Name: ecotoxicological method with marine bacteria vibrio anguillarum to evaluate the acute toxicity of environmental contaminants
Uploaded: 2017-05-26T13:00:00.000+00:00
Duration: 11 min 26 s
Description: Watch this Scientific Journal Video about Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants at JoVE.com

这项工作由研究项目资助:"NanoBioTech ambiente e salute。Progetto 2:Ambiente。Strumenti e metodi per il monitoraggio ecotossicologico delle Nanoparticelle"（ "Nano-BioTechnology:environment and health。Project 2:Environment。Tools and methods for ecotoxicological监测纳米粒子" ）授予LM来自Regione Lazio-Consorzio Hypatia的LM。 AR在以前引用的项目的框架下由Tor Vergata / Regione Lazio-Consorzio Hypatia大学的博士后资助。与ISPRA-Tor Vergata大学（N. 2015/52857）的协议允许相互使用设施和研究人员的交流。 作者感谢Maria Cristina Thaller教授担任所有微生物活动的守护天使，以提高对微生物世界的兴趣，并大力帮助改善我们对该问题的研究。作者衷心感谢AndreaTornambè和Erika Magaletti与浮游植物生态和生态毒理学ISPRA实验室的宝贵协作。

1.试剂/材料的制备<ol><li>准备（约300）无菌1.5 mL管，用于连续稀释细菌悬浮液，以及15 mL无菌管作为测试浓度标记的测试容器。 </li><li>准备2％NaCl溶液作为曝光介质并灭菌。或者，使用灭菌的合成或天然海水，盐度范围为5至40。 </li><li>根据标签方向准备具有2％NaCl的胰蛋白酶大豆琼脂（TSA）生长培养基，并考虑培养基中已经存在的NaCl量。 </li><li>将TSA（冷却但仍然是液体的）倒入以前用测试浓度和暴露时间，重复数量和稀释因子标记的90-mm培养皿中; 19 mL是适当的体积。 </li><li>根据标签方向制备胰蛋白胨大豆肉汤（TSB）生长培养基。加入适量的NaCl以获得与曝光介质相同的盐度。 </li><li>准备具有双蒸水的CuSO 4储备溶液，并使用0.22μm注射器过滤器消毒（必需）等分试样。在环境样品的情况下，准备适当的样品稀释间隔，并使用0.22μm注射器过滤器进行灭菌。 </li><li>在测试浓度标记的15 mL管中制备测试溶液。用5 mL曝光介质（2％NaCl）填充阴性对照。用适量的暴露介质和CuSO 4储备液填充其他管，以获得最终体积为5 mL的测试浓度。 </li></ol>细菌接种物制备<ol><li>试验前12-18 h，制备鳗弧菌液体新鲜培养物。使用无菌环，从固体培养基（TSA）上的过夜培养中选择一个单独的，分离良好的菌落。接种填充有10mL TSB的管，并将细菌培养物在25℃下孵育12-18小时。 </li&gt; <li> 12-18小时后，用分光光度法估计接种物的细菌浓度。涡旋接种物并测量600 nm波长的光密度，使用TSB作为空白。 </li><li>为了获得已知的细菌浓度，通过加入通过以下公式计算的TSB量稀释2mL的涡旋接种物: TSB mL = [（OD / 0.14）* 2] -2。 </li><li>验证稀释接种物的光密度是0.140（±0.005），这对应于麦克法兰德比浊法标准的0.5分。 </li><li>将稀释的接种物以3,000g离心10分钟。消除上清液并将微生物沉淀物重新悬浮在1mL的2％NaCl溶液（曝光介质）中。 </li></ol> 3.测试曝光<ol><li>加入150μL重悬细菌接种物到每个管，包括对照。在黑暗和连续条件下，在25℃下孵育鳗弧菌悬浮液6小时摇晃以避免沉淀。 </li><li>在暴露时间的开始（T 0 ）和结束（T 6 ），使用菌落形成单位（CFU）计数法在所有暴露的细菌悬浮液中进行细菌计数。 </li><li>准备连续稀释每种暴露的细菌悬浮液一式三份，应用十倍稀释因子（最多10 -5 ）。将100μL每个细菌悬浮液加入到已经填充有900μL曝光介质（2％NaCl）的相应管中。继续进行连续稀释，每个步骤涡旋以重新悬浮细菌。 </li><li>在对应的片段中的TSA培养皿上10μL的10 -4和10 -5稀释液。通过旋转盘子快速让液滴滑入一个小圆圈。将板在25℃孵育48小时。 </li></ol> 4. CFU计数<ol><li> 48小时后，计数在培养皿上生长的菌落;藏有b的盘子5和50个菌落是准确计数的最佳选择。 </li><li>通过应用以下公式计算每毫升暴露的每个细菌悬浮液的活细菌数: 10 -4 →CFU / mL = n°CFU×100×10,000 10 -5 →CFU / mL = n°CFU×100×100,000 </li><li>使用并行重复获得的计数的平均值来评估与对照相比的死亡率。以下列公式计算死亡率百分比: M％= 100 - [（N / C）* 100] 注意:其中N =暴露于毒物后生长的CFU / mL数; C =在对照培养基中生长的CFU的数量。 </li><li>使用适当的统计软件（参见材料表），使用非线性回归分析计算LC 50 （ 即，将活性复制细菌数量减少50％/ mL的CFU / mL）。运行单向方差分析（ANOVA）通过事后成对t检验来评估治疗之间的显着差异。 </li></ol>

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作者宣称他们没有竞争的经济利益。

这项工作描述了一种新的生物测定与海洋细菌V. anguillarum成功应用于评估CuSO 4 （一种参考毒物）的毒性作用，显示出明确的剂量反应关系。海洋细菌V. anguillarum被选为模式生物，因为它是无害的，无所不在的，代表海洋生态系统。 测试可以在广泛的盐度值（5-40）下进行，并且可以使用盐水溶液和合成或天然海水作为曝光介质，只要微生物能够在整个测试期间容易地存活。这样可以分析不同种类的样品，包括咸水和海洋环境。 在暴露阶段不需要生长培养基，避免干扰污染物8及其对生物反应的可能影响。钍e协议可靠，快速，成本有效，而且比较容易。液 - 板微计数9的过程具有使用小（样品）体积的优点，尽管这意味着高度的精度和鲁棒性。三项独立试验的结果和每次治疗的重复显示了该方法的高重复性。使用细菌作为生物学模型，以及该技术的适应性，有利于此过程的生态和环境相关性。其他关键的技术问题是在细菌接种物的制备中的准确性和在该步骤的一些步骤中所需的不育性。 提出的测试比其他海洋生态毒理学测定（24-96小时）更快（6小时），并且不会引起使用高等生物所带来的伦理问题。此外，参考毒物的数据显示LC 50值与用急性t获得的值相当<html>对其他海洋物种的遗传10,11表现出良好的敏感性。在细菌生物测定中， V. fischeri发光抑制测试是最常见和标准化的12 。该生物测定非常快速（15-30分钟），并且对于固相样品的测试是有效的，但它可能受到着色和混浊样品的影响，这些样品干扰了发光测量。盐度是使用上述试验的限制因素，2％NaCl需要13 。相反，这里提出的具有V. illar illar the the um um um um um um um um um um um um um um。。。。。。。。。。。。。。。。。。。。。。。。。。。我们的研究结果与V. fisheri的文献中的比较 14 ，ss ="xref"&gt; 15,16表示可比较的EC 50值，进一步支持此生物测定的有效性。 该生物测定评估了在目前可用于微生物的测试中使用的细菌培养性（通常称为死亡率）的降低，而不是群体生长速率或酶活性抑制。 LC 50计算允许与通常应用于通常以生存/死亡率为终点的海洋环境的生态毒理学评估的其他生物测定法进行比较。为了评估/确认该测试的可靠性和可重复性，并且支持其在规范协议中的标准化和使用，迫切需要进行相互校验。 纳米材料的越来越多的使用及其在环境中的潜在释放意味着需要进行风险评估17 。但是，克拉斯这些新兴污染物的sical（eco）毒理学方法似乎没有提供可承受的结果，可能需要一些适应性18 。这种新的生物测定的特征使其易于有效的应用于纳米颗粒的毒性评估。事实上，在不同盐度下进行测定的可能性将会说明不同离子强度下的纳米粒子行为，一个可以显着影响毒性的环境参数变量。此外，在纳米颗粒的生态毒性评估中，特别推荐使用生长培养基和营养素，因为有机物质可以通过增加毒性作用促进它们的吸收，或者可能引起聚集，降低生物利用度，从而降低其毒性21 。 总之，鳗弧菌的生物测定是ap古典和新兴污染物风险评估的浪漫工具，以及海洋和咸水环境状况的评估。

生态毒理学生物测定评估化学品或环境样品与标准生物学模型的毒性，将物理，化学和生物应激物的作用整合到生态系统上。由于生态系统的复杂性，生态毒理学风险评估必须考虑一系列涉及不同营养水平的生物体的生物测定。实验室动物的毒性测定可能是昂贵的，耗时的，并且在道德上是有问题的。限制动物测试和开发替代方法（ 例如细菌和非脊椎动物）的动力现在是当前欧洲立法框架中所报告的关键问题，其中包括欧盟动物保护指令，第七修正案欧盟化妆品指令和REACH。 甲壳类动物，鱼类和藻类主要用于海洋环境中的毒性测量1 。细菌是重要的成分生态系统的变化以及对微生物群落的改变可能对生物地球化学循环和其他关键生态系统服务产生重大影响。基于微生物的毒性测试越来越受欢迎，因为它们相对快速，可重现和便宜，不会引起伦理问题2 。这项工作的目的是描述一种生态毒理学方案，以评估当暴露于环境污染物时海洋细菌鳗弧菌 （ 鳗 弧菌 ）的响应。 鳗弧菌是具有极性鞭毛的革兰氏阴性短曲线状细菌（0.5×1.5μm）。通常在咸水或盐水中发现，它是耐盐的，最佳盐度约为20，最佳温度在25至30℃之间。由于其普遍存在及其在海洋中的重要生态作用，被选为生物体模型全球4 。已知鳗弧菌的一些血清型可导致各种海洋或微咸鱼类物种5,6的弧菌病 。为此，实验的一些步骤需要标准的微生物学实践，但不需要特殊的安全设备或预防措施。拟议的毒性试验方案使用细菌培养性（ 即，复制和形成菌落的能力）作为终点，并允许LC 50的测定，导致细菌积极复制和形成菌落50％的浓度的浓度，之后6小时曝光。在弧菌中 ，与其他微生物一样，我们通常表示为死亡率的这种减少可部分归因于可行但不可培养（VBNC）阶段7中的个体。在本研究中，我们应用这种方法来测量硫酸铜（CuSO 4）的毒性作用），一种参考毒物。 该方法被开发用于为污染物/化合物的生态毒性评估提供适合的基于微生物的测试，包括新兴的污染物如纳米材料。与现有的用于微生物的方法相比，该方案的新颖性主要与暴露介质和终点有关。事实上，暴露在盐溶液中进行，避免生长培养基与被研究的污染物的任何可能的干扰，这可能影响生物反应8 。终点是细菌培养性的降低，这可以容易地与基于生存/死亡率的海洋/微咸水环境中用于生态毒理学筛选的其它急性终点相比较。此外，该方案使用已经在大肠杆菌 9上使用的液 - 板微量计数技术，减少体积，因此实验效率ort（有关详细信息，请参阅协议的步骤3.3和3.4）。

<table><tbody><tr><td>&lt;i&gt;Vibrio anguillarum&lt;/i&gt; (strain AL 102, serotype O1)</td><td></td><td></td><td>Obtained from the laboratory collection of NOIFMA (Norway)</td></tr><tr><td>Tryptic Soy Agar&amp;nbsp;</td><td>Liofilchem</td><td>610052</td><td>Dehydrated Culture Media</td></tr><tr><td>Tryptic Soy Broth growth medium</td><td>Liofilchem</td><td>610053</td><td>Dehydrated Culture Media</td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>209198</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S-3014</td><td></td></tr><tr><td>&lt;strong&gt;Name of Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Biosafety Laminar Flow Hood&amp;nbsp;</td><td>ESCO</td><td></td><td></td></tr><tr><td>Incubator&amp;nbsp;</td><td>Fratelli Galli</td><td>Mod. 2100</td><td></td></tr><tr><td>&lt;strong&gt;Name of Software&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Benchmark Dose Software&amp;nbsp;</td><td>US EPA</td><td>Benchmark Dose 2.4.0&amp;nbsp;</td><td>2012</td></tr></tbody></table>

ecotoxicological method with marine bacteria vibrio anguillarum to evaluate the acute toxicity of environmental contaminants

细菌是生态系统的重要组成部分，微生物群落的改变可以对生物地球化学循环和食物网产生重大影响。基于微生物的毒性测试被广泛使用，因为它们相对快速，可重复，便宜，并且与伦理问题无关。在这里，我们描述了一种生态毒理学方法来评估海洋细菌鳗弧菌的生物反应。该方法评估化学化合物的急性毒性，包括新型污染物如纳米颗粒以及环境样品。终点是由于暴露于有毒物质而导致细菌培养性降低（ 即复制和形成菌落的能力）。这种减少通常可以称为死亡率。该测试允许测定LC 50 ，导致细菌积极复制和形成菌落的50％减少的浓度，之后6小时曝光。以菌落形成单位（CFU）计数培养的细菌，并评估"死亡率"并与对照进行比较。在这项工作中，评估了硫酸铜（CuSO 4 ）的毒性。观察到明确的剂量反应关系，三次独立试验后平均LC 50为1.13 mg / L。该方案与现有的微生物方法相比，适用于更宽的盐度范围，对彩色/混浊样品没有限制。它使用盐水溶液作为曝光介质，避免生长培养基与被研究污染物的任何可能的干扰。 LC 50计算有助于与通常用于海洋环境生态毒理学评估的其他生物测定法进行比较。

将鳗弧菌暴露于四种浓度的CuSO 4的三个独立试验的结果显示出明显的剂量 - 反应关系和显着减少的细菌积极地复制和形成集落，随着参考毒物浓度的增加（ 图1 ; ANOVA，F = 20.28，p &lt;0.001）。与对照（CNTR）相比，CFU / mL的数量以1.25mg / L（事后Tukey检验，p &lt;0.05）显着降低。在最高测试浓度下检测到任何可培养的细菌。在暴露介质的宽盐度范围（ 即 5,20或35g / L NaCl，数据未显示）中，没有发现CuSO 4毒性的差异。 补充图1中报告了显示非线性回归的统计分析的代表性输出。 LC 50值计算为三项独立试验（&lt;strong&gt;表1）突出了该方法的可行性和可重复性。 <img alt="图1" src="/files/ftp_upload/55211/55211fig1.jpg" /> 图1:暴露于CuSO的鳗弧菌 4.暴露于不同浓度的CuSO 4 6小时的CFU / mL（CFU =菌落形成单位）的平均数。这些值代表三次独立试验的平均值（±SD）。相对于对照（CNTR）积极复制和形成菌落的细菌的减少报告在黄色框中（以百分比表示）。与基于事后Tukey测试的对照的显着差异用星号表示（* = p &lt;0.05; ** = p &lt;0.01）。 <a href="http://ecsource.jove.com/files/ftp_upload/55211/55211fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 表1:暴露于CuSO 4的 鳗弧菌的致死浓度（LC50）值。报告了三项独立试验和手段（±SD）的结果。 补充图1:非线性回归分析。对测试结果进行非线性回归分析（Logit-Hill模型）的代表性输出。 <a href="http://ecsource.jove.com/files/ftp_upload/55211/suppl_fig_1.tif" target="_blank">请点击这里下载此图。</a>

Watch this Scientific Journal Video about Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants at JoVE.com

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

这项工作描述了一种新的方案来评估污染物的生态毒性，包括新兴的污染物，如纳米材料，使用海洋细菌鳗弧菌 。该方法允许在暴露6小时后，确定导致细菌培养性降低50％的浓度的LC 50或死亡率。

生物毒理学方法与海洋细菌<em&gt;鳗弧菌</em&gt;评估环境污染物的急性毒性

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

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JoVE Journal

Environment

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

10.9K 次观看.  Institute for Environmental Protection and Research (ISPRA), Rome (Italy). 这项工作描述了一种新的方案来评估污染物的生态毒性，包括新兴的污染物，如纳米材料，使用海洋细菌鳗弧菌 。该方法允许在暴露6小时后，确定导致细菌培养性降低50％的浓度的LC 50或死亡率。 

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

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

科研

环境科学

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

对污水处理工艺的疗效评估利用化学和生态毒理学方法的电池，以消除雌激素潜能

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

<meta charset="utf8"></head><body>评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

使用 卤虫 盐L的致死性生物测定。

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

化学

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

对于检测到的敏感性和特异性显色琼脂培养基发展<em&gt;副溶血性弧菌</em&gt;及其他<em&gt;弧菌</em&gt;物种

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

免疫与感染

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

生物工程

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

生物学

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

生物毒理学方法与海洋细菌

摘要

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此视频中的章节

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

对污水处理工艺的疗效评估利用化学和生态毒理学方法的电池，以消除雌激素潜能

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

使用卤虫盐L的致死性生物测定。

对于检测到的敏感性和特异性显色琼脂培养基发展

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

致命的甲壳类动物长期毒性试验卤虫franciscana

用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定

生物毒理学方法与海洋细菌

摘要

探索更多视频

此视频中的章节

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

对污水处理工艺的疗效评估利用化学和生态毒理学方法的电池，以消除雌激素潜能

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

使用 卤虫 盐L的致死性生物测定。

对于检测到的敏感性和特异性显色琼脂培养基发展

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

致命的甲壳类动物长期毒性试验卤虫franciscana

用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定

使用卤虫盐L的致死性生物测定。