笔者想感谢所有的食品安全实验室的学生和志愿者谁帮助了这项研究。这项研究已经由美国农业部国家综合食品安全倡议（NIFSI）补助金（奖＃10507316）。

1。标准曲线<ol><li>称取37.5毫克荧光粉末，溶于6毫升200标准乙醇。使在200标准乙醇此溶液的2倍连续稀释。 </li><li>涡旋10秒所有样品并测量吸光度在370 nm处。 </li><li>计算荧光粉末的量每1ml 200标准乙醇。 </li><li>制备标准曲线（浓度在y轴上和吸光度在x轴370纳米毫克/毫升），以证明根据荧光粉浓度的吸光度。 </li></ol> 2。接种熟食肉类（博洛尼亚）用荧光粉<ol><li>切熟肉成用干净刀，菜板大约100毫米厚的样品。准备标记为A，B和C三个样本</li><li>均匀地涂样品A使用微湿的干净的海绵荧光粉。分散粉末的肉样品的均匀。不接种的样品B和C与荧光粉末。 </LI&gt; <li>包装样品用保鲜膜和荧光粉涂层的样品标记为“A”和其余两个样品为B和C用胶带。 </li><li>将样品在熟食店情况下或在4°C的模拟厨房，实验将开展举行冰箱。 </li></ol> 3。模拟德利厨房准备<ol><li>在厨房里提供干净的手套和70％的乙醇喷雾。 </li><li>安装黑色紧凑型荧光灯与周围的切片机区13瓦的灯泡。 </li><li>制备铝箔5 5cm×5cm的切口，作为擦拭模板。 </li><li>制备含6毫升95％乙醇15ml试管。 </li></ol> 4。视频设置在战略位置登上三个视频摄像机来观察模拟熟食店的各个领域的同时。开启模拟切片机程序前，相机（如协议5解释），并同时分析结果（如在协议7解释）。 5。模拟德利切片过程来跟踪交叉污染为了提高样本量，并纳入变化，由各自机构的伦理审查委员会的批准协议后，由招募参加者进行这项研究。参与者可以是一个大学生谁可以或不可以在零售熟食店环境中工作的经验。确保每个参与者戴着手套，一旦他们进入熟食店。采取其次是“之前”下的黑色荧光图像的研究完成后，画面“后”。提供清晰的书面和口头说明，每个参与者如下： <ol><li>去冰箱。 </li><li>肉取出标记为“A”。 </li><li>拆开包装肉类和保存保鲜膜。 </li><li>将肉切片机的运输托盘。 </li><li>安全肉与肉的抓地力。 </li><li>打开电源开关。 </li><li>调整SLICER指数旋钮为“2”。 </li><li>切片和免除5片肉到熟食纸上。 </li><li>打开电源开关，并松开肉抓地力。 </li><li>将肉切片在塑料袋中标记为“A”。 </li><li>重新包装肉的“A”，并返回到冰箱。 </li><li>重复步骤2-11与肉类标有“B”和“C”。 </li></ol> 6。量化荧光粉在模拟切片（5.4节）在熟食店厨房完成后，量化荧光粉。对于这个请执行以下操作： <ol><li>用灭菌的5 5cm×5cm的铝箔模板并将其放置在标记为图1中的区域。 </li><li>使用无菌的藻酸钙棉签浸泡在95％乙醇中，擦拭面积，并将其放置在6ml 95％乙醇中的无菌聚丙烯管中。 </li><li>涡管彻底转移到玻璃比色皿来读取吸光度在370 nm处。 </li><li>参阅STANDAR3D曲线使用下面的公式来确定荧光粉末的量（Y）为：y = mx + b中 ，其中x是吸湿性米的标准曲线的斜率，b为截距。 </li><li>供的数据的归一化，计算每单位面积使用下列公式计算样品的荧光粉末的百分比为： </li></ol><img src="/files/ftp_upload/51402/51402eq1.jpg" alt="式（1）" /> 7。视频分析录像带的参与者，因为他们完成任务5.1-5.12。同步和视频记录保存到DVD光盘，使得所有角度可以在同一时间被观看。观看视频录制的手摸的数量上在熟食店环境（4个不同的研究人员将分析独立设置的完整数据）各种表面（次志愿者碰在模拟熟食区的数量）。

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没有利益冲突的声明。

本研究的总体目标是要证明细菌交叉污染可能发生如何在零售熟食店环境，以便制定战略，以控制和减少食源性疾病的风险。零售熟食店环境具有特别高的风险L。菌污染。美国农业部食品安全检验局报告说，熟肉切片在零售商店有七次更高的协会与李斯特菌病的熟肉切片和包装在一个联邦检查的工厂9。 荧光为基础的方法分别报告了专利，检测粪便污染屠体<sup...

食源性致病菌在食品制备环境，特别是在零售部的交叉污染，是由于食源性疾病的从各种来源，包括肉以及蔬菜1-5风险增加的一个主要关注的问题。大多数情况下，细菌病原体通过污染的食物产品6进入零售环境。在零售层面准备随时可以吃的食物是特别关注的，因为通常在消费前7没有进一步的干预或治疗（ 如烹煮或加热）。此外，病原体存在于污染的即食食物可以被转移到其他食品或食品接触面的零售环境。 虽然交叉污染可以发生在几乎所有类型的食品零售环境，熟食店是特别感兴趣的，特别是因为即食熟食肉类与病原体李斯特菌monocytog协会埃内斯2。根据风险评估分析由美国食品和药物管理局（FDA）和农业部食品安全检验局（USDA-FSIS）美国能源部进行的，受污染的熟食肉类很可能负责90％的李斯特菌病的病例 - 一个罕见的造成L.但严重的人类疾病菌-在美国8。一般情况下，在切片零售和食品服务场所熟肉制品已与L的高风险相关菌相比，在美国农业部检查植物presliced ​​熟食肉类制造商9。这可能是因为引进病原体的食物处理，传入的原材料，或加工食品可能被污染后处理7的可能性增加。总体而言，李斯特菌病占每年所造成的食源性疾病的死亡人数估计在美国约28％ 因为在L的折痕风险菌污染切片在零售层面熟食肉类，熟食切片机本身已被确定为食源性致病菌在零售环境中2,10表面转移的重要因素。如由辛1，切片是最后的处理步骤之前，消费，因此，熟食切片机，应考虑为了解和防止交叉污染1关键控制点。 本研究的总体目标是开发一种方法来使用荧光粉如格洛胚芽零售熟食店环境跟踪污染。这是一个已经被用来模拟细菌交叉污染和卫生实践11,12的荧光化合物。本研究旨在追踪来自受污染的肉类病原体转移到切片机，周边环境，和其他熟食肉类。关键是要理解续的途径胺化发展既有效干预和零售员工和管理人员的教育材料。类似的方法可以用来模拟其它处理环境（ 如禽肉和牛肉），农场和医院病原体转移。

<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

食源性致病菌在零售环境中的交叉污染是一个显著的公共卫生问题造成的风险增加食源性疾病。现成的吃（RTE）加工食品，如熟食肉类，奶酪，而且在某些情况下新鲜农产品，已经介入，由于病原体污染，如李斯特菌食源性疾病的爆发。相对于L。单核细胞增生 ，熟食切片机往往是交叉污染的主要来源。本研究的目的是利用一种荧光化合物，以模拟的细菌污染并在零售环境跟踪这种污染。有模拟熟食店厨房被设计来模拟零售环境。德利肉接种荧光化合物和志愿者招募完成一组任务相似，预期食品零售员工。这些志愿者被指示切片，封装，肉类存放在一家熟食店冰箱。潜在的交叉污染是通过擦拭特定区域和测量用分光光度计在擦拭区域的光密度跟踪在模拟零售环境。结果表明，冰箱（ 即熟食的情况下）的抓地力和各个领域的切片机有风险最高的交叉污染。这项研究的结果可能被用于开发零售员工更集中的培训材料。此外，类似的方法也可以用于食品生产的环境中（ 如小型养殖场），医院，养老院，游船，和酒店追踪微生物污染。

图1表示被擦拭后的志愿者完成指定任务的限幅器的区域。这些志愿者被录像来分析手接触这些不同的表面上的平均频率。手接触的频率是由四个不同的观察家分析，取平均值。这些结果显示在图2。数据显示，肉的抓地力，熟食肉制品的包装，熟食肉类和熟食纸具有手接触（平均为8-14芯）的发病率最高。 在此之后，执行的分析来定量研究荧光粉的量...

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

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

本研究的总体目标是要证明的食源性致病菌单核细胞增生李斯特氏菌在零售熟食店设置的潜在交叉污染的机制。这种方法可以应用到各种不同的环境跟踪病原体污染。

跟踪微生物污染在零售环境中使用荧光粉 - 零售熟食店环境范例

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

tracking-microbial-contamination-retail-environments-using

Research

JoVE Journal

Environment

11.0K 次观看.  University of Houston. 本研究的总体目标是要证明的食源性致病菌单核细胞增生李斯特氏菌在零售熟食店设置的潜在交叉污染的机制。这种方法可以应用到各种不同的环境跟踪病原体污染。 

视频: 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.

在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的

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

科研

环境科学

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.

技术树内组织学习节肢动物和微生物群落

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.

水生微生物Metaproteomics工作流程：从细胞对胰蛋白酶肽适合串联质谱为基础的分析

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.

提取及土壤微生物磷脂脂肪酸分析

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.

高悬浮泥沙荷载和大型漂浮碎屑水生环境中 Microplastics 的取样、分类和表征

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).

利用高效液相色谱-电化学检测仪检测大气环境中的 3-硝基酪氨酸

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.

荧光标记细菌作为追踪器揭示水生生态系统中有机碳流动的新途径

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.

零售食品样品中丁酸二氮的浓缩和检测

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.

在西图化学分析中，以检验水生生态系统中的微生物行为

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.

使用微生物分子特征评估水力压裂对溪流的影响

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