , (1997).</li><li>Ashby, K. D., Wen, 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=Fluorescence+of+dietary+porphyrins+as+a+basis+for+real-time+detection+of+fecal+contamination+on+meat.">Fluorescence of dietary porphyrins as a basis for real-time detection of fecal contamination on meat.</a> J. Agric. Food Chem. 51, 3502-3507 (2003).</li><li>Cabrera-Diaz, E., Moseley, T. M., 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=Fluorescent+protein+marked+Escherichia+coli+biotype+I+strains+as+surrogates+for+enteric+pathogens+in+validation+of+beef+carcass+interventions.">Fluorescent protein marked Escherichia coli biotype I strains as surrogates for enteric pathogens in validation of beef carcass interventions.</a> J. Food Protect. 72, 295-303 (2009).</li><li>Byrd, J., Hargis, B., 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=Fluorescent+marker+for+the+detection+of+crop+and+upper+gastrointestinal+leakage+in+poultry+processing+plants.">Fluorescent marker for the detection of crop and upper gastrointestinal leakage in poultry processing plants.</a> Poult. Sci. 81, 70-74 (2002).</li><li>Vorst, K. L., Todd, E. C., 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=Transfer+of+Listeria+monocytogenes+during+mechanical+slicing+of+turkey+breast+bologna,+and+salami..">Transfer of Listeria monocytogenes during mechanical slicing of turkey breast bologna, and salami..</a> J. Food Protect. 69, 619-626 (2006).</li></ol>

The overall goal of this study was to demonstrate how bacterial cross contamination could occur in a retail deli environment in order to develop strategies to control and reduce the risk of foodborne illness. The retail deli environment has an especially high risk for L. monocytogenes contamination. The USDA-FSIS reported that deli meat sliced at the retail store has a seven times higher association with listeriosis that deli meat sliced and packaged at a federally inspected plant9.
<p class="jove...

Cross contamination of foodborne pathogens in the food preparation environment, especially in the retail section, is a major concern due to an increased risk of foodborne illness from a variety of sources including the meats as well as the vegetables1-5. Most often, bacterial pathogens enter the retail environment through contaminated food products6. Ready-to-eat foods prepared at the retail level are of particular concern as there is usually no further intervention or treatment (i.e. cooking or heating) prior to consumption7. Moreover, pathogens present on contaminated RTE food can then be transferred to other food products or food contact surfaces in the retail environment.
While cross contamination can occur within virtually any type of retail food environment, delis are of particular interest specifically because of the association of RTE deli meats with the pathogen Listeria monocytogenes2. Based on a risk assessment analyses conducted by the U.S. Food and Drug Administration (FDA) and U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS), contaminated deli meats are likely responsible for 90% of the cases of listeriosis -&#160;a rare yet severe human disease caused by L. monocytogenes -&#160;in the United States8. In general, deli meats sliced at retail and food service establishments have been associated with a higher risk of L. monocytogenes compared to presliced deli meats in a USDA inspected plant by the manufacturer9. This could be because of the increased likelihood of introduction of pathogens by food handlers, incoming raw ingredients, or processed foods that may become contaminated post-treatment7. Overall, listeriosis accounts for approximately 28% of the estimated deaths caused by foodborne illness per year in the U.S.
Because of the increased risk of L. monocytogenes contamination in deli meats sliced at the retail level, the deli slicer itself has been identified as an important factor for surface transfer of foodborne pathogens in the retail environment2,10. As indicated by Sheen1, slicing is the last processing step prior to consumption; therefore,&#160;the deli slicer should be considered a critical control point for both understanding and preventing cross contamination1.
The overall goal of this study was to develop a methodology to track contamination in the retail deli environment using a fluorescent powder such as Glo Germ. This is a fluorescent compound that has been used to simulate bacterial cross contamination and sanitary practices11,12. This study was designed to track pathogen transfer from contaminated meats to the slicer, the surrounding environment, and other deli meats. It is crucial to understand the pathways of contamination to develop both effective interventions and educational material for retail employees and managers. A similar approach may be employed to simulate pathogen transfer in other processing environments (e.g. poultry and beef), farms, and hospitals.

<table border="0" cellpadding="0" cellspacing="0" width="637">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glo-Germ Powder</td>
			<td style="width:71px;">Glo Germ Co</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Purchased from vendor</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Ethanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E7023</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Permanent markets</td>
			<td style="width:71px;">Sharpie</td>
			<td style="width:119px;"></td>
			<td colspan="2">Purchased from stationary store</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Gloves</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">82026-424</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Deli Meat</td>
			<td style="width:71px;">NA</td>
			<td style="width:119px;">NA</td>
			<td colspan="2">Bologna Chub from regular grocery store</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Cutting Board</td>
			<td style="width:71px;">NA</td>
			<td style="width:119px;">NA</td>
			<td colspan="2">A regukar kitchen cutting board</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Knife</td>
			<td style="width:71px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>A regular kitchen knife&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">5 x 5 cm sterile templates</td>
			<td style="width:71px;">NA</td>
			<td style="width:119px;">NA</td>
			<td colspan="2">Aluminium foil templates cut into 5x 5 cm templates and sterilized&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">15 ml polypropylene centrifuge tubes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">89039-664</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Cotton swabs</td>
			<td style="width:71px;">Puritan</td>
			<td style="width:119px;">25-806</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Glass cuvettes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">470019-186</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Vortex</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">58816-121</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Flip camera</td>
			<td style="width:71px;">Flip Ultra HD</td>
			<td style="width:119px;">NA</td>
			<td>Purchased online</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Deli slicer</td>
			<td style="width:71px;">Bizerba</td>
			<td style="width:119px;">SE-12</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Deli refrigerator</td>
			<td style="width:71px;">True company</td>
			<td style="width:119px;">TDBD-722</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Scale</td>
			<td style="width:71px;"></td>
			<td style="width:119px;">NA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Spectrophotometer</td>
			<td style="width:71px;">Milton Roy Spectronic 20D</td>
			<td style="width:119px;">NA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

tracking microbial contamination in retail environments using fluorescent powder - a retail delicatessen environment example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

Figure 1 represents the areas of the slicer that were swabbed after the volunteers completed the tasks assigned. The volunteers were videotaped to analyze the average frequency of hand contact on these various surfaces. The frequency of hand contact was analyzed by four different observers and averaged. These results are demonstrated in Figure 2. The data shows that the meat grip, deli meat wrapper, deli meat, and deli paper had the highest rates of hand contact (average of 8-14 contacts...

Watch this Scientific Journal Video about Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example at JoVE.com

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

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Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

An Aquatic Microbial Metaproteomics Workflow: From Cells to Tryptic Peptides Suitable for Tandem Mass Spectrometry-based Analysis

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and metabolism. Although powerful, metagenomics alone can only elucidate functional potential. On the other hand, metaproteomics enables the description of the expressed in situ metabolism and function of a community. Here we describe a protocol for cell lysis, protein and DNA isolation, as well as peptide digestion and extraction from marine microbial cells collected on a cartridge filter unit (such as the Sterivex filter unit) and preserved in an RNA stabilization solution (like RNAlater). In mass spectrometry-based proteomics studies, the identification of peptides and proteins is performed by comparing peptide tandem mass spectra to a database of translated nucleotide sequences. Including the metagenome of a sample in the search database increases the number of peptides and proteins that can be identified from the mass spectra. Hence, in this protocol DNA is isolated from the same filter, which can be used subsequently for metagenomic analysis.

This protocol is for the extraction and concentration of protein and DNA from microbial biomass collected from seawater, followed by the generation of tryptic peptides suitable for tandem mass spectrometry-based proteomic analysis.

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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 ...

Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris

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Most microplastic research to date has occurred in marine systems where suspended solid levels are relatively low. Focus is now shifting to freshwater systems, which may feature high sediment loads and floating debris. This protocol addresses collecting and analyzing microplastic samples from aquatic environments that contain high suspended solid loads.

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only ...

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

Fluorescently Labeled Bacteria as a Tracer to Reveal Novel Pathways of Organic Carbon Flow in Aquatic Ecosystems

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial communities has many limitations, and determining a predator, prey, and predatory rates is often difficult. Presented here is an optimized method based on the addition of fluorescently labelled prey as a tracer, which allows for reliable quantitation of the grazing rates in aquatic predatory eukaryotes and estimation of nutrient transfer to higher trophic levels.

Presented here is a protocol for a single-cell, epifluorescence microscopy-based technique to quantify grazing rates in aquatic predatory eukaryotes with high precision and taxonomic resolution.

Elucidating trophic interactions, such as predation and its effects, is a frequent task for many researchers in ecology. The study of microbial ...

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized into five different toxinotypes, A through E, based on the carriage of four major toxin genes. The prevalence and distribution of these various toxinotypes is understudied, especially their pervasiveness in American retail food. Of particular interest to us are the type B and D strains, which produce epsilon toxin, an extremely lethal toxin suggested to be the environmental trigger of multiple sclerosis in humans. To evaluate the presence of different C. perfringens toxinotypes in various food samples, we developed an easy method to selectively culture these bacteria without the use of an anaerobic container system only involving three culturing steps. Food is purchased from local grocery stores and transported to the laboratory under ambient conditions. Samples are minced and inoculated into modified rapid perfringens media (RPM) and incubated overnight at 37 &#176;C in a sealed, airtight conical tube. Overnight cultures are inoculated onto a bottom layer of solid Tryptose Sulfite Cycloserine (TSC) agar, and then overlaid with a top layer of molten TSC agar, creating a &#34;sandwiched&#34;, anaerobic environment. Agar plates are incubated overnight at 37 &#176;C and then evaluated for appearance of black, sulfite-reducing colonies. C. perfringens-suspected colonies are removed from the TSC agar using sterile eye droppers, and inoculated into RPM and sub-cultured overnight at 37 &#176;C in an airtight conical tube. DNA is extracted from the RPM subculture, and then analyzed for the presence of C. perfringens toxin genes via polymerase chain reaction (PCR). Depending on the type of food sampled, typically 15&#8211;20% of samples test positive for C. perfringens.

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples

The objective of this protocol is to detect different Clostridium perfringens toxinotypes in locally purchased foods, particularly epsilon toxin producing strain types B and D, without the use of anaerobic chambers.

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized ...

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil and gas. This process revolutionized the U.S. energy industry, as it gives access to resources that were previously unobtainable and now produces two-thirds of the total natural gas in the United States. Although fracking has had a positive impact on the U.S. economy, several studies have highlighted its detrimental environmental effects. Of particular concern is the effect of fracking on headwater streams, which are especially important due to their disproportionately large impact on the health of the entire watershed. The bacteria within those streams can be used as indicators of stream health, as the bacteria present and their abundance in a disturbed stream would be expected to differ from those in an otherwise comparable but undisturbed stream. Therefore, this protocol aims to use the bacterial community to determine if streams have been impacted by fracking. To this end, sediment, and water samples, from streams near fracking (potentially impacted) and upstream or in a different watershed of fracking activity (unimpacted) must be collected. Those samples are then subjected to nucleic acid extraction, library preparation, and sequencing to investigate microbial community composition. Correlational analysis and machine learning models can subsequently be employed to identify which features are explanative of variation in the community, as well as identification of predictive biomarkers for fracking&#39;s impact. These methods can reveal a variety of differences in the microbial communities among headwater streams, based on the proximity to fracking, and serve as a foundation for future investigations on the environmental impact of fracking activities.

Here, we present a protocol to investigate the impacts of hydraulic fracturing on nearby streams by analyzing their water and sediment microbial communities.

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil ...