Os autores gostariam de agradecer a todos os alunos do laboratório de segurança alimentar e voluntários que ajudaram com este estudo. Esta pesquisa foi financiada por uma concessão Safety Initiative USDA Nacional Integrado de Alimentos (NIFSI) (Prêmio # 10507316).

1. Curva padrão <ol><li> Pesar 37,5 mg pó fluorescente e dissolver em 6 ml de etanol de 200 provas. Fazer diluições em série de 2 vezes desta solução em etanol 200 de prova. </li><li> Vortex todas as amostras de 10 segundos e medir a absorvância a 370 nm. </li><li> Calcula-se a quantidade de pó fluorescente por 1 ml de etanol prova 200. </li><li> Preparar a curva padrão (concentração em mg / ml sobre o eixo y e a absorvância a 370 nm, no eixo dos x) para demonstrar a absorvência com base na concentração do pó fluorescente. </li></ol> 2. Inocular Deli Carnes (Bologna) com pó fluorescente <ol><li> Corte a carne deli em aproximadamente 100 milímetros de espessura amostras usando uma faca limpa e tábua de corte. Preparar três amostras rotulados A, B, e C. </li><li> Brasão amostra A uniformemente com pó fluorescente usando uma esponja limpa ligeiramente úmido. Espalhar o pó na amostra de carne de forma uniforme. Não inocular as amostras B e C com o pó fluorescente. </li&gt; <li> Enrole as amostras com filme plástico e rotular a amostra revestida pó fluorescente como &#39;A&#39; e permanecendo duas amostras como B e C com fita adesiva. </li><li> Coloque as amostras em processo deli frigorífico ou mantidas a 4 ° C na cozinha simulado, onde a experiência será realizada. </li></ol> 3. Mock Deli Cozinha Preparação <ol><li> Fornecer luvas limpas e 70% spray de etanol na cozinha. </li><li> Monte negras lâmpadas fluorescentes compactas, com 13 W lâmpadas ao redor da área de segmentação de dados. </li><li> Prepare a 5 cm x 5 cm do entalhe de folha de alumínio para servir como um modelo para o esfregaço. </li><li> Preparar tubos de 15 ml contendo 6 ml de etanol 95%. </li></ol> 4. Configuração de vídeo Monte três câmeras de vídeo em locais estratégicos para observar todas as áreas da deli falsa ao mesmo tempo. Ligue as câmeras antes de simular o processo de cortador (como explicado no protocolo 5) e analisar os resultados ao mesmo tempo (comoexplicadas no protocolo 7). 5. Simulando Procedimentos Deli Cortando a Track Contaminação Cruzada A fim de melhorar o tamanho da amostra e incorporar variação, realizar este estudo pelos participantes recrutamento após a aprovação do protocolo pelo Conselho de Revisão Institucional da respectiva instituição. O participante pode ser um estudante universitário que pode ou não ter experiência de trabalho em ambientes de varejo deli. Certifique-se que cada participante usa luvas, uma vez que eles entram nas delicatessen. Dê um &quot;antes&quot; imagem sob luz fluorescente preto seguido por uma imagem &quot;depois&quot; após a conclusão do estudo. Fornecer instruções escritas e verbais claras para cada participante da seguinte forma: <ol><li> Ir para geladeira. </li><li> Retirar carne marcado &quot;A&quot;. </li><li> Desembrulhe a carne e salvar filme plástico. </li><li> Coloque a carne na bandeja transporte de fatiador. </li><li> Proteja carne com aperto carne. </li><li> Coloque o interruptor ON. </li><li> Ajuste slibotão índice cer para &quot;2&quot;. </li><li> Slice e dispensar 5 pedaços de carne em papel delicatessen. </li><li> Coloque o interruptor OFF e libertar a pega carne. </li><li> Coloque fatias de carne em saco plástico rotulado como &quot;A&quot;. </li><li> Embrulhe a carne &quot;A&quot; e retornar à geladeira. </li><li> Repita os passos 2-11 com carnes rotulados como &quot;B&quot; e &quot;C&quot;. </li></ol> 6. Quantificar fluorescente Pó Quantificar o pó fluorescente após a conclusão do corte simulada (Seção 5.4) na cozinha delicatessen. Para isso faça o seguinte: <ol><li> Use o estéril 5 centímetros x 5 cm modelo folha de alumínio e coloque-o sobre as áreas marcadas na Figura 1. </li><li> Utilize um cotonete de alginato de cálcio de algodão estéril embebido em etanol a 95%, limpe a área e coloque em 6 ml de etanol 95% em um tubo de polipropileno estéril. </li><li> Vortex do tubo completamente e transferir para uma cuvete de vidro para ler a absorvância a 370 nm. </li><li> Consulte o standard curva para determinar a quantidade de pó fluorescente (y) utilizando a seguinte equação: y = mx + b, onde x é a absorção, m é o declive da curva padrão, e b é a intercepção. </li><li> Para a normalização dos dados, calcular a percentagem de pó fluorescente por área de amostra utilizando a seguinte fórmula: </li></ol><img src="/files/ftp_upload/51402/51402eq1.jpg" alt="Fórmula 1" /> 7. Análise de Vídeo Videotape os participantes como eles completar tarefas 5,1-5,12. Sincronize e guardar as gravações de vídeo para discos DVD, de modo que todos os ângulos podem ser vistos ao mesmo tempo. Assista aos vídeos para registrar o número de toques de mão (o número de vezes que os voluntários tocam áreas no deli simulada) em várias superfícies no ambiente deli (4 diferentes pesquisadores vão analisar o conjunto de dados de forma independente completos).

<ol>
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Não há conflitos de interesse declarados.

O objetivo geral deste estudo foi demonstrar como a contaminação cruzada de bactérias pode ocorrer em um ambiente de varejo deli, a fim de desenvolver estratégias para controlar e reduzir o risco de doenças transmitidas por alimentos. O ambiente deli varejo tem um risco especialmente alto para L. monocytogenes contaminação. O USDA-FSIS informou que a carne deli cortado na loja de varejo tem sete vezes maior associação com listeriose que a carne deli cortado e embalado em uma fábrica federal inspeciona...

A contaminação cruzada de patógenos de origem alimentar no ambiente de preparo de alimentos, especialmente na seção de varejo, é uma grande preocupação, devido a um aumento do risco de doenças transmitidas por alimentos a partir de uma variedade de fontes, incluindo as carnes, bem como os legumes 1-5. Na maioria das vezes, patógenos bacterianos entrar no ambiente de varejo, através de produtos alimentares contaminados 6. Alimentos prontos-a-comer preparados no varejo são particularmente preocupantes como geralmente há mais nenhuma intervenção ou tratamento (ou seja, cozinhar ou aquecimento) antes do consumo 7. Além disso, agentes patogénicos presentes em alimentos contaminados RTE podem, então, ser transferidos para outros produtos alimentares ou superfícies em contacto com alimentos, no ambiente de retalho. Enquanto a contaminação cruzada pode ocorrer dentro de praticamente qualquer tipo de ambiente de varejo de alimentos, delis são de particular interesse especificamente por causa da associação da RTE frios com o patógeno Listeria monocytogenes 2. Com base em uma avaliação de risco analisa conduzido por os EUA Food and Drug Administration (FDA) e os EUA Departamento de Agricultura Segurança Alimentar e Serviço de Inspeção (USDA-FSIS), frios contaminados são provavelmente responsáveis ​​por 90% dos casos de listeriose - uma rara doença humana ainda grave causada por L. monocytogenes - nos Estados Unidos 8. Em geral, frios fatiados em estabelecimentos de serviços de varejo e alimentos têm sido associados com um risco maior de L. monocytogenes em comparação com frios presliced ​​em uma planta inspecionada USDA pelo fabricante 9. Isso pode ser por causa do aumento da probabilidade de introdução de patógenos por manipuladores de alimentos, matérias-primas recebidas, ou alimentos processados, que podem tornar-se contaminados pós-tratamento 7. No geral, a listeriose é responsável por aproximadamente 28% das mortes estimadas causadas por doenças transmitidas por alimentos por ano em os EUA Por causa da emrisco acrescido de L. monocytogenes contaminação em frios fatiados ao nível do retalho, o próprio cortador deli tem sido identificada como um fator importante para a transferência de superfície de patógenos de origem alimentar no ambiente de varejo 2,10. Como indicado por Sheen 1, corte é a última etapa de processamento antes do consumo, portanto, o cortador de delicatessen deve ser considerado um ponto crítico de controle, tanto para a compreensão e prevenção de contaminação cruzada 1. O objetivo geral deste estudo foi desenvolver uma metodologia para monitorar a contaminação no ambiente deli varejo usando um pó fluorescente como Glo Germ. Este é um composto fluorescente que tem sido usado para simular a contaminação cruzada bacteriana e práticas sanitárias 11,12. Este estudo foi desenhado para controlar a transferência de patógenos de carnes contaminadas com o cortador, o meio ambiente, e de outros produtos de charcutaria. É crucial para entender os caminhos de contaminao desenvolver ambas as intervenções eficazes e material educativo para os empregados de varejo e gestores. Uma abordagem semelhante pode ser empregado para simular a transferência do patógeno em outros ambientes de processamento (por exemplo, aves e carne bovina), fazendas e hospitais.

<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

A contaminação cruzada de patógenos de origem alimentar no ambiente de varejo é um problema de saúde pública significativo contribuindo para um aumento do risco de doenças transmitidas por alimentos. Alimentos processados ​​prontos-a-comer (RTE), como frios, queijos, e em alguns casos, produtos frescos, ter sido envolvido em surtos de doenças transmitidas por alimentos devido à contaminação com patógenos, como Listeria monocytogenes. Com respeito a L. monocytogenes, cortadores de deli são muitas vezes a principal fonte de contaminação cruzada. O objetivo deste estudo foi a utilização de um composto fluorescente para simular a contaminação bacteriana e acompanhar esta contaminação em um ambiente de varejo. Uma cozinha deli simulado foi projetado para simular o ambiente de varejo. Deli carne foi inoculado com o composto fluorescente e voluntários foram recrutados para completar um conjunto de tarefas semelhantes aos esperados de um empregado do varejo de alimentos. Os voluntários foram instruídos a cortar, embalar, e armazenar a carne no frigorífico delicatessen. O potencialcontaminação cruzada foi localizado no ambiente de varejo simulada limpando áreas específicas e medir a densidade óptica da área limpo com um espectrofotômetro. Os resultados indicaram que o refrigerador (isto é deli caso) aderência e diversas áreas na fatiador teve o maior risco para a contaminação cruzada. Os resultados deste estudo podem ser usados ​​para desenvolver material de treinamento mais focado para os empregados de varejo. Além disso, as metodologias semelhantes também podem ser usados ​​para rastrear a contaminação microbiana em ambientes de produção de alimentos (por exemplo, pequenas fazendas), hospitais, lares de idosos, cruzeiros e hotéis.

A Figura 1 representa as zonas do moinho de facas que foram esfregadas após os voluntários completaram as tarefas atribuídas. Os voluntários foram filmados para analisar a freqüência média de contato da mão sobre essas várias superfícies. A freqüência de contato manual foi analisado por quatro observadores diferentes e média. Estes resultados estão demonstrados na Figura 2. Os dados mostram que a aderência carne, deli invólucro, carne deli, deli e papel tiveram as maiore...

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

O objetivo geral deste estudo foi demonstrar o mecanismo de contaminação cruzada potencial de patógenos de origem alimentar Listeria monocytogenes em um ambiente deli varejo. Esta metodologia pode ser aplicada a uma variedade de ambientes diferentes para controlar a contaminação do patógeno.

Rastreamento contaminação microbiana em ambientes de varejo com fontes fluorescentes Powder - Um ambiente de exemplo Retail Delicatessen

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 visualizações.  University of Houston.  O objetivo geral deste estudo foi demonstrar o mecanismo de contaminação cruzada potencial de patógenos de origem alimentar Listeria monocytogenes em um ambiente deli varejo. Esta metodologia pode ser aplicada a uma variedade de ambientes diferentes para controlar a contaminação do patógeno. 

Vídeo: 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.

Determinação da Microbial Extracelular Enzyme actividade nas águas, solos e sedimentos, utilizando alta taxa de transferência de microplaca Ensaios

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

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 de Estudos de Artrópodes e comunidades microbianas dentro Árvore Tecidos

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.

Um Aquatic Microbial metaproteômica fluxo de trabalho: a partir de células para Tryptic Peptides adequado para análise baseada em espectrometria de massa em tandem

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.

Extração e Análise de Microbial Phospholipid Fatty Acids em solos

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.

Amostragem, classificação e caracterização Microplastics em ambientes aquáticos com cargas de sedimentos suspensos altos e grandes destroços flutuantes

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).

Deteção de 3-Nitrotyrosine em ambientes atmosféricos através de alta performance sistema Detector eletroquímico-cromatografia 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.

Bactérias rotuladas fluorescently como um Tracer para revelar novas vias de fluxo de carbono orgânico em ecossistemas aquáticos

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.

Enriquecimento e detecção de toxinotipos de Clostridium perfringens em amostras de alimentos para varejo

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.

In Situ Chemotaxis Assay para examinar o comportamento microbiano em ecossistemas aquáticos

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.

Avaliando o impacto da fratura hidráulica nos córregos usando assinaturas 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 ...