The authors would like to acknowledge the BAM for funding through the focus area Analytical Sciences: https://www.bam.de/Navigation/EN/Topics/Analytical-Sciences/Rapid-Oil-Test/rapid-oil-test.html.

1. Fluorescent Dyes (Figure 1A)
<ol>
	<li>Purchase commercially available 4-DNS and 4-DNS-OH. 
	Note: 4-DNS-COOH is not commercially available and is prepared from 4-DNS-OH as described hereafter.</li>
	<li>Place 50 mg (0.16 mmol) of 2-[ethyl[4-[2-(4-nitrophenyl)ethenyl]phenyl]amino]ethanol, 2 mg (0.016 mmol) of 4-dimethylaminopyridine and 19.2 mg (0.192 mmol) of succinic anhydride in a 10 mL round bottom flask.</li>
	<li>Dissolve the reagents in 2 mL of dry dichloromethane under argon atmosphere.</li>
	<li>Add 11.6 &#181;L (0.08 mmol) of triethylamine and let the mixture react for 20 h.</li>
	<li>Monitor the reaction by thin layer chromatography until quantitative conversion of the starting materials (Rf = 0.61) into the product (Rf = 0.27) is indicated (hexane/EtOAc, 4/6, v/v)</li>
	<li>Add 2 mL of water to the mixture before acidification to pH 2 with acetic acid (approx. 10 &#181;L).</li>
	<li>Extract the mixture by performing two successive liquid-liquid extractions, with 10 mL of dichloromethane each time.</li>
	<li>Wash once the reunited organic phases with 10 mL of saturated NaCl (&#62; 359 g L&#8211;1).</li>
	<li>Dry the organic phases by adding Na2SO4 powder until some fine drying agent powder remains visible.</li>
	<li>Purify the crude product by flash silica column chromatography with petroleum ether:ethylacetate 1:9 as the eluent. 
	Note: The yield achieved were 49 mg (74%) of the desired product.</li>
	<li>Perform 1H NMR analysis of the purified product in DMSO-d6 to validate the structure (&#948; 8.17 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 16.3 Hz, 1H), 7.10 (d, J = 16.3 Hz, 1H), 6.75 (d, J = 8.9 Hz, 2H), 4.18 (t, J = 6.0 Hz, 2H), 3.58 (t, J = 6.0 Hz, 2H), 3.43 (q, J = 7.0 Hz, 2H), 2.50 &#8211; 2.45 (m, 4H), 1.10 (t, J = 7.0 Hz, 3H) ppm).</li>
	<li>Perform 13C NMR analysis of the purified product in DMSO-d6 to validate the structure (&#948; 173.36, 172.20, 147.99, 145.23, 145.13, 133.89, 128.76, 126.30, 124.03, 123.67, 120.95, 111.58, 61.52, 48.05, 44.57, 28.73, 28.63, 12.00 ppm).</li>
	<li>Perform high resolution mass spectrometry with positive electro spray ionization of the purified product, corresponding to the calculated value (C22H25N2O6 [M+H]+: 413.1707) m/z ratio of 413.1713.</li>
</ol>
2. Synthesis of the Reference Dye
Note: The synthetic procedure of 8-(phenyl)-1,3,5,7-tetramethyl-2,6-diethyl-4,4-difluoro-4 bora-3a,4a-diaza-s-indacene was adopted from Coskun et al.18.
<ol>
	<li>Purify the crude product by column chromatography on silica with toluene as eluent. 
	Note: The yield achieved were 441 mg (29 %) of bright reddish crystals.</li>
	<li>Perform 1H NMR analysis of the pure product at 600 MHz in DMSO-d6 to validate the structure (&#948; 0.98 (t, 6H, J = 7.6 Hz), 1.27 (s, 6H), 2.29 (q, 4H, J = 7.6 Hz), 2.53 (s, 6H), 7.27-7.29 (m, 2H), 7.46-7.48 (m, 3H) ppm).</li>
	<li>Perform high resolution mass spectrometry with positive electro spray ionization of the purified product, corresponding to the calculated value (C23H28BF2N2 [M+H]+: 381.2314) m/z ratio of 381.2267.</li>
</ol>
3. TEST STRIP FABRICATION, METHOD 1.
<ol>
	<li>Prepare 1 mM solutions of the reference dye and dyes 4-DNS, 4-DNS-OH and 4-DNS-COOH in toluene.</li>
	<li>Cut cellulose strips of 30 &#215; 5 mm from filter paper.</li>
	<li>Place approximately 50 of those strips (611 mg) in a sealable 5 mL vial together with 4.5 mL of the desired dye solution from step 3.1.</li>
	<li>Shake the strips inside the vial with a vertical rotator for 20 min at 30 rpm.</li>
	<li>Pour the toluene solution out of the vial, and immediately fill with 4 mL of cyclohexane and rotate for 1 min at 30 rpm to wash off excess dyes.</li>
	<li>Repeat the washing operation from Step 3.5 three times.</li>
	<li>Dry the obtained test strips on a filter paper for 10 min in air at room temperature.</li>
</ol>
4. Test Strip Fabrication, Method 2.
<ol>
	<li>Amination of the paper strips.
	<ol>
		<li>Cut cellulose strips of 30 &#215; 5 mm from filter paper.</li>
		<li>Under a fume hood, place approximately 20 of those strips (308 mg) in a flask containing 40 mL of toluene.</li>
		<li>Add 960 &#181;L of 3-aminopropyltriethoxysilane (APTES) in the flask and stir the mixture for 24 h at 80 &#176;C.</li>
		<li>Remove the strips from the flask and wash thoroughly with 50 mL of ethanol.</li>
		<li>Dry the strips for 2 h at 50 &#176;C.</li>
	</ol>
	</li>
	<li>Grafting of the dye.
	<ol>
		<li>Under a fume hood, dissolve 5 mg of 4-DNS-COOH (13 &#181;mol) in 10 mL of dry dichloromethane under argon atmosphere in a 25 mL flask.</li>
		<li>Add N,N&#39;-dicyclohexylcarbodiimide (DCC, 3.3 mg, 16 &#181;mol) and allow the carboxylic acid to be activated for 15 min.</li>
		<li>Add triethylamine (2.2 &#181;L, 16 &#181;mol) and 18 aminated paper strips (278 mg).</li>
		<li>Stir the mixture for additional 2 h.</li>
		<li>Remove the strips from the solution and wash with 25 mL of dichloromethane and 25 mL of ethanol.</li>
	</ol>
	</li>
</ol>
5. Sample Pre-Treatment.
<ol>
	<li>Laboratory treatment
	<ol>
		<li>Place 10 mL of a fresh diesel/kerosene blend in a 25 mL vial.</li>
		<li>Suspend 10 wt% of active charcoal in the blend.</li>
		<li>Stir the vial for 1 h, centrifuge (400 x g, 10 min) and filter to remove the charcoal.</li>
	</ol>
	</li>
	<li>On-site treatment
	<ol>
		<li>Purchase circular activated carbon loaded filters of 47 mm diameter.</li>
		<li>Place four of the filters in a 47 mm stainless steel in-line filter holder.</li>
		<li>Flush 5 mL of a fresh diesel/kerosene blend through the filters with a standard 10 mL syringe; approximately 2 mL of polycyclic aromatic hydrocarbon-free solution was obtained.</li>
	</ol>
	</li>
</ol>
6. Smartphone Reader Implementation
Note: An Android based smartphone with a centered front camera was used as the core of the smartphone measurement system. All the necessary optical elements and 3D-printed accessory were custom-made for this device. However, any other smartphone with a CMOS (Complementary Metal Oxide Semiconductor) camera can be used.19,20
<ol>
	<li>Purchase a standard 5 mm epoxy LED at 460 nm, a 100 &#937; resistor and a USB on-the-go (OTG) cable with an ON/OFF switch and a micro USB port.</li>
	<li>Cut the USB cable on the opposite of the OTG side to isolate the red wire powering +5 V (up to 300 mA) and the black wire corresponding to the ground.</li>
	<li>Cut the black wire of the USB cable and solder the 100 &#937; resistor on the back of the switch. Solder the LED anode to the +5V red wire and the LED cathode to the ground black wire.</li>
	<li>Purchase a diffuser and two filters for the LED and the camera, typically a short pass filter for the excitation channel (LED) and a band pass filter for the emission collection (camera).</li>
	<li>3D-print a smartphone case that fits on the smartphone and integrates the different optical parts consisting of a black chamber (20 x 30 x 40 mm)21 as described in Figure 2.</li>
	<li>3D-print a strip holder as described in Figure 2 to hold a reference and a test strip.</li>
	<li>Implement the excitation channel by placing the LED, the diffuser and the filter to illuminate the paper strips at an angle of 60&#176;.</li>
	<li>Implement the reading channel by placing the filter in front of the smartphone CMOS camera.</li>
	<li>Insert the test strip holder containing the strips to start a measurement.</li>
</ol>
7. Sample Analysis Using the Smartphone-Based Detector
Note: Analyses were carried out by running a Java app(lication) for Android that finally displayed the adulteration level on the screen. Without the app, pictures can be taken, exported to a computer and analyzed with a standard image analysis software.
<ol>
	<li>Select the adequate calibration file, here diesel/kerosene, from the software memory by clicking on the Menu button in the upper-right corner of the software window.</li>
	<li>Dip the test strip into the diesel sample for a couple of seconds by holding the test strip with tweezers.</li>
	<li>Remove excess fuel by simple patting with a drying paper.</li>
	<li>Place the test strip inside the strip holder besides the reference strip and introduce the holder into the smartphone case. 
	Note: An image of the strips&#8217; fluorescence is then immediately displayed on the smartphone&#8217;s screen.</li>
	<li>Press the SHOOT button to record the fluorescence intensities of test and reference strips. 
	Note: The degree of adulteration is immediately calculated by the internal algorithm and displayed on the screen.</li>
</ol>

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The authors have nothing to disclose.

A fluorescent probe, based on a molecular rotor dye that is sensitive to viscosities in the range of those measured for diesel and its different blends with kerosene, was used to obtain simple and efficient test strips for the detection of diesel fuel adulteration. The emission intensity of 4-DNS at 550 nm in various diesel/kerosene blends correlates with a reduction in viscosity when the proportion of kerosene increases. At a temperature of 24 &#176;C, a nonlinear fluorescence quenching of up to 55% was observed for up ...

Fuel adulteration is a serious problem in many different parts of the world, simply due to the enormous relevance of fuel as an energy source. Running engines on adulterated fuel reduces their performance, leads to earlier engine failure and entails environmental pollution1. Increased SOx emissions occur if diesel is adulterated with kerosene that usually contains a higher amount of sulfur2,3. Although the problem exists for decades, sustainable fuel management that uncovers such criminal activity at its point of origin is still rare, because simple and reliable tests for fuel adulteration are largely lacking4. Despite substantial progress in laboratory-based mineral oil analysis in the past decades5,6,7, approaches to on-site measurements are still scarce. Various methods for the use outside of the laboratory have recently been devised, using fiber optics8, field-effect transistors9 or mechano-chromic materials10. Although they overcome some of the drawbacks of conventional methods, robust, user-friendly and portable methods are still lacking largely. Fluorescent viscosity probes based on molecular rotors are an interesting alternative11,12, because mineral oils are comprised of a great variety of hydrocarbons that differ in chain length and cyclicity, being often reflected in different viscosities. Because fuels are complex mixtures without specific lead compounds to act as tracers, the measurement of the change of a macroscopic property like viscosity or polarity seems very promising. The latter can be addressed by fluorescent molecular rotors for which the fluorescence quantum yields depend on environmental viscosity. After photoexcitation, deactivation commonly involves a twisted intramolecular charge transfer (TICT) state, the population of which is determined by the viscosity of its surrounding microenvironment13. Highly viscous solvents hinder molecular rotors to adopt a TICT state, entailing bright emission. In low-viscous solvents, the rotor can much better access the TICT state, accelerating non-radiative decay and thus quenched fluorescence. The addition of kerosene, with a viscosity of 1.64 mm2&#8729;s-1 at 27 &#176;C, to diesel, with respective viscosities of 1.3-2.4, 1.9-4.1, 2.0-4.5 or 5.5-24.0 mm2&#8729;s -1 at 40 &#176;C for grades 1D, 2D, EN 950 and 4D14,15,16, reduces the kinematic viscosity of the mixture and potentially leads to a proportional quenching of the fluorescence of a molecular rotor probe. The family of 4-dimethylamino-4-nitrostilbenes (4-DNS) seemed most promising to us because of their strong fluorescence variation over a kinematic viscosity range of 0.74-70.6 mm2&#8729;s -1. This range matches well with the known values of kerosene and diesel.
We therefore explored the ability of 4DNS, 2-[ethyl[4-[2-(4-nitrophenyl)ethenyl]phenyl]amino]ethanol (4DNSOH) and (E)-4-(2-(ethyl(4-(4-nitrostyryl)phenyl)amino)ethoxy)-4-oxobutanoic acid (4DNSCOOH) to indicate the viscosity of diesel-kerosene mixtures through their fluorescence, depending on intramolecular rotation and finally yielding a rapid test for diesel adulteration with kerosene. The disposable test is easy to use, precise, reliable, cost-effective and dimensionally small. The adsorption of the probes onto the filter paper as a solid support was investigated and the analysis was accomplished with an embedded smartphone-based fluorescence reader. Today, ubiquitously available smartphones are equipped with high-quality cameras, rendering the detection of optical changes such as color and fluorescence straightforward, and paving the way for powerful on-site analyses. We demonstrate here that the measurement of the emission of fluorescent probes adsorbed on paper strips with a smartphone can be used for fraud detection on combustion fuels in a reliable manner17.

<table>
	<tbody>
		<tr>
			<td>4-dimethylamino-4-nitrostilbene (CAS Number: 2844-15-7)</td>
			<td>Sigma-Aldrich</td>
			<td>39255</td>
			<td>4-DNS Dye</td>
		</tr>
		<tr>
			<td>2-[ethyl[4-[2-(4-nitrophenyl)ethenyl]phenyl]amino]ethanol (CAS Number: 122258-56-4)</td>
			<td>Sigma-Aldrich</td>
			<td>518565</td>
			<td>4-DNS-OH Dye</td>
		</tr>
		<tr>
			<td>Whatman&#160;qualitative filter paper, Grade 1</td>
			<td>Sigma-Aldrich</td>
			<td>Z274852</td>
			<td>Test strips support</td>
		</tr>
		<tr>
			<td>Whatman&#160;application specific filter, activated carbon loaded paper, Grade 72</td>
			<td>Sigma-Aldrich</td>
			<td>WHA1872047</td>
			<td>Fuel pre-treatment filters</td>
		</tr>
		<tr>
			<td>Pall reusable in-line filter holders stainless steel, diam. 47&#160;mm</td>
			<td>Sigma-Aldrich</td>
			<td>Z268453&#160;</td>
			<td>Holder pre-treatment filters</td>
		</tr>
		<tr>
			<td>(3-Aminopropyl)triethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>919-30-2</td>
			<td>APTES</td>
		</tr>
		<tr>
			<td>4-(Dimethylamino)pyridine</td>
			<td>Sigma-Aldrich</td>
			<td>1122-58-3</td>
			<td>DMAP</td>
		</tr>
		<tr>
			<td>Succinic anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>108-30-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma-Aldrich</td>
			<td>121-44-8</td>
			<td>Et3N</td>
		</tr>
		<tr>
			<td>N,N&#39;-dicyclohexylcarbodiimide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>538-75-0</td>
			<td>DCC</td>
		</tr>
		<tr>
			<td>Stuart Tube Rotators</td>
			<td>Cole-Parmer</td>
			<td>SB3</td>
			<td>Rotator</td>
		</tr>
		<tr>
			<td>FreeCAD</td>
			<td>freecadweb.org</td>
			<td>-</td>
			<td>Freeware - 3D design</td>
		</tr>
		<tr>
			<td>Ultimaker Cura</td>
			<td>Ultimaker</td>
			<td>-</td>
			<td>Freeware - 3D printing</td>
		</tr>
		<tr>
			<td>Android Studio</td>
			<td>Google</td>
			<td>-</td>
			<td>Freeware - App programming</td>
		</tr>
		<tr>
			<td>Renkforce SuperSoft OTG-Mirror Micro-USB Cable 0,15 m</td>
			<td>Conrad.de</td>
			<td>1359890&#160;- 62</td>
			<td>Smartphone setup electronic part</td>
		</tr>
		<tr>
			<td>Black Cord Switch 1 x Off / On</td>
			<td>Conrad.de</td>
			<td>1371835&#160;- 62</td>
			<td>Smartphone setup electronic part</td>
		</tr>
		<tr>
			<td>Carbon Film Resistor 100 &#937;</td>
			<td>Conrad.de</td>
			<td>1417639&#160;- 62</td>
			<td>Smartphone setup electronic part</td>
		</tr>
		<tr>
			<td>492 nm blocking edge BrightLine&#160;short-pass filter</td>
			<td>Semrock</td>
			<td>FF01-492/SP-25</td>
			<td>Filter excitation</td>
		</tr>
		<tr>
			<td>550/49&#160;nm BrightLine single-band bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-550/49-25</td>
			<td>Filter emission</td>
		</tr>
		<tr>
			<td>&#216;1/2&#34; Unmounted N-BK7 Ground Glass Diffuser, 220 Grit</td>
			<td>Thorlabs</td>
			<td>DG05-220</td>
			<td>Diffuser excitation</td>
		</tr>
		<tr>
			<td>LED 465 nm, 9 cd, 20 mA, &#177;15&#176;, 5 mm clear epoxy</td>
			<td>Roithner</td>
			<td>RLS-B465</td>
			<td>LED excitation</td>
		</tr>
	</tbody>
</table>

fluorescent paper strips for the detection of diesel adulteration with smartphone read-out

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate the content of kerosene in diesel/kerosene blends, a wide-spread activity to adulterate fuel. In solvents with low viscosity, the dyes rapidly deactivate via a so-called twisted intramolecular charge transfer state, efficiently quenching the fluorescence. Measurements of diesel/kerosene blends revealed a good linear correlation between the decrease in fluorescence and the increase of the fraction of the less viscous kerosene in diesel/kerosene blends. Immobilization of the hydroxy derivative 4-DNS-OH in cellulose paper yielded test strips that preserve the fluorescent indicator&#39;s behavior. Combination of the strips with a reader based on a smartphone and a controlling app allowed to create a simple field test. The method can reliably detect the presence of kerosene in diesel from 7 to 100%, outperforming present standard methods for diesel adulteration.

The three structures of the two commercial dyes 4-DNS and 4-DNS-OH and the synthesized dye 4-DNS-COOH contain a stilbene core element substituted with a donor (-NR2) and an acceptor (-NO2) group at both ends, the central double bond constituting the hinge of the so called &#39;molecular rotor&#39; (Figure 1A). The structures differ in amino group substitution pattern with short alkyl groups for 4-DNS, two slightly longer groups including...

Watch this Scientific Journal Video about Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out at JoVE.com

Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out

Here, we present a protocol to detect the adulteration of diesel with kerosene using test strips coated with a fluorescent viscosity probe together with a smartphone-based analysis system.

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate ...

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9.3K Views.  Bundesanstalt für Materialforschung und –prüfung (BAM). Here, we present a protocol to detect the adulteration of diesel with kerosene using test strips coated with a fluorescent viscosity probe together with a smartphone-based analysis system.

Video: Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.

Single-plant, Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Growth of Medicago truncatula plants in symbiosis with the nitrogen-fixing bacteria Sinorhizobium meliloti in individual, sterile microcosms made from standard laboratory plates permits frequent examination of root systems and nodules without compromising sterility. Plants can be maintained in these growth chambers for up to 9 weeks.

Rhizobial  bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host  legume plants.  One  of the most well-developed model systems ...

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.

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 ...

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Building Double-decker Traps for Early Detection of Emerald Ash Borer

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions of forest and landscape ash (Fraxinus spp.) trees. Several artificial trap designs to attract and capture EAB beetles have been developed to detect, delineate, and monitor infestations. Double-decker (DD) traps consist of two corrugated plastic prisms, one green and one purple, attached to a 3 m tall polyvinyl chloride (PVC) pipe supported by a t-post. The green prism at the top of the PVC pipe is baited with cis-3-hexenol, a compound produced by ash foliage. Surfaces of both prisms are coated with sticky insect glue to capture adult EAB beetles. Double-decker traps should be placed near ash trees but in open areas, exposed to sun. Double-decker trap construction and placement are presented here, along with a summary of field experiments demonstrating the efficacy of DD traps in capturing EAB beetles. In a recent study in sites with relatively low EAB densities, double-decker traps captured significantly more EAB than green or purple prism traps or green funnel traps, all of which are designed to be suspended from a branch in the canopy of ash trees. A greater percentage of double decker traps were positive, i.e., captured at least one EAB, than the prism traps or funnel traps that were hung in ash tree canopies.

Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

Emerald ash borer (EAB) (Agrilus planipennis Fairmaire), the most destructive forest insect to have invaded North America, has killed hundreds of millions ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Characterization of Aquatic Biofilms with Flow Cytometry

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond quickly to environmental changes and can be thus used as indicators of water quality. Currently, biofilm assessment is mostly based on integrative and functional endpoints, such as photosynthetic or respiratory activity, which do not provide information on the biofilm community structure. Flow cytometry and computational visualization offer an alternative, sensitive, and easy-to-use method for assessment of the community composition, particularly of the photoautotrophic part of freshwater biofilms. It requires only basic sample preparation, after which the entire sample is run through the flow cytometer. The single-cell optical and fluorescent information is used for computational visualization and biological interpretation. Its main advantages over other methods are the speed of analysis and the high-information-content nature. Flow cytometry provides information on several cellular and biofilm traits in a single measurement: particle size, density, pigment content, abiotic content in the biofilm, and coarse taxonomic information. However, it does not provide information on biofilm composition on the species level. We see high potential in the use of the method for environmental monitoring of aquatic ecosystems and as an initial biofilm evaluation step that informs downstream detailed investigations by complementary and more detailed methods.

Flow cytometry in combination with visual clustering offers an easy-to-use and fast method for studying aquatic biofilms. It can be used for biofilm characterization, detection of changes in biofilm community structure, and detection of abiotic particles embedded in the biofilm.

Biofilms are dynamic consortia of microorganism that play a key role in freshwater ecosystems. By changing their community structure, biofilms respond ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

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.