ai_agent

这项工作得到了NSF DBI生物整合研究所（BII）资助（第2119968号;PI-Ceballos）和阿肯色州INBRE计划，由国家普通医学科学研究所（NIGMS）资助，P20 GM103429来自美国国立卫生研究院。作者还感谢瓦奇塔浸会大学帕特森暑期本科生研究项目的支持。

虽然该协议是为Gordonia terrae编写的，但它也已成功用于Gordonia lacunae，Gordonia rubripertincta和Gordonia westfalica。
1. 细菌制备
<ol><li>在生物安全柜中，使用良好的微生物学规范17 将戈 登氏 菌 CAG3 的单个菌落接种到 1 L 无菌挡板烧瓶中，其中装有 200 mL 蛋白胨酵母钙肉汤18（PYCa），含有 0.01 mg/mL 环己酰亚胺（参见 材料表）。</li>
	<li>将烧瓶在30°C下以250rpm振荡孵育，直到培养物饱和或3天7。</li>
	<li>在 PYCa 肉汤中连续稀释饱和的 G. terrae 培养物，并在 PYCa 平板19，20 上铺平板 100 μL 10−4、10−5 和 10−6 稀释液。将未稀释的饱和培养物在4°C下冷藏。</li>
	<li>将倒置的展开板在30°C下孵育3天。</li>
	<li>孵育后，确定一个"可数板"，即具有20-200个菌落的板。计算该板上的菌落数，并计算每毫升的菌落形成单位（cfu/mL）19，20。</li>
	<li>用 PYCa 肉汤将饱和培养物稀释至 4.0 x 106 cfu/mL 细菌。</li>
</ol>2. 噬菌体制备
注意:报告的代表性结果是用 Gordonia 噬菌体DelRio 21获得的，DelRio21是在 G. terrae上分离的温带噬菌体。这些方法也已成功与其他 戈登菌 体一起使用。
<ol><li>从分离的噬菌体开始，将噬菌体样品在噬菌体缓冲液7中连续稀释至1 x 10−8稀释。通过用 10 μL 的每种噬菌体稀释液感染 0.3 mL 饱和 G. terrae 培养物来进行双层琼脂噬菌体滴度测定7。室温孵育5-10分钟后，将细菌-噬菌体混合物与3mL PYCa顶级琼脂混合，倒入PYCa琼脂平板上。</li>
	<li>将板在30°C下倒置孵育3天或直到形成斑块7。</li>
	<li>孵育后，确定一个"可数板"，即具有20-200块斑块的板。计算该板上的斑块数量，并计算每毫升的斑块形成单位（pfu/mL）7。</li>
</ol>3. 微孔板制备
注意:此方法应使用平底96孔微孔板（参见 材料表）。所有板的制备和装载必须在生物安全柜中完成，并且应使用良好的微生物学规范17。
<ol><li>通过混合 100 μL Triton-X 100、40 mL 100% 异丙醇和 160 mL 去离子水制备防雾盖涂层溶液22.搅拌混合，并在室温下储存。</li>
	<li>在生物安全柜中，将 6 mL 的盖子涂层溶液添加到无菌 96 孔微孔板盖的内表面，握住盖子的边缘并旋转以确保其被溶液覆盖。让溶液在盖子上静置20秒，然后将溶液倒出，并将盖子倒在高压灭菌的纸巾上以一定角度，直到盖子完全干燥，这通常需要35-45分钟。小心握住盖子的边缘。</li>
	<li>为每个 96 孔微孔板准备 20 mL 0.1% 琼脂糖水溶液，微波融化琼脂糖。</li>
	<li>琼脂糖冷却至50-60°C后，将100μL的0.1%琼脂糖移液到板孔之间的所有空间中，将200μL琼脂糖移液到A行和H行以及第1列，第2列，第11列和第12列23 的孔中（图1）。</li>
</ol>4.用细菌和噬菌体装载板
注意:所有板的制备和装载必须在生物安全柜中完成，并且应使用良好的微生物规范17 。
<ol><li>96 孔板每孔中将包含 75 μL 的 2.0 x 106 cfu/mL 细菌9。将 4.0 x 106 cfu/mL 细菌培养物与 2x PYCa 肉汤 1:1 稀释至 2.0 x 106 cfu/mL。每 96 孔板制备 5 mL。</li>
	<li>使用噬菌体缓冲液7 稀释噬菌体裂解物，制成 2.0 x 106 pfu/mL、2.0 x 105 pfu/mL 和 2.0 x 104 pfu/mL 浓度各 1 mL。这将允许微孔板9 内的感染多重性 （MOI） 为 1、0.1 和 0.01。</li>
	<li>要加载微孔板，取用防雾溶液和琼脂糖制备的板，并将 75 μL 的 2.0 x 106 cfu /mL 细菌移液到 3-10 列中，如图 1 所示。</li>
	<li>如图 1所示，向第3列和第4列中加入75 μL无菌噬菌体缓冲液，作为无噬菌体阳性对照，并在每次加入后上下移液混合。向第 5 列和第 6 列中加入 75 μL 2.0 x 106 pfu/mL 噬菌体，向第 7 列和第 8 列中加入 75 μL 2.0 x 105 pfu/mL 噬菌体，向第 9 列和第 10 列中加入 75 μL 2.0 x 104 pfu /mL 噬菌体，每次加入后上下移液混合。</li>
	<li>用 0.5 英寸的标签胶带将板的两个短边都粘住，以部分密封板，同时允许气体交换。</li>
</ol>5. 孵育和吸光度测量
<ol><li>一旦板装载细菌和噬菌体，将它们以250rpm的速度放在微孔板振荡器上，并在30°C下孵育。</li>
	<li>孵育板96小时，从第16小时开始每8小时在酶标仪上以600nm处进行光密度测量。在测量之间将板放回摇床。 注意:从第0小时开始评估4，h 6 h，8 h和12 h的测量周期，并确定从感染后16小时开始的8小时采样周期最好地捕获 了Gordonia-噬菌体相互作用。</li>
	<li>在整个实验过程中监测盖子是否有冷凝。如果观察到冷凝，请根据步骤3.2用另一个涂覆的盖子更换盖子。</li>
	<li>按照协议第6节生成对照和感染的生长曲线。</li>
</ol>6. 数据分析
<ol><li>使用电子表格程序按照 Stacy-Ceballos-Index GitHub 存储库 （https://github.com/eichristenson/Stacy-Ceballos-Index） 中的电子表格布局计算每种噬菌体稀释液的平均值和标准偏差。</li>
	<li>创建对照和感染生长曲线，并使用 R（请参阅材料表）和 DescTools 24、dplyr 25、ggplot226 和 readxl 27 软件包，并按照 Stacy-Ceballos-Index GitHub 存储库 （https://github.com/eichristenson/Stacy-Ceballos-Index） 中的 R 脚本计算感染指标。<ol><li>AUCcon 是未感染对照曲线下的面积，而 AUCinf 是感染曲线16 下的面积。计算AUC，然后使用以下公式根据曲线下面积值PIAUC16计算生长抑制百分比: （1 - [AUCinf/AUCcon]） × 100</li>
		<li>每条曲线上的水平虚线显示生长峰值，未感染的生长峰标记为N渐近线（con）， 感染的生长峰标记为N渐近线（inf）。 确定 N渐近 线值，然后使用以下公式根据这些峰值生长值 PImax16 计算生长抑制百分比: （1 - [N 渐近线（inf）/ N渐近线（con）]） × 100</li>
		<li>根据 PIAUC 和 PI最大值计算 Stacy-Ceballos 指数 I SC 16，如下所示: （PIAUC ×PI 最大值）0，5 通过整合 Stacy-Ceballos 指数随时间变化来计算相对毒力16.</li>
	</ol></li>
</ol>

使用适应性光密度测定法表征放线杆菌噬菌体感染

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作者没有什么可透露的。

这种基于光密度的微孔板方法允许研究噬菌体宿主范围和感染动力学11，并显示了Stacy-Ceballos指数16作为噬菌体毒力的衡量标准。虽然该方法可以与任何噬菌体宿主系统一起使用，但它专门设计用于适应快速微孔板生长测定9，10，11，以用于生长较慢的细菌，如放线菌。快速微孔板测定不能用于缓慢生长的细菌，除非进行修改以解决蒸发和盖子冷凝。该方法描述了这些必要的修改，并首次证明了使用Stacy-Ceballos指数和相关指标16来描述噬菌体感染。
在多天 96 孔板生长曲线测定中，蒸发可能是一项重大挑战;该方法通过将琼脂糖添加到边界孔和孔之间的空间来解决此问题。琼脂糖边缘与防雾盖处理22相结合，在微孔板内提供必要的湿度，并允许可靠的光密度测量。在不增加湿度的情况下，在所需的漫长孵育期内发生大量的边缘效应蒸发23 ，导致人为的高光密度读数。防雾盖处理是必要的修改，因为盖子冷凝也会人为地提高光密度值。在孵育期间摇动平板是推荐的修改，因为放线菌细菌在生长过程中可能会聚集，从而人为地提供高光密度值并有效降低感染的多重性。
在表征感染动力学的实验中，细菌与噬菌体的比例至关重要，因为必须有足够的噬菌体来显示感染效果，但不能太多，以至于宿主细菌种群立即崩溃9或溶原频率显着增加28。在这种方法中，发现对获得一致结果最有效的比率是MOI为1，但MOI为0.1和0.01时也获得了可用结果。实施该方法时，建议选择一种细菌浓度，并在0.01-19，10，11的MOI范围内测试多种噬菌体浓度。
这里描述的这种技术允许在高通量微孔板测定中评估噬菌体 - 宿主相互作用中生长缓慢的细菌，而不是在每个测量间隔29从较大的培养瓶中子采样。此外，通过演示如何适应微孔板生长测定9，10，11，该技术增加了其他基于微孔板的噬菌体测定对生长较慢的细菌的效用，包括噬菌体表征5，6，12和进化研究30，31。最后，该方法演示了使用Stacy-Ceballos指数16来描述噬菌体感染。该指标最初是使用来自古菌病毒模型系统的数据开发的，并根据光密度值计算得出，因此在不同的病毒系统中具有广泛的实用性。

噬菌体或噬菌体是感染细菌的病毒。它们是地球上数量最多的生物实体1，人们普遍认为噬菌体会影响细菌进化和生态系统过程2，3，4。有几种方法可以描述、测量和分析噬菌体宿主范围8和感染动力学5，6，包括基于琼脂的方法，例如双层琼脂方法7和基于光密度的微孔板方法8，9，10，11，12.每种方法都有自己的优点和缺点。由于其效率，使用双层琼脂法的铺板测试是宿主范围测定的"金标准"，但这种方法是时间和劳动密集型的9。快速微孔板方法可在 24 小时或更短的时间内返回结果，对于快速生长的细菌宿主（例如大肠杆菌9，10，11，12）提供出色的结果，但太短而无法显示生长较慢的细菌宿主（如放线菌7，13，14，15）中的噬菌体感染进展。
专为快速生长的细菌设计的基于光密度的微孔板测定无法运行数天来表征生长缓慢的宿主细菌中的感染动力学，而不会发生蒸发并产生人为的高细菌密度。因此，获得有关缓慢生长细菌物种的噬菌体感染动态的可比高通量数据需要适用于这些细菌的专门技术。
这里介绍的微孔板方法减少了蒸发，从而允许缓慢生长的细菌与噬菌体共培养96小时，并能够研究噬菌体感染动力学和宿主范围。该方法还展示了Stacy-Ceballos指数16，这是一种基于光密度的指标，可以在不同的宿主病毒系统之间进行毒力比较。

<table><tbody><tr><td>Agarose</td><td>Omni-Pur</td><td>2090</td><td>for filling border wells of microplate&amp;nbsp;</td></tr><tr><td>Costar 96 Well Lid Low Evaporation Corner Notch</td><td>Corning</td><td>3931</td><td>replacement microplate lid</td></tr><tr><td>Isopropanol</td><td>Fisher Chemical</td><td>A461-4</td><td>for lid coating</td></tr><tr><td>Microplate reader</td><td>Tecan Spark 20M</td><td></td><td></td></tr><tr><td>Microplate Shaker with 4-Place Platform</td><td>Thermo Fisher Scientific</td><td>88-861-023</td><td>to shake plates during incubation</td></tr><tr><td>Non-Tissue Culture-Treated Plate 96 well</td><td>Falcon (a Corning Brand)</td><td>351172</td><td>microplate for growth curve assay</td></tr><tr><td>Peptone yeast calcium (PYCa) agar</td><td>Homemade</td><td></td><td>1 g peptone&lt;br/&gt; 15 g yeast extract&lt;br/&gt; 15 g agar&lt;br/&gt; 990 mL dd H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 4.5 mL 1 M CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 2.5 mL 40% dextrose&lt;br/&gt; 1 mL 10 mg/mL cycloheximide</td></tr><tr><td>Peptone yeast calcium (PYCa) broth</td><td>Homemade, from Reference 16</td><td></td><td>1 g peptone&lt;br/&gt; 15 g yeast extract&lt;br/&gt; 990 mL dd H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 4.5 ml 1 M CaCl&lt;sub&gt;2 &lt;/sub&gt;&lt;br/&gt; 2.5 mL 40% dextrose&lt;br/&gt; 1 mL 10 mg/mL cycloheximide</td></tr><tr><td>Peptone yeast calcium (PYCa) top agar</td><td>Homemade</td><td></td><td>1 g peptone&lt;br/&gt; 15 g yeast extract&lt;br/&gt; 4 g agar&lt;br/&gt; 990 mL dd H&lt;sub&gt;2&lt;/sub&gt;O&lt;br/&gt; 4.5 mL 1M CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 2.5 mL 40% dextrose</td></tr><tr><td>Petri plates</td><td>Thermo Fisher Scientific</td><td>FB0875713</td><td>for determination of bacterial concentration and phage titer assay</td></tr><tr><td>Phage Buffer</td><td>Homemade, from Reference 7</td><td></td><td>10 mL 1 M Tris, pH 7.5&lt;br/&gt; 10 mL 1 M MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;br/&gt; 4 g NaCl&lt;br/&gt; 980 ml dd H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>R software</td><td>https://www.r-project.org/</td><td>version 4.3.0</td><td></td></tr><tr><td>Sterile Disposable PETG Flask Baffled Bottom w/Vented Closure</td><td>Thermo Fisher Scientific</td><td>4116-1000</td><td>for bacterial culture</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>9036-19-5</td><td>for lid coating</td></tr></tbody></table>

an adapted optical density-based microplate assay for characterizing actinobacteriophage infection

噬菌体是自然环境的关键部分，它们具有塑造细菌种群的强大能力。为了了解单个噬菌体如何与缓慢生长的细菌宿主（如放线菌）相互作用，需要一种简单可靠的方法来量化噬菌体存在下的长期细菌生长。分光光度酶标仪可实现高通量重复测量，但长时间孵育小体积可能会带来技术挑战。该程序适应标准的96孔微孔板，以允许噬菌体和细菌的共同培养而无需子采样96小时，每8小时使用分光光度法吸光度值记录细菌生长。使用R分析这些光密度值以产生感染指标，包括生长抑制百分比，相对毒力和Stacy-Ceballos指数。这里概述的方法提供了一种有效的方法来进行和分析延长持续时间的微孔板生长曲线实验，并包括减少蒸发和盖冷凝的修改。这些协议有助于基于微孔板的缓慢生长细菌宿主与其噬菌体之间相互作用的测定。

如果所得生长曲线显示阳性对照细菌种群随时间增加且吸光度没有突然波动，则实验成功。在MOI为1的情况下，有和没有生产性噬菌体感染的成功实验示例分别显示在图 2 和 图3中。 MOI 为 0.01 的生产性感染如图 4 所示。在所有三个图中都可以看到的阳性对照生长模式（绿色曲线）表明细菌正在生长，它们在生长过程中没有结块，并且不存在污染物。结块和污染表现为单个时间点异常高的吸光度。标准偏差通常会随着实验的时间过程而增加;然而，对照曲线和感染曲线之间的急剧增加或标准偏差重叠可能表明一个或多个孔中存在污染或结块。
描述生产性噬菌体感染的生长曲线（ 如图2所示）显示，添加噬菌体的孔中的细菌吸光度随时间推移而降低。如果细菌超出噬菌体的宿主范围，则不会看到细菌密度的降低，如图 3所示。
显示了所有代表性实验的感染指标，图2和图4中的生产性感染的I SC相对较大，图3中没有有效感染宿主细菌的噬菌体的ISC非常小。 
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65482/65482fig01.jpg"> 图1:微孔板布局。 灰色区域填充0.1%琼脂糖。第 3 列和第 4 列中的空白孔是仅含有噬菌体缓冲液和 2.0 x 106 cfu/mL 细菌的无噬菌体阳性对照孔。虚线孔含有噬菌体和 2.0 x 106 cfu/mL 细菌;第 5 列和第 6 列中的大点表示 MOI 为 1，2.0 x 106 pfu/mL 噬菌体;第 7 列和第 8 列中的培养基点表示 2.0 x 105 pfu/mL 噬菌体的 MOI 为 0.1;第 9 列和第 10 列中的小点表示 MOI 为 0.01，噬菌体为 2.0 x 104 pfu/mL。 <a href="https://www.jove.com/files/ftp_upload/65482/65482fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65482/65482fig02.jpg"> 图 2:使用MOI为1的噬菌体进行成功的生长曲线实验，该噬菌体可有效感染宿主细菌。 显示未感染细菌（绿色）和添加噬菌体的细菌（蓝色）的平均吸光度值（±标准偏差）。缩写:AUC = 曲线下的面积;PIAUC = 根据曲线下面积计算的生长抑制百分比;N渐近 线=峰值生长值;PImax = 根据峰值生长值计算的生长抑制百分比;ISC = Stacy-Ceballos 指数。这张图代表了温带 戈登尼亚 噬菌体DelRio感染G . terrae，它是被分离的细菌。 <a href="https://www.jove.com/files/ftp_upload/65482/65482fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65482/65482fig03.jpg"> 图 3:使用MOI为1的噬菌体进行成功的生长曲线实验，该噬菌体不能有效感染宿主细菌。 显示未感染细菌（绿色）和添加噬菌体的细菌（蓝色）的平均吸光度值（±标准偏差）。缩写:AUC = 曲线下的面积;PIAUC = 根据曲线下面积计算的生长抑制百分比;N渐近 线=峰值生长值;PImax = 根据峰值生长值计算的生长抑制百分比;ISC = Stacy-Ceballos 指数。该图代表DelRio的 G. rubripertincta 感染。 <a href="https://www.jove.com/files/ftp_upload/65482/65482fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65482/65482fig04.jpg"> 图 4:使用 MOI 为 0.01 的噬菌体进行成功的生长曲线实验。 显示未感染细菌（绿色）和添加噬菌体的细菌（蓝色）的平均吸光度值（±标准偏差）。缩写:AUC = 曲线下的面积;PIAUC = 根据曲线下面积计算的生长抑制百分比;N渐近 线=峰值生长值;PImax = 根据峰值生长值计算的生长抑制百分比;ISC = Stacy-Ceballos 指数。这张图代表DelRio的 G. terrae 感染。 <a href="https://www.jove.com/files/ftp_upload/65482/65482fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection at JoVE.com

作者聚焦:一种用于表征放线菌噬菌体感染的基于光密度的微孔板检测  

描述了一种基于光密度的微孔板方法，用于量化噬菌体存在下的长期细菌生长，适用于放线菌和其他生长缓慢的细菌。此方法包括减少蒸发和盖冷凝的修改，以及用于计算病毒感染指标的 R 代码，包括曲线下面积、生长最大值和相对毒力。

一种适用于表征放线菌噬菌体感染的基于光密度的微孔板测定

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual ...

最终，我们对噬菌体 - 宿主相互作用感兴趣。该协议能够测量生长缓慢的细菌中的噬菌体感染，生成描述性的感染指标。该方法有助于研究生长缓慢的放线菌细菌中的噬菌体-宿主协同进化。快速微孔板方法对快速生长的细菌宿主提供了极好的结果，但太短，无法显示生长较慢的细菌宿主（如放线菌）中的细菌噬菌体感染进展。我们的方法适用于生长缓慢的细菌，允许细菌和噬菌体在表征噬菌体感染动态所需的多天内共培养。我们的协议允许评估噬菌体 - 宿主相互作用以评估微孔板中生长缓慢的细菌，而不是要求从较大的培养瓶中进行子采样。该方案使用现成的材料来适应延长的培养时间，并允许在单个微孔板中接种多个重复。

an-adapted-optical-density-based-microplate-assay-for-characterizing

Research

JoVE Journal

Biology

An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection

2.5K 次观看.  Ouachita Baptist University. 最终，我们对噬菌体 - 宿主相互作用感兴趣。该协议能够测量生长缓慢的细菌中的噬菌体感染，生成描述性的感染指标。该方法有助于研究生长缓慢的放线菌细菌中的噬菌体-宿主协同进化。快速微孔板方法对快速生长的细菌宿主提供了极好的结果，但太短，无法显示生长较慢的细菌宿主（如放线菌）中的细菌噬菌体感染进展。我们的方法适用于生长缓慢的细菌，允许细...

视频: 作者聚焦:一种用于表征放线菌噬菌体感染的基于光密度的微孔板检测  

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases' is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease's inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant "light-tagged" reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage ΦA1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage Wβ::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Y. pestis interaction with luxAB phage diagram; bacterial luminescence gene process." src="/files/ftp_upload/2740/2740fig1.jpg" data-altupdatedat="1761809329000" data-alt="Figure 1"> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

用于检测 A 类细菌病原体的"生物发光"报告噬菌体

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

"发光法"的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

科研

免疫与感染

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

用于评估巨噬细胞吞噬作用和肌动蛋白聚合的高通量荧光技术

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

噬菌体表型组学：表征新型病毒蛋白的生理学方法

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学:生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

生物淘选 T7 噬菌体的定量 PCR

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

以此 T7 噬菌体的定量 PCR 研究

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant bacteria, novel antibacterial agents, are urgently needed. A bacteriophage-based biocontrol strategy is the therapeutic use of phages to control bacterial pathogens in agricultural settings. Phage biocontrol is increasingly accepted as a sustainable technology, effective at decontaminating foodborne pathogens. To ensure effective biocontrol outcomes, systematic screening of phage combinations against targeted bacteria under required environmental conditions is crucial. Antibacterial efficacy of phage cocktails may be affected by phage genera and combination, targeted bacterial strains, the multiplicity of infection, temperature, and time. To formulate a phage cocktail with superior efficacy, the proposed method was to systematically evaluate the effectiveness of individual phages and phage cocktails in killing foodborne bacterial pathogens under targeted conditions. Bacterial killing efficacy was monitored by measuring optical density at desired temperatures and durations. Superior phage efficacy was determined by complete inhibition of bacterial growth. The proposed method is a robust, evidence-based approach to facilitate formulating phage cocktails with superior antibacterial efficacy.

通过高通量设置评估噬菌体对食源性病原体的生物防治效果

This protocol describes a robust method for using high-throughput settings to screen the antibacterial efficacy of bacteriophage cocktails.

噬菌体对食源性病原体生物防治的有效性通过高通量设置进行评估

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant ...

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the detection cost, an intelligent high-throughput AST/phage screening system, including a 96-dot matrix inoculator, image acquisition converter, and corresponding software, was developed according to AST criteria and the breakpoints of resistance (R) formulated by the Clinical &amp; Laboratory Standards Institute (CLSI). AST and statistics of minimum inhibitory concentration (MIC) distributions (from R/8 to 8R) of 1,500 Salmonella strains isolated from poultry in Shandong, China, against 10 antimicrobial agents were carried out by the intelligent high-throughput AST/phage screening system. The Lar index, meaning "less antibiosis, less resistance and residual until little antibiosis", was obtained by calculating the weighted average of each MIC and dividing by R. This approach improves accuracy in comparison with using the prevalence of resistance to characterize the antimicrobial resistance (AMR) degree of highly resistant strains. For the strains of Salmonella with high AMR, lytic phages were efficiently screened from the phage library by this system, and the lysis spectrum was computed and analyzed. The results showed that the intelligent high-throughput AST/phage screening system was operable, accurate, highly efficient, inexpensive, and easy to maintain. Combined with the Shandong veterinary antimicrobial resistance monitoring system, the system was suitable for scientific research and clinical detection related to AMR.

智能高通量药敏检测/噬菌体筛选系统及抗菌素耐药性 Lar 指数的应用

Here we introduce the principle, structure, and instruction of the intelligent high-throughput antimicrobial sensitivity testing/phage screening system. Its application is illustrated by using Salmonella isolated from poultry in Shandong, China, as an example. The Lar index is calculated, and its significance in evaluating antimicrobial resistance is discussed comprehensively.

智能高通量抗菌药敏检测/噬菌体筛选系统及Lar耐药性指数的应用

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the ...

环境科学

使用 T4 噬菌体探索小鼠模型中的肠道噬菌体-细菌动力学

T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host&#39;s health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli,&#160;in the context of the murine gastrointestinal tract. The methods described here outline&#160;how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels.&#160;This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

作者聚焦:研究噬菌体诱导的 Gnotobiotic 小鼠免疫反应

Bacteriophages (phages), viruses that infect bacteria, are an integral component of the gut microbiome. Though these symbiotic inhabitants drive bacterial fitness and population dynamics, little is understood about how they impact gut homeostasis and disease. This protocol studies isolated T4 phages within a mouse model, adaptable to other phage-bacterial pairs.

小鼠肠道中 T4 噬菌体和 大肠杆菌 相互作用:研究体内宿主-噬菌体动力学的原型模型

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across ...

<meta charset="utf8"></head><body>可视化细菌病原体中的抗生素持久性

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to&#160;underlie clinical treatment failure.&#160;Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited.
This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus)&#160;during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.

<meta charset="utf8"></head><body>铜绿假单胞菌和金黄色葡萄球菌异质表型的延时落射荧光显微镜成像

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize&#160;phenotypic variants, such as persisters, by time-lapse epifluorescence&#160;microscopy.

铜绿假单胞菌和金黄色葡萄球菌异质表型的延时落射荧光显微镜成像

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment ...

生物学

Environmental Sampling of Photosynthetic Microbes and Their Viruses: From Field to Lab

Environmental sampling of photosynthetic microorganisms and their viruses plays a critical role in understanding contemporary marine and freshwater biodiversity and ecosystem dynamics as well as the impacts of climate change-related factors (e.g., rising temperatures and acidification) on evolutionary trajectories of species and community composition. Unfortunately, the diversity of the virosphere does not support a single universal sampling and experimental workflow. Indeed, each virus system has unique features, which require modifications to standard protocols in virology to accomplish research goals. Although virus discovery and characterization require approaches that are specific to the target system, for all viruses, the research aims are similar: isolate the virus; determine host range; confirm productive infection; and characterize the virus, the host, and virus-host dynamics. Robust descriptions of virus-host systems consist minimally of elucidating morphology, physiology, biochemistry, and omics profiles. Further information may be obtained by manipulating the system by changing factors such as multiplicity of infection, temperature, pH, host-switch, directed evolution, or applying drugs to observe virus-host system response. Our laboratory studies viruses across domains of life (Archaea, Bacteria, and Eukarya). In this report, we detail methods for sampling photosynthetic microbes from the euphotic zone of freshwater and marine environments with focus on isolating bacteriophage (i.e., cyanophage) of cyanobacteria. Cyanobacteria are keystone species critical to primary production and nutrient cycling in these aquatic ecosystems. The described workflow extends from sampling waters at different depths to characterizing virus-host system features using liquid and solid media culture, advanced molecular/genetic methods, and analytical approaches. The methods described are adaptable to bacteriophage and virus discovery in virus-host systems across domains of life.

Here, we describe methods of sampling from the euphotic zones of marine and freshwater ecosystems to isolate cyanophages (and their hosts) and post-sampling processes that enrich single genotypes and characterize virus-host interaction dynamics.

光合微生物及其病毒的环境采样：从田间到实验室

Environmental sampling of photosynthetic microorganisms and their viruses plays a critical role in understanding contemporary marine and freshwater ...

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

显微镜的正确保养和清洁

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

一种适用于表征放线菌噬菌体感染的基于光密度的微孔板测定

Transcript

Summary

Explore More Videos

"发光法"的分类检测细菌病原体的记者噬菌体

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

噬菌体型组学:生理途径来表征新型病毒蛋白

以此 T7 噬菌体的定量 PCR 研究

噬菌体对食源性病原体生物防治的有效性通过高通量设置进行评估

智能高通量抗菌药敏检测/噬菌体筛选系统及Lar耐药性指数的应用

小鼠肠道中 T4 噬菌体和大肠杆菌相互作用:研究体内宿主-噬菌体动力学的原型模型

铜绿假单胞菌和金黄色葡萄球菌异质表型的延时落射荧光显微镜成像

光合微生物及其病毒的环境采样：从田间到实验室

适当的照顾和清洁的显微镜

一种适用于表征放线菌噬菌体感染的基于光密度的微孔板测定

Transcript

Summary

Explore More Videos

"发光法"的分类检测细菌病原体的记者噬菌体

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

噬菌体型组学:生理途径来表征新型病毒蛋白

以此 T7 噬菌体的定量 PCR 研究

噬菌体对食源性病原体生物防治的有效性通过高通量设置进行评估

智能高通量抗菌药敏检测/噬菌体筛选系统及Lar耐药性指数的应用

小鼠肠道中 T4 噬菌体和 大肠杆菌 相互作用:研究体内宿主-噬菌体动力学的原型模型

铜绿假单胞菌和金黄色葡萄球菌异质表型的延时落射荧光显微镜成像

光合微生物及其病毒的环境采样：从田间到实验室

适当的照顾和清洁的显微镜

小鼠肠道中 T4 噬菌体和大肠杆菌相互作用:研究体内宿主-噬菌体动力学的原型模型