著者らは、この研究を手伝ってくれました食品安全研究室の学生やボランティアのすべてに感謝したいと思います。この研究は、米国農務省国立統合型食品安全イニシアチブ（NIFSI）補助金（賞＃10507316）によって資金を供給されています。

1。標準曲線<ol><li> 37.5 mgの蛍光粉を計量し、6ミリリットル200プルーフエタノールに溶解する。 200プルーフのエタノール中のこの溶液を2倍希釈系列を作る。 </li><li>ボルテックス10秒のすべてのサンプルおよび370 nmの吸光度を測定する。 </li><li> 1ミリリットル200プルーフエタノールあたりの蛍光粉の量を計算する。 </li><li>蛍光粉の濃度に基づいて吸光度を実証する（x軸上の370 nmでのy軸上に吸光度ミリグラム/ mlの濃度）の標準曲線を調製する。 </li></ol> 2。蛍光粉とデリの肉（ボローニャ）の接種<ol><li>きれいなナイフとまな板を使用して、約100ミリメートルの厚さのサンプルにデリの肉​​をカット。 A、B、Cのラベル3サンプルを調製</li><li>少し湿った、清潔なスポンジを使用して蛍光粉に均一に被覆のサンプルA。均等に肉のサンプルに粉を広げた。蛍光粉の試料BとCを接種しないでください。 </李&gt; <li>ラップでサンプルをラップし、「A」のような蛍光粉体塗装サンプルにラベルを付け、テープで、BとCと2のサンプルを残り。 </li><li>実験が実施される模擬キッチンで4℃に保っデリケースや冷蔵庫内のサンプルを置きます。 </li></ol> 3。モックデリキッチンの準備<ol><li>キッチンで清潔な手袋、70％エタノールスプレーを提供する。 </li><li>スライサー面積約13 Wの電球と黒のコンパクト蛍光灯を取り付けます。 </li><li>拭き取り用のテンプレートとして機能するようにアルミ箔を5センチ×5cmのカットアウトを準備します。 </li><li> 6ミリリットルの95％エタノールを含む15ミリリットルのチューブを準備します。 </li></ol> 4。ビデオ設定マウント同時にモックデリのすべての領域を観察するための戦略的な場所にある3台のビデオカメラ。 （プロトコル5で説明したように）スライサー手順をシミュレートする前に、カメラの電源を入れ、同時に結果を分析する（ように）プロトコル7で説明した。 5。交差汚染を追跡するためにシミュレートデリスライス手続きサンプルサイズを改善し、変化を取り入れる、それぞれの機関の施設内倫理委員会によるプロトコルの承認後に参加者を募集し、この研究を行うためには。参加者はや経験は、小売デリ環境で作業していない場合があります大学生することができます。彼らはデリが入ると、各参加者が手袋を身に着けていることを確認してください。調査が完了すると「後」画像に続いて黒蛍光灯の下で絵 &quot;の前に&quot;を取る。以下のように各参加者に明確な書面と口頭の指示を提供します。 <ol><li>冷蔵庫に移動します。 </li><li> 「A」と表示された肉を削除します。 </li><li>肉のラップを解除し、ラップを保存します。 </li><li>スライサーの搬送トレイに肉を置きます。 </li><li>肉グリップ付き肉を固定します。 </li><li>電源スイッチをONにします。 </li><li> SLIを調整「2」のcerインデックスノブ。 </li><li>スライスとは、デリ紙に肉の5個を分配。 </li><li>オフ電源スイッチを切り、肉のグリップを離します。 </li><li> 「A」と表示され、ビニール袋に肉のスライスを配置します。 </li><li>肉 &quot;A&quot;を再ラップして冷蔵庫に戻ります。 </li><li>リピートは「B」と「C」と表示された肉を2-11繰り返します。 </li></ol> 6。蛍光粉を定量化するデリキッチンでモックスライス（5.4節）終了後の蛍光粉を定量化する。これは次の手順を実行します。の場合： <ol><li>無菌のセンチ×5cmのアルミ箔のテンプレートを使用し、 図1にマークされた領域の上に置きます。 </li><li>領域をスワブ、95％エタノールに浸した無菌のアルギン酸カルシウムの綿棒を使用し、無菌のポリプロピレンチューブ内に6ミリリットルの95％エタノール中に置く。 </li><li>徹底的にチューブをボルテックスし、370 nmの吸光度を読み取ることがガラスキュベットに移す。 </li><li> standarを参照してください。以下の式を用いて蛍光粉末の量（y）を決定するために、曲線dは、xは吸光度ではy = mx + bの、mは、標準曲線の傾きであり、bは切片である。 </li><li>データの正規化については、以下の式を用いてサンプリングされた面積当たりの蛍光粉末のパーセントを計算する： </li></ol><img src="/files/ftp_upload/51402/51402eq1.jpg" alt="フォーミュラ1" /> 7。ビデオ分析ビデオテープの参加者、彼らの完全なタスク5.1から5.12など。 SYNCおよび全ての角度を同時に見ることができるようにDVDディスクにビデオ録画を保存します。手のタッチ数デリ環境（4異なる研究者が独立して完全なデータセットを分析し）での様々な表面上で（ボランティアがモックデリの領域に触れる回数）を記録するためにビデオを見る。

<ol>
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利害の対立が宣言されていません。

本研究の全体的な目標は、交差汚染が食中毒のリスクをコントロールし、削減するための戦略を開発するために、小売デリ環境で発生する可能性がどのように細菌を証明することであった。デリ小売環境は、L.のために特に高いリスクを有する汚染をリステリア 。米国農務省食品安全検査局は、小売店でスライスデリ肉はデリ肉はスライスして、連邦政府検査所9でパッ?...

特に小売セクションの調理環境における食品由来病原体の交差汚染は、原因肉だけでなく、野菜を1-5など、さまざまなソースからの食中毒のリスクの増加に主要な関心事である。ほとんどの場合、細菌性病原体は、汚染された食品の6を通じて小売環境を入力してください。消費前7へのさらなる介入または治療（ すなわち 、調理または加熱）は通常存在しないように、小売レベルでの準備すぐに食べられる食品は特に懸念される。また、汚染されたRTE食品に存在する病原体は、次いで、小売環境における他の食品または食品接触表面に転写することができる。 交差汚染は、小売、食品、環境の事実上すべてのタイプの中で発生する可能性がありますが、デリが原因病原体リステリアmonocytogとのRTEデリ肉の協会特に特に重要であるエン2。米国食品医薬品局（FDA）農業食品安全検査局（USDA-FSIS）米国商務省が実施したリスクアセスメントの分析に基づき、汚染されたデリの肉は、リステリア症の症例の90％を担当している可能性が高い - まれL.によって引き起こさまだ厳しい人間の病気リステリア-米国8。一般的には、小売·食品サービス施設でスライスデリ肉はLの高いリスクと関連している米国農務省にあるあらかじめスライスされたデリの肉に比べてリステリアは、メーカー9で工場を視察した。これは、食品取扱着信原材料、あるいは治療後7汚染されることがあり、加工食品による病原体の導入の可能性の増大の可能性があります。全体的に、リステリア症は、米国では年間食中毒に起因すると推定死亡者の約28％を占めるなぜなら中のLのしわのリスク小売段階でスライスデリ肉の汚染をリステリア 、デリスライサー自体は小売環境2,10における食品由来の病原体の表面移動のための重要な因子として同定されている。シーン1で示すように、スライスは、消費前の最後の処理工程であるため、デリスライサーは、理解と相互汚染を防止するため1の両方の重要管理点を考慮する必要があります。 本研究の全体的な目標は、このようなグロ胚芽などの蛍光粉を使用して、小売デリ環境の汚染を追跡する方法論を開発することでした。これは、細菌の交差汚染及び衛生慣行11,12をシミュレートするために使用されている蛍光化合物である。この研究は、スライサに汚染された肉、周囲の環境、およびその他のデリ肉からの病原体の移動を追跡するように設計した。それは、CONTの経路を理解することが重要です小売従業員と管理職のための効果的な介入と教育材料の両方を開発するためのアミノ化。同様のアプローチは、他の処理環境（ 例えば、鶏肉、牛肉）、農場、病院内の病原体伝達をシミュレートするために使用することができる。

<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

小売環境での食中毒病原体の交差汚染は食中毒のリスク増加に貢献して重要な公衆衛生問題である。このようなデリ肉、チーズ、そしていくつかのケースでは、生鮮食品などのすぐに食べられる（RTE）加工食品は、 リステリア菌などの病原体による汚染に起因する食品媒介疾患のアウトブレイクに関与している。 L.に関してリステリアは 、デリスライサーは、多くの場合、交差汚染の主な情報源である。この研究の目的は、細菌汚染をシミュレートし、小売環境でこの汚染を追跡するために蛍光化合物を使用することであった。モックデリキッチンは小売環境をシミュレートするように設計されました。デリの肉は、蛍光化合物を接種し、ボランティアは、食品小売業従業員に期待されるものと同様の一連のタスクを完了するために募集された。志願者は、スライスパッケージ、およびデリ冷凍肉を保存するように指示した。潜在的な交差汚染は、特定の領域をスワブし、分光光度計を用いて綿棒領域の光学密度を測定することによって模擬​​小売環境で追跡した。結果は、スライサーの上、冷蔵庫（ すなわちデリケース）グリップと様々な分野が交差汚染のリスクが最も高いを持っていたことが示された。この研究の結果は、小売店の従業員のより集中訓練材料を開発するために使用されてもよい。また、同じような方法論は、食品生産環境（ 例えば 、小農場）、病院、老人ホーム、クルーズ船、ホテルなどでの微生物汚染を追跡するために使用することができる。

図1に、ボランティアが割り当てられたタスクを完了した後にふいたスライサーの領域を表します。ボランティアは、これらの様々な表面上で、手で直接触れるの平均周波数を分析するためにビデオテープに録画されました。手の接触頻度は、4つの異なる観察者により分析し、平均した。これらの結果を図2に実証される。データは、肉のグリップ、デリの肉ラッパ?...

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

本研究の全体的な目標は、小売デリ設定で食中毒病原体リステリア菌の潜在的なクロス汚染メカニズムを実証することであった。この方法は、病原体汚染を追跡するために、異なる様々な環境に適用することができる。

蛍光粉を使用した小売環境における微生物汚染を追跡する - 小売デリカ環境の例

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 ビュー.  University of Houston. 本研究の全体的な目標は、小売デリ設定で食中毒病原体リステリア菌の潜在的なクロス汚染メカニズムを実証することであった。この方法は、病原体汚染を追跡するために、異なる様々な環境に適用することができる。 

ビデオ: 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.

ハイスループットマイクロプレートアッセイを用いてウォーターズ、土壌、および底質中の微生物細胞外酵素活性の測定

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

環境学

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.

ツリー組織内節足動物と微生物群集を研究するための技術

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ワークフロー：細胞からのトリプシンペプチドのタンデム質量分析ベースの分析に適し

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.

土壌中の微生物のリン脂質脂肪酸の抽出と分析

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.

サンプリング、並べ替え、および高い浮遊負荷と大きい浮遊残骸水圏環境における Microplastics の特性

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

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

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

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

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

3-ニトロチロシン大気環境を介して高性能液体クロマトグラフィー-電気化学検出器システムの検出

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.

水生生態系における有機炭素流の新しい経路を明らかにするトレーサーとしての蛍光標識細菌

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.

小売食品サンプルにおけるクロストリジウム・パーフリンゲントキシノタイプの濃縮と検出

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.

水生生態系における微生物行動を調べるSitu chemotaxisアッセイで

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.

微生物分子シグネチャを用いた河川における水圧破砕の影響評価

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