Name: a rapid image-based bacterial virulence assay using amoeba
Uploaded: 2018-06-27T15:00:00
Description: Watch this Scientific Journal Video about A Rapid Image-based Bacterial Virulence Assay Using Amoeba at JoVE.com

KP 和写和修订的手稿。KP 进行了实验和分析。这项工作得到了国家卫生研究院 (NIH) 职业过渡奖 (K22AI112816) 的支持。

所有实验程序都是在加利福尼亚大学欧文分校进行的。
1. 缓冲器和解决方案
<ol><li>在1升玻璃瓶中加入25克的溶源性汤 (磅), 在1升的双蒸馏水 (ddH2O) 中添加一个磅米勒混合物。对于培养皿, 添加额外的20克琼脂。灭菌釜。将25毫升的熔融琼脂培养基倒入10厘米直径的培养皿中, 并允许室温固化。将液体介质存放在室温和琼脂板上, 温度为4摄氏度。</li>
	<li>在1升玻璃瓶中混...

<ol>
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本协议描述一种快速、定量的方法来测定铜绿假单胞菌的毒力。此协议可与其他细菌进行测试。然而, 重要的是要记住, 生长培养基应与变形虫生长条件兼容。特别是, 我们已经优化的协议使用 PS: DB 作为细菌生长培养基。如果使用其他介质, 则可能需要执行仅生长介质控制, 在该控件中没有细菌细胞来验证介质是否与阿米巴兼容。
这一方法可以扩展, 以测定毒力的生长阶...

细菌感染是人类和动物死亡的主要原因之一,1,2。检测细菌在培养或生物膜中的毒力的能力在医疗保健和研究环境中很重要。在这里, 我们描述了一种通用的, 快速的, 相对简单的方法来量化细菌的毒性。以真核生物盘基柄柄(变形虫) 为模型宿主有机体。柄已被用作鉴定铜绿假单胞菌致病因子的宿主 (绿脓杆菌)3,4,5和其他细菌6,7 ,8 , 并且很容易受到同样的毒力因素的影响, 杀死哺乳动物细胞, 包括 III. 型分泌物9,10。以前使用柄的毒力测定, 在3、4、5小时的过程中, 会使细菌与d. 柄细胞长期接触。在这里, 该协议提出了一种快速的方法来确定毒力使用这种变形虫。本协议 (图 1) 描述了如何: (1) 生长阿米巴 axenically (在没有细菌的情况下), (2) 为化验培养细菌, (3) 为显微镜准备细菌和宿主细胞, (4) 执行荧光显微术, (5) 分析变形虫荧光。
阿米巴最初是从冰冻的储存和生长在大肠杆菌 (大肠杆菌) 的草坪上, 阿米巴产生孢子。这些孢子被采摘并接种成富含培养基无菌生长。阿米巴是通过无菌生长营养丰富的条件来维持的, 直到它们准备与细菌混合以评估细菌的毒力。阿米巴的生存或死亡通过测量 calcein-乙酰甲基 (calcein AM) 的荧光量量化, 这是由细胞内酯, 从而激活荧光11,12。活阿米巴表现很少或没有荧光, 而压力和垂死的细胞荧光强烈。这一结果是由于很少或没有纳入 calcein 的健康阿米巴和成立和分裂的基质在强调阿米巴13。这种行为明显有别于哺乳动物细胞11、14、15、16的 calcein 荧光。
将被评估为毒力的细菌分别生长。在这里, 我们描述如何测量机会病原菌的毒力, 并详细说明如何量化的毒性的浮游 (游泳) 和表面附着的亚人口。本议定书可用于测试其他细菌的毒力。在有代表性的结果部分, 我们表明, 毒力是激活的表面附着细胞和低的浮游细胞, 这是以前报告13。毒力活化的表面附着的铜绿假单胞菌杀死阿米巴, 而非毒性浮游细胞消耗的阿米巴。如果浮游细菌的毒力仅被测定, 细菌可以在普通的试管中培养, 而不是按照议定书中所描述的那样使用培养皿。
阿米巴和铜绿假单胞菌培养的生长必须协调, 以使铜绿假单胞菌培养达到预期的生长阶段, 而阿米巴在营养丰富的条件下生长在稳定的状态。这种情况通常要求阿米巴文化在与细菌混合之前至少1天被稀释。阿米巴和细菌是固定使用琼脂垫, 共孵化1小时, 并成像使用低分辨率 (10X, 数字光圈 0.3) 目标, 绿色荧光蛋白 (GFP) 过滤器, 和成像相机。可以使用自由可用的 ImageJ 软件或自定义图像分析软件进行分析。我们的分析是使用我们自己的软件编写的, 使用一个科学的分析包13。该软件应创建一个面具使用相对比图像和提取荧光值从蒙面区域的荧光图像。荧光值平均在至少100个细胞, 导致一个数字主机杀死指数。

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			<td height="21" style="height:21px;width:220px;">Reagents</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Bacto agar, dehydrated</td>
			<td style="width:108px;">BD Difco</td>
			<td style="width:91px;">214010</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Antibiotic-Antimycotic (100X)</td>
			<td style="width:108px;">Life Technologies</td>
			<td style="width:91px;">15240062</td>
			<td style="width:297px;">Aliquot &#60; 1 mL and store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Calcein-acetoxymethyl ester (calcein-AM)</td>
			<td style="width:108px;">Life Technologies</td>
			<td style="width:91px;">C34852</td>
			<td style="width:297px;">Calcein Acetoxymethyl (AM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Calcium chloride, anhydrous</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">C1016</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">D-Glucose</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">D16500</td>
			<td style="width:297px;">Dextrose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Dimethyl sulfoxide</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">D5879</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Folic acid</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">F8758</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">LB-Miller</td>
			<td style="width:108px;">BD Difco</td>
			<td style="width:91px;">244620</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Magnesium chloride</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">M8266</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Yeast extract</td>
			<td style="width:108px;">Oxoid</td>
			<td style="width:91px;">LP0021</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Special peptone</td>
			<td style="width:108px;">Oxoid</td>
			<td style="width:91px;">LP0072</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Potassium hyroxide</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">P250</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Potassium phosphate monobasic&#160;</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">P0662</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Sodium phosphate dibasic heptahydrate</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">S373</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Vitamin B12</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">V2876</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Strains</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:220px;">Dictyostelium discoideum</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">Strain AX318</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Escherichia coli</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">Strain B/r17</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Pseudomonas aeruginosa</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">PA14</td>
			<td style="width:297px;">PA14 strain19</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Pseudomonas aeruginosa &#916;lasR</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">AFS20.1</td>
			<td style="width:297px;">PA14-derived strain20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Supplies</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">0.22 &#181;m filter&#160;</td>
			<td style="width:108px;">Millipore</td>
			<td style="width:91px;">SCGPT01RE</td>
			<td style="width:297px;">For filter sterilization</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:220px;">Conical tube, 15 mL</td>
			<td style="width:108px;">Corning</td>
			<td style="width:91px;">352097</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Glass storage bottles</td>
			<td style="width:108px;">Pyrex</td>
			<td style="width:91px;">13951L</td>
			<td style="width:297px;">250 mL, 500 mL, 1000 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Petri dish, 6 cm diameter</td>
			<td style="width:108px;">Corning</td>
			<td style="width:91px;">351007</td>
			<td style="width:297px;">60 x 15 mm polystyrene plates</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:220px;">Petri dish, 10 cm diameter</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">FB0875712</td>
			<td style="width:297px;">100 x 15 mm polystyrene plates</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:220px;">Plastic containers with lid</td>
			<td style="width:108px;">Ziploc</td>
			<td style="width:91px;">2.57E+09</td>
			<td style="width:297px;">Square 3-cup containers</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:220px;">Glass plates</td>
			<td style="width:108px;">Bio-Rad</td>
			<td style="width:91px;">1653308</td>
			<td style="width:297px;">For preparing agar pads. Other glass plates may be used with similar dimensions.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Wooden sticks</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">23-400-102&#160;</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Eclipse Ti-E microscope&#160;</td>
			<td style="width:108px;">Nikon</td>
			<td style="width:91px;">MEA53100</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">10X Plan Fluor Ph1 objective&#160; 0.3 NA</td>
			<td style="width:108px;">Nikon</td>
			<td style="width:91px;">MRH20101</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Fluorescence excitation source</td>
			<td style="width:108px;">Lumencor</td>
			<td style="width:91px;">Sola light engine</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:220px;">Fluorescence filter set</td>
			<td style="width:108px;">Semrock</td>
			<td style="width:91px;">LED-DA/FI/TX-3X3M-A-000&#160;</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Orca Flash 4.0 V2 Camera</td>
			<td style="width:108px;">Hamamatsu</td>
			<td style="width:91px;">77054098</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">ImageJ</td>
			<td style="width:108px;">NIH</td>
			<td style="width:91px;">v. 1.49</td>
			<td style="width:297px;">Software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">MATLAB</td>
			<td style="width:108px;">Mathworks</td>
			<td style="width:91px;">R2013</td>
			<td style="width:297px;">Software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Orbital shaker incubator</td>
			<td style="width:108px;">VWR</td>
			<td style="width:91px;">89032-092</td>
			<td style="width:297px;">For growth of bacteria at 37 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Platform shaker</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">13-687-700</td>
			<td style="width:297px;">For growth of amoebae at 22 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Spectrophotometer</td>
			<td style="width:108px;">Biochrom</td>
			<td style="width:91px;">Ultrospec 10</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Undercounter refrigerated incubator</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">97990E</td>
			<td style="width:297px;">For growth of amoebae at 22 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Isotemp waterbath&#160;</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">15-462-21Q</td>
			<td style="width:297px;">For cooling media to 55 &#176;C</td>
		</tr>
	</tbody>
</table>

a rapid image-based bacterial virulence assay using amoeba

传统的细菌毒力检测包括在几个小时内长期暴露细菌, 以宿主细胞。在这段时间内, 由于接触宿主生长环境和宿主细胞的存在, 细菌可以经受生理变化。我们开发了一种快速测量细菌的毒力状态的检测方法, 以最大限度地减少细菌在宿主细胞存在时的生长程度。细菌和阿米巴混合在一起, 并固定在一个单一的成像平面上使用琼脂垫。该程序使用单细胞荧光成像与 calcein-乙酰甲基酯 (calcein) 作为一个指标的宿主细胞健康。利用荧光显微术对宿主细胞的1小时暴露后的宿主细胞进行荧光分析。图像分析软件用于计算主机的杀戮指数。该方法用于测定生物膜形成初期浮游和表面附着铜绿假单胞菌亚群中的毒力, 并可适应其他细菌和生物膜生长的其他阶段。该协议提供了一种快速和稳健的方法来测量毒力, 并避免了许多与生长和维护哺乳动物细胞系相关的复杂性。使用阿米巴测定的毒力表型也得到了使用小鼠巨噬细胞的验证。特别是, 这一检测被用来建立的表面附着上调毒力在铜绿假单胞菌。

我们在6厘米直径的培养皿中生长野生型铜绿假单胞菌 PA1419或ΔlasR菌株20 , 并测定浮游和表面附着细胞的毒力。文化从单一殖民地接种了入 PS: DB 文化, 在养殖管子隔夜生长在37°c 到饱和, 稀释1:100 入 ps: DB, 增长 8 h 在 6 cm 直径培养皿颤抖在100转, 并且浮游物和表面附上铜绿假单胞菌亚群被隔离, 如3节所述。
<p class="jo...

Watch this Scientific Journal Video about A Rapid Image-based Bacterial Virulence Assay Using Amoeba at JoVE.com

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

一种基于变形虫的快速图像细菌毒力检测方法

在这里, 我们提出了一个协议, 以测量浮游或表面附着细菌的毒力, 使用柄(变形虫) 作为宿主。在1小时内对毒力进行测量, 并使用荧光显微镜和图像分析对宿主杀死进行量化。我们用铜绿假单胞菌证明了这个协议。

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Research

JoVE Journal

Bioengineering

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

使用辅助电喷雾质谱（SESI - MS）表征细菌挥发

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

科研

生物工程

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Bacterial Detection & Identification Using Electrochemical Sensors

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

医疗器械的使用3D印刷模具的液体注射成型快速，低成本原型

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

活细胞层的快速消减带图案微流控探头

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

一种快速和无农药血红蛋白检测法与光热角光散射

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

仿生纳米羟基磷灰石的快速混合制备生物医学应用

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

高通量识别对伪单鱼的电阻syringae pv。番茄中的番茄使用苗子洪水测定

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

使用模块化金门克隆的多基因结构快速组装

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

通过交替粘性 - 惯性力喷射进行 体外 水凝胶微载体的3D打印

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...