Los autores desean agradecer a todos los estudiantes del laboratorio de seguridad alimentaria y de los voluntarios que ayudaron con este estudio. Esta investigación ha sido financiada por la Iniciativa de Seguridad USDA Nacional Integrado de Alimentos (NIFSI) subvención (Award # 10507316).

1. Curva Estándar <ol><li> Pesar el polvo fluorescente 37,5 mg y disolver en 6 ml etanol 200 proof. Hacer 2 veces diluciones seriadas de esta solución en etanol 200 proof. </li><li> Vortex todas las muestras durante 10 segundos y medir la absorción a 370 nm. </li><li> Calcular la cantidad de polvo fluorescente por 1 ml de etanol prueba 200. </li><li> Preparar la curva estándar (concentración en mg / ml en el eje y, y la absorbancia a 370 nm en el eje x) para demostrar la absorbancia sobre la base de la concentración de polvo fluorescente. </li></ol> 2. Inocular Deli Meat (Bologna) con el polvo fluorescente <ol><li> Cortar la carne deli en aproximadamente 100 mm muestras gruesas con un cuchillo limpio y tabla de cortar. Prepare tres muestras marcadas A, B, y C. </li><li> Muestra Escudo Un uniformemente con polvo fluorescente usando un poco húmedo, esponja limpia. Corre el polvo en la muestra de carne uniformemente. No inocular las muestras B y C con el polvo fluorescente. </li&gt; <li> Envuelva las muestras con papel plástico y la etiqueta de la muestra con recubrimiento de polvo fluorescente como &quot;A&quot; y el restante dos muestras como B y C con cinta adhesiva. </li><li> Coloque las muestras en el caso de deli o refrigerador mantenido a 4 ° C en la cocina maqueta donde se llevará a cabo el experimento. </li></ol> 3. Mock Deli Preparación Cocina <ol><li> Proporcione guantes limpios y el 70% de pulverización de etanol en la cocina. </li><li> Montar las luces fluorescentes compactas negras con 13 bombillas W alrededor del área de la máquina de cortar. </li><li> Preparar un recorte de 5 cm x 5 cm de papel de aluminio para servir como una plantilla para limpiando. </li><li> Preparar tubos de 15 ml que contienen 6 ml de etanol al 95%. </li></ol> 4. Configuración de vídeo Mount tres cámaras de video en lugares estratégicos para observar todas las áreas del deli simulacro, al mismo tiempo. Encienda las cámaras antes de la simulación de la máquina de cortar de procedimiento (como se explica en el Protocolo 5) y analizar los resultados de forma simultánea (comose explica en el Protocolo n º 7). 5. Simulación de Procedimientos Deli rebanar a la pista Contaminación Cruzada Con el fin de mejorar el tamaño de la muestra e incorporar la variación, lleve a cabo este estudio los participantes de reclutamiento después de la aprobación del protocolo por el Comité de Ética de la institución respectiva. El participante puede ser un estudiante universitario que pueden o no tener experiencia de trabajo en entornos de delicatessen al por menor. Asegúrese de que cada participante lleva guantes una vez que entran las delicatessen. Tome una imagen &quot;anterior&quot; bajo una luz fluorescente negro seguido por un &quot;después&quot; de la imagen tras la finalización del estudio. Proporcionar instrucciones por escrito y verbales a cada participante de la siguiente manera: <ol><li> Ir al refrigerador. </li><li> Retire la carne con la etiqueta &quot;A&quot;. </li><li> Quite el envoltorio de la carne y guardar una envoltura de plástico. </li><li> Coloque la carne en la bandeja de transporte de la máquina de cortar. </li><li> Asegure la carne con el apretón de la carne. </li><li> Gire el interruptor de encendido en ON. </li><li> Ajuste sliperilla de CER en &quot;2&quot;. </li><li> Slice y dispensar 5 trozos de carne sobre papel delicatessen. </li><li> Gire el interruptor de alimentación en OFF y liberar la presión de la carne. </li><li> Coloque las rebanadas de la carne en una bolsa de plástico con la etiqueta &quot;A&quot;. </li><li> Vuelva a envolver la carne &quot;A&quot; y volver a la nevera. </li><li> Repita los pasos 2-11 con las carnes con la etiqueta &quot;B&quot; y &quot;C&quot;. </li></ol> 6. La cuantificación fluorescente Polvo Cuantificar el polvo fluorescente después de la finalización de la maqueta corte en lonchas (Sección 5.4) en la cocina delicatessen. Para ello haga lo siguiente: <ol><li> Utilice la 5 cm x 5 cm de plantilla de papel de aluminio estéril y colóquelo en las áreas marcadas en la Figura 1. </li><li> Use un hisopo de algodón estéril de alginato de calcio empapado en etanol al 95%, limpie el área y colocar en 6 ml de etanol al 95% en un tubo de polipropileno estéril. </li><li> Vortex el tubo a fondo y la transferencia a una cubeta de vidrio para leer la absorbancia a 370 nm. </li><li> Consulte el standarcurva d para determinar la cantidad de polvo fluorescente (y) usando la siguiente ecuación: y = mx + b, donde x es la absorbencia, m es la pendiente de la curva estándar, y b es la intersección. </li><li> Para la normalización de los datos, calcular el porcentaje de polvo fluorescente por área de muestra usando la siguiente fórmula: </li></ol><img src="/files/ftp_upload/51402/51402eq1.jpg" alt="Fórmula 1" /> 7. Análisis de vídeo Videotape los participantes a medida que completas tareas 5.1 a 5.12. Sincroniza y guardar las grabaciones de vídeo en discos DVD para que todos los ángulos se pueden ver al mismo tiempo. Vea los videos para registrar el número de toques manuales (el número de veces que los voluntarios tocan áreas en el deli simulacro) en varias superficies en el entorno de vitrina (4 investigadores diferentes analizarán los establecidos de forma independiente los datos completos).

<ol>
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No hay conflictos de interés declarado.

El objetivo general de este estudio fue demostrar cómo podría producirse una contaminación cruzada de bacterias en un ambiente de delicatessen al por menor con el fin de desarrollar estrategias para controlar y reducir el riesgo de enfermedades transmitidas por alimentos. El entorno de vitrina minorista tiene un riesgo especialmente alto para L. monocytogenes. El USDA-FSIS informó que la carne de deli en rodajas en la tienda al por menor tiene un siete veces mayor asociación con listeriosis que la carne de...

La contaminación cruzada de patógenos en el ambiente de preparación de alimentos, especialmente en la sección de venta al por menor, es una gran preocupación, debido a un mayor riesgo de enfermedades transmitidas por alimentos de una variedad de fuentes, incluyendo las carnes, así como las verduras 1-5. Muy a menudo, los patógenos bacterianos entran al medio ambiente al por menor a través de productos alimenticios contaminados 6. Alimentos listos para el consumo preparados a nivel minorista son de particular preocupación, ya que no suele haber más intervención o tratamiento (es decir, la cocción o calentamiento) antes de su consumo 7. Por otra parte, los agentes patógenos presentes en los alimentos LPC contaminada a continuación pueden ser transferidos a otros productos de alimentos o superficies de contacto con alimentos en el entorno minorista. Mientras que la contaminación cruzada puede ocurrir dentro de prácticamente cualquier tipo de entorno comercial de comida, tiendas de delicatessen son de particular interés debido específicamente a la asociación de RTE carnes frías con el patógeno Listeria monocytogenes 2. Sobre la base de una evaluación de los análisis de riesgos realizado por la Food and Drug Administration de EE.UU. (FDA) y el Departamento de Agricultura de Seguridad e Inspección de Alimentos (USDA-FSIS) EE.UU., embutidos contaminados son probables responsables del 90% de los casos de listeriosis - una rara enfermedad humana todavía grave causada por L. monocytogenes - en los Estados Unidos 8. En general, embutidos en rodajas en establecimientos de servicios minoristas y de alimentos han sido asociados con un mayor riesgo de L. monocytogenes en comparación con las carnes frías presliced ​​en una planta inspeccionada por el USDA el fabricante 9. Esto podría ser debido a la mayor probabilidad de introducción de patógenos por los manipuladores de alimentos, materias primas entrantes, o los alimentos procesados ​​que pueden contaminarse después del tratamiento 7. En general, la listeriosis representa aproximadamente el 28% de las muertes estimadas causadas por enfermedades transmitidas por alimentos cada año en los EE.UU. Debido a la enriesgo aumentado de L. monocytogenes en embutidos en rodajas en el nivel minorista, la propia máquina de cortar delicatessen ha sido identificada como un factor importante para la transferencia de la superficie de los patógenos transmitidos por alimentos en el entorno minorista 2,10. Según lo indicado por Sheen 1, rebanar es el último paso del proceso antes de su consumo, por lo tanto, la máquina de cortar deli debe considerarse como un punto crítico de control, tanto para comprender y prevenir la contaminación cruzada 1. El objetivo general de este estudio fue desarrollar una metodología para el seguimiento de la contaminación en el medio ambiente deli menor usando un polvo fluorescente tal como Glo Germ. Este es un compuesto fluorescente que se ha utilizado para simular la contaminación cruzada bacteriana y prácticas sanitarias 11,12. Este estudio fue diseñado para realizar un seguimiento de la transferencia de patógenos de las carnes contaminadas con la máquina de cortar, el medio ambiente circundante, y otros embutidos. Es crucial entender los caminos de contaminación para desarrollar tanto las intervenciones eficaces y material educativo para los empleados del sector minorista y los administradores. Un enfoque similar se puede emplear para simular la transferencia de patógenos en otros entornos de procesamiento (por ejemplo, las aves de corral y carne), granjas y hospitales.

<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

La contaminación cruzada de patógenos transmitidos por los alimentos en el entorno minorista es un problema de salud pública importante que contribuye a un mayor riesgo de enfermedades transmitidas por alimentos. Alimentos procesados ​​listos para comer (RTE), tales como carnes frías, queso, y en algunos casos, los productos frescos, han estado involucrados en los brotes de enfermedades transmitidas por los alimentos debido a la contaminación con patógenos como Listeria monocytogenes. Con respecto a L. monocytogenes, cortadoras de fiambres son a menudo la principal fuente de contaminación cruzada. El objetivo de este estudio fue utilizar un compuesto fluorescente para simular la contaminación bacteriana y seguimiento de esta contaminación en un entorno minorista. Una cocina deli simulacro fue diseñado para simular el entorno minorista. Carne de Deli se inoculó con el compuesto fluorescente y voluntarios fueron reclutados para completar un conjunto de tareas similares a las que se espera de un empleado menor de alimentos. Los voluntarios fueron instruidos para cortar, empaquetar y almacenar la carne en el refrigerador delicatessen. El potencialcontaminación cruzada fue localizado en el entorno minorista maqueta en muestras de secreciones áreas específicas y medir la densidad óptica de la zona limpia con un espectrofotómetro. Los resultados indicaron que el refrigerador (es decir deli caso) agarre y diversas zonas de la máquina de cortar tenían el mayor riesgo de contaminación cruzada. Los resultados de este estudio pueden ser utilizados para desarrollar material de formación más especializada para empleados del sector minorista. Además, metodologías similares también podrían ser utilizados para rastrear la contaminación microbiana en ambientes de producción de alimentos (por ejemplo, las pequeñas granjas), hospitales, hogares de ancianos, cruceros y hoteles.

La figura 1 representa las áreas de la máquina de cortar que se limpió después de los voluntarios completaron las tareas asignadas. Los voluntarios fueron grabados en vídeo para analizar la frecuencia media de contacto con las manos sobre estos diferentes superficies. La frecuencia de contacto de las manos fue analizado por cuatro observadores diferentes y promedió. Estos resultados se demuestran en la Figura 2. Los datos muestran que la agarre carne, envoltura de carnes frías, c...

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

El objetivo general de este estudio fue demostrar el potencial mecanismo de contaminación cruzada de patógenos de origen alimentario Listeria monocytogenes en un entorno minorista delicatessen. Esta metodología se puede aplicar a una variedad de diferentes entornos para rastrear la contaminación con patógenos.

El seguimiento de la contaminación microbiana en los entornos comerciales Usando fluorescente Polvo - Un menor Charcutería Ambiente Ejemplo

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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Research

JoVE Journal

Environment

11.0K vistas.  University of Houston.  El objetivo general de este estudio fue demostrar el potencial mecanismo de contaminación cruzada de patógenos de origen alimentario Listeria monocytogenes en un entorno minorista delicatessen. Esta metodología se puede aplicar a una variedad de diferentes entornos para rastrear la contaminación con patógenos. 

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

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.

Determinación de la actividad enzimática extracelular microbiana en aguas, suelos y sedimentos de los que utilizan y alto rendimiento de microplacas Ensayos

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

Investigación

Medio ambiente

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.

Técnica para el estudio de los artrópodos y las comunidades microbianas dentro de los tejidos del árbol

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.

Un Aquatic Microbial metaproteomics Flujo de trabajo: a partir de células a tríptico péptidos adecuados para el análisis basado en la espectrometría de masas en tándem

Meta-omic technologies such as metagenomics, metatranscriptomics and metaproteomics can aid in the understanding of microbial community structure and ...

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.

Extracción y análisis de ácidos grasos de fosfolípidos microbiana en suelos

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

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only recently have microplastics in freshwater systems, such as rivers and lakes, been quantified. Microplastic sampling at the surface usually consists of deploying drift nets behind either a stationary or moving boat, which limits the sampling to environments with low levels of suspended sediments and floating or submerged debris. Previous studies that employed drift nets to collect microplastic debris typically used nets with &#8805;300 &#181;m mesh size, allowing plastic debris (particles and fibers) below this size to pass through the net and elude quantification. The protocol detailed here enables: 1) sample collection in environments with high suspended loads and floating or submerged debris and 2) the capture and quantification of microplastic particles and fibers &lt;300 &#181;m. Water samples were collected using a peristaltic pump in low-density polyethylene (PE) containers to be stored before filtering and analysis in the lab. Filtration was done with a custom-made microplastic filtration device containing detachable union joints that housed nylon mesh sieves and mixed cellulose ester membrane filters. Mesh sieves and membrane filters were examined with a stereomicroscope to quantify and separate microplastic particulates and fibers. These materials were then examined using a micro-attenuated total reflectance Fourier transform infrared spectrometer (micro ATR-FTIR) to determine microplastic polymer type. Recovery was measured by spiking samples using blue PE particulates and green nylon fibers; percent recovery was determined to be 100% for particulates and 92% for fibers. This protocol will guide similar studies on microplastics in high velocity rivers with high concentrations of sediment. With simple modifications to the peristaltic pump and filtration device, users can collect and analyze various sample volumes and particulate sizes.

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.

Muestreo, clasificación y caracterización de Microplastics en ambientes acuáticos con cargas de sedimento en suspensión alta y basura flotante grande

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).

Detección de 3-nitrotirosina en ambientes atmosféricos a través de un alto rendimiento sistema Detector electroquímico en cromatografía líquida

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.

Bacterias etiquetadas fluorescentes como un rastreador para revelar nuevos caminos de flujo de carbono orgánico en ecosistemas acuáticos

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.

Enriquecimiento y detección de toxinatipos de Clostridium perfringens en muestras de alimentos al por menor

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.

Ensayo de Chemotaxis Situ para Examinar el Comportamiento Microbiano en Ecosistemas Acuáticos

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.

Evaluación del impacto de la fracturación hidráulica en las corrientes utilizando firmas moleculares microbianas

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