Name: visualizing bacterial motility based on a color reaction
Uploaded: 2022-02-15T15:00:00.000+00:00
Duration: 4 min 44 s
Description: Watch this Scientific Journal Video about Visualizing Bacterial Motility Based on a Color Reaction at JoVE.com

本研究得到了江苏省高校优先学术项目开发（PAPD）和中国药科大学教学改革研究项目（2019XJYB18）的支持。

1. 半固体培养基的制备
<ol><li>传统半固体琼脂
	<ol><li>根据细菌运动测试培养基配方，使用基本成分制备传统的半固体琼脂11。将 10 g 色糖、15 g NaCl、4 g 琼脂溶解在足够的蒸馏水中，将 pH 值调节至 7.2 ± 0.2，并将最终体积补足至 1，000 mL。</li>
		<li>将琼脂在121°C高压灭菌20分钟，并将其作为3cm高的半固体培养基分配到10mL试管中。</li>
	</ol></li>
	<li>含TTC的传统半固体琼脂
	<ol><li>将常规半固体培养基高压灭菌后，冷却至50°C，将5mL无菌1%TTC溶液加入100mL培养基中，混合，并将其分配到10 mL试管中，形成3cm高的半固体培养基。</li>
	</ol></li>
</ol>2. 细菌菌株
注意:从水生环境中分离出80株菌株，并使用自动细菌鉴定仪器（见材料表）进行鉴定，包括大肠杆菌，铜绿假单胞菌，沙门氏菌属，弧菌属，肺炎克雷伯菌和嗜水气单胞菌（表1）。 金黄色葡萄球菌（见材料表）被用作阴性非运动对照;大肠杆菌、铜绿假单胞菌和鼠伤寒沙门氏菌（见材料表）用作阳性对照菌株。
<ol><li>确定用于运动分析的测试细菌菌株。</li>
	<li>包括阴性非运动对照和运动阳性对照菌株。</li>
</ol>3. TTC增强细菌运动观察
<ol><li>从琼脂平板中挑选测试细菌的单个菌落，并使用接种针穿刺将它们接种到上述两种半固体培养基中（步骤1.1.2和1.2.1）。</li>
	<li>将管在培养箱中于37°C培养24-48小时以观察结果。</li>
	<li>观察生长状态:如果只有穿刺线是红色的，则将细菌表征为非运动（-）。如果红色沿穿刺线12 向外轻微扩散，则将细菌表征为运动 （+）。</li>
</ol>4. 不同琼脂浓度对细菌运动的影响
<ol><li>如上所述，制备含有0.3%，0.5%和0.8%琼脂的半固体培养基，并通过穿刺接种它们。孵育24-48小时后观察结果。</li>
</ol>

<ol>
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作者没有利益冲突需要披露。

半固体培养基法细菌运动的检测受许多因素13，14的影响。细菌生长条件，例如氧气（琼脂表面有氧，半固体培养基在管底部无氧），pH和温度，会影响细菌鞭毛的活力，从而导致运动能力降低甚至运动力丧失15。此外，一些粘液型细菌作为其运动性会受到鬼臼结合物产生的影响。 
将TTC添加到半固体介质中有助于观...

细菌运动在细菌致病性、生物膜形成和耐药性中起关键作用1。细菌运动性与致病性密切相关，是宿主细胞早期感染期间细菌定植所必需的2。生物膜的形成与细菌运动密切相关，细菌通过运动粘附在固体培养基的表面。长期以来，人们一直认为细菌运动与生物膜形成呈正相关。由于生物膜引起的高度细菌耐药性可导致对人类健康构成威胁的持续感染3，4，5。因此，检测细菌运动非常重要。细菌运动性测试主要用于检查不同形式细菌在生活状态下的运动性，可以间接确定鞭毛的存在与否，因此对细菌的鉴定具有重要作用。
有直接和间接的方法来检测细菌运动6。由于带有鞭毛的细菌表现出运动性，因此可以通过检测是否存在鞭毛来间接检测细菌是否具有运动性。例如，可以通过电子显微镜和鞭毛染色间接检测运动性，以表明细菌是运动的。也可以通过直接方法进行检测，例如悬浮液滴和半固体穿刺方法。 
本科微生物学实验室常用的半固体穿刺法检测细菌运动性，根据细菌生长方向，将细菌接种到含有0.4-0.8%琼脂的半固体琼脂培养基中穿刺中。如果细菌沿着穿刺线生长到周围扩散，则会出现云状（刷状）生长痕迹，表明存在鞭毛，因此存在运动性。如果没有穿刺线生长痕迹，则细菌既不鞭毛也不运动。 
然而，这种方法有其缺点:细菌无色透明，鞭毛活性受活细菌的生理特性和其他因素的影响，以及琼脂的浓度和试管的小直径。而且，需氧菌只适合在琼脂表面生长，影响细菌运动的观察。因此，为了改进该实验，将2，3，5-三苯基氯化四唑（TTC）（无色）添加到培养基中，以建立比目前使用细胞内脱氢酶催化形成TTC7，8，9，10红色产物的直接穿刺方法更可靠，更直观的细菌运动性测定方法。

<table><tbody><tr><td>Bacto Agar</td><td>Difco</td><td></td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>ATCC</td><td>ATCC25922</td><td>Positive control</td></tr><tr><td>&lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt;</td><td>ATCC</td><td>ATCC27853</td><td>Positive control</td></tr><tr><td>&lt;em&gt;Salmonella typhimurium&lt;/em&gt;</td><td>ATCC</td><td>ATCC14028</td><td>Positive control</td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt;</td><td>ATCC</td><td>ATCC25923</td><td>Negative nonmotile control</td></tr><tr><td>Tryptose</td><td>&amp;nbsp;OXOID</td><td></td><td></td></tr><tr><td>TTC</td><td>Sigma</td><td>298-96-4</td><td></td></tr><tr><td>VITEK 2 automated microbial identification system</td><td>Bio M&amp;eacute;rieux</td><td></td><td></td></tr></tbody></table>

visualizing bacterial motility based on a color reaction

细菌运动性对细菌致病性、生物膜形成和耐药性至关重要。细菌运动对于许多致病物种的入侵和/或传播至关重要。因此，检测细菌运动非常重要。细菌生长条件，如氧气、pH和温度，会影响细菌的生长和细菌鞭毛的表达。这可能导致运动能力降低甚至失去运动能力，导致对细菌运动性的评估不准确。基于2，3，5-三苯基氯化四唑（TTC）与活细菌细胞内脱氢酶的显色反应，将TTC添加到传统的半固体琼脂中用于细菌运动检测。结果表明，该TTC半固体琼脂法检测细菌运动性简单，操作方便，不涉及大型且昂贵的仪器。结果还表明，在用0.3%琼脂制备的半固体培养基中观察到最高的运动性。与传统的半固体介质相比，结果更容易评估，也更准确。

比较标准菌株和分离菌株的运动检测，结果如 表1所示。由于没有鞭毛， 金黄色葡萄球菌 和 肺炎克雷伯菌 仅在传统和TTC半固体培养基上沿接种线生长。相比之下， 铜绿假单胞菌、 大肠杆菌 和 鼠伤寒沙门氏菌 在TTC半固体培养基上培养24 h后，在接种系周围各方向均显示生长。培养48小时后，这一点更加明显（图1）。尽管细菌?...

Watch this Scientific Journal Video about Visualizing Bacterial Motility Based on a Color Reaction at JoVE.com

Visualizing Bacterial Motility Based on a Color Reaction

在这里，我们提出了一种基于颜色反应检测细菌运动的方案。这种方法的主要优点是易于评估且更准确，并且不需要专门的设备。

基于颜色反应可视化细菌运动

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

visualizing-bacterial-motility-based-on-a-color-reaction

Research

JoVE Journal

Biology

3.9K 次观看.  China Pharmaceutical University. 在这里，我们提出了一种基于颜色反应检测细菌运动的方案。这种方法的主要优点是易于评估且更准确，并且不需要专门的设备。

视频: Visualizing Bacterial Motility Based on a Color Reaction

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

一个微流体装置量化细菌趋稳定的浓度梯度

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

科研

生物学

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

量化诱导剂梯度板上细菌表面聚集运动

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

生物化学

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

生成受控动态化学景观，研究微生物行为

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

生物工程

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光抗生素探针对细菌耐药性的可视化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

免疫与感染

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

单细胞物种间细菌相互作用的动力学可视化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

准备，成像和定量的细菌表面运动性测定

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

鞭毛马达功能的生物物理表征

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

通过在系列中应用三种既定技术，调查大 肠杆菌 的弗拉盖拉驱动的动性

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Source: Smith, S., et al., <a href="https://app.jove.com/v/62851">Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging.</a> J. Vis. Exp. (2021).
This video demonstrates a fluorescence microscopy-based method to study bacterial competition at the single-cell level. This method provides insights into the structure and function of bacterial communities.

基于颜色反应可视化细菌运动

摘要

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此视频中的章节

一个微流体装置量化细菌趋稳定的浓度梯度

量化诱导剂梯度板上细菌表面聚集运动

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

生成受控动态化学景观，研究微生物行为

使用荧光抗生素探针对细菌耐药性的可视化

单细胞物种间细菌相互作用的动力学可视化

准备，成像和定量的细菌表面运动性测定

鞭毛马达功能的生物物理表征

通过在系列中应用三种既定技术，调查大肠杆菌的弗拉盖拉驱动的动性

细菌共孵育试验:一种基于荧光显微镜的技术，可在单细胞水平上可视化种内细菌竞争

基于颜色反应可视化细菌运动

摘要

探索更多视频

此视频中的章节

一个微流体装置量化细菌趋稳定的浓度梯度

量化诱导剂梯度板上细菌表面聚集运动

从 酿酒酵母 细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

生成受控动态化学景观，研究微生物行为

使用荧光抗生素探针对细菌耐药性的可视化

单细胞物种间细菌相互作用的动力学可视化

准备，成像和定量的细菌表面运动性测定

鞭毛马达功能的生物物理表征

通过在系列中应用三种既定技术，调查大 肠杆菌 的弗拉盖拉驱动的动性

细菌共孵育试验:一种基于荧光显微镜的技术，可在单细胞水平上可视化种内细菌竞争

从酿酒酵母细胞中纯化的双向驱动蛋白-5 Cin8的单分子和簇的运动性

通过在系列中应用三种既定技术，调查大肠杆菌的弗拉盖拉驱动的动性