この研究は、江蘇省高等教育機関の優先学術プログラム開発(PAPD)および中国薬科大学の教育改革研究プロジェクト(2019XJYB18)の支援を受けました。

1.半固体培地の調製
<ol><li>従来の半固体寒天
	<ol><li>基本成分11を使用して、細菌運動性試験培地レシピに従って伝統的な半固体寒天を調製する。トリプトース10 g、NaCl15 g、寒天4 gを十分な蒸留水に溶解し、pHを7.2±0.2に調整し、最終容量を1,000 mLにします。</li>
		<li>寒天を121°Cで20分間オートクレーブし、高さ3cmの半固体培地として10mLの試験管に分注します。</li>
	</ol></li>
	<li>TTCを含む従来の半固体寒天
	<ol><li>従来の半固体培地をオートクレーブした後、50°Cに冷却し、100mLの培地に5mLの滅菌1%TTC溶液を加えて混合し、10mLの試験管に分注して高さ3cmの半固体培地を形成します。</li>
	</ol></li>
</ol>2.細菌株
注:水生環境から80株を分離し、大腸菌、緑膿菌、サルモネラ属菌、ビブリオ属菌、肺炎桿菌、およびアエロモナス親水性菌を含む自動細菌識別装置(資料表を参照)を使用して同定しました(表1)。黄色ブドウ球菌(材料の表を参照)は、負の非運動性対照として使用されました。大腸菌、緑膿菌、およびネズミチフス菌(材料表参照)をポジティブコントロール株として使用しました。
<ol><li>運動性分析に使用する試験細菌株を特定します。</li>
	<li>負の非運動性対照および運動性陽性対照株を含める。</li>
</ol>3. TTC増強細菌運動性観察
<ol><li>寒天プレートから被験菌の単一コロニーを選び、接種針を用いた穿刺により、上記の2つの半固体培地(ステップ1.1.2および1.2.1)に接種します。</li>
	<li>チューブをインキュベーター内で37°Cで24〜48時間培養し、結果を観察した。</li>
	<li>成長状態を観察する:穿刺線だけが赤の場合は、細菌を非運動性(-)として特徴付けます。赤色が穿刺線12に沿ってわずかに外側に広がる場合、細菌を運動性(+)として特徴付ける。</li>
</ol>4. 寒天濃度の違いが細菌の運動に及ぼす影響
<ol><li>0.3%、0.5%、0.8%の寒天を含む半固体培地を調製し、上記のように穿刺して接種する。インキュベーションの24-48時間後に結果を観察してください。</li>
</ol>

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著者は開示する利益相反を持っていません。

半固体培地法による細菌の運動性の検出は、多くの要因の影響を受けます13,14。酸素(寒天表面での好気性、半固体培地を含むチューブの底部での非好気性)、pH、温度などの細菌増殖条件は、細菌のべん毛の生存率に影響を与える可能性があり、運動性の低下または運動性の喪失につながる可能性があります15。さらに、それらの運動性としてのいくつかの粘液型細菌は、ポドコンジュゲートの産生によって影響を受ける可能性がある。
半固体培地へのTTCの添加は、運動性の観察に役立つ。原動力を持つ発酵細菌は、インキュベーション後に穿刺線に沿ってあらゆる方向に成長する可能性があります。したがって、穿刺線の周りの媒体は赤くなります。原動力を有する非発酵性細菌は、培地下部の酸素含有量が低い。したがって、これらの細菌は増殖が乏しいため、培地の上層のみが赤くなります。原動力のない細菌は接種ライン上でしか増殖できず、穿刺ラインのみが赤く見えます。 
TTC半固体培地で細菌の運動性を検出する場合、培養時間が長いほど、特に寒天濃度が低い場合の結果はより明白になります。結果の解釈が困難な場合は、培養時間を適切に延長する必要があります。これは、半固体培地の寒天濃度、菌数、およびそれらの運動性16に関係している可能性があります。また、この方法では、大腸 菌 や緑 膿 菌の一部の菌株は、従来の半固体寒天培地では生育できず、培地表面にかすかな赤色しか見られないことが明らかになりました。これは、そのような株によるカプセルの産生による可能性があり、それはそれらの運動性に影響を及ぼす17。この現象は、そのカプセルのために 髄膜炎菌18 でも発生します。結論として、TTCを含む発色半固体培地を用いた細菌の運動性の検出は、試験結果に対する細菌因子の影響を低減し、肉眼で結果を観察しやすくする。高い検出率の利点により、これは細菌の運動性を検出するための従来の半固体培地を置き換えることができる効果的な方法になります。

細菌の運動性は、細菌の病原性、バイオフィルム形成、および薬剤耐性において重要な役割を果たします1。細菌の運動性は病原性と密接に関連しており、宿主細胞の早期感染中の細菌のコロニー形成に必要です2。バイオフィルムの形成は細菌の運動性と密接に関連しており、細菌は運動性を介して固体培地の表面に付着します。細菌の運動性は、バイオフィルム形成と正の相関があると長い間考えられてきました。バイオフィルムによる高度の細菌薬剤耐性は、人間の健康を脅かす持続感染につながる可能性があります3,4,5。したがって、細菌の運動性を検出することが重要です。細菌の運動性試験は、主に生きている状態にあるさまざまな形態の細菌の運動性を調べるために使用され、べん毛の有無を間接的に決定できるため、細菌の同定に重要な役割を果たします。
細菌の運動性を検出するための直接的および間接的な方法があります6。べん毛を有する細菌は運動性を示すため、べん毛の有無を検出することで間接的に細菌が運動性であるかどうかを検出することができる。例えば、細菌が運動性であることを示すために電子顕微鏡観察およびべん毛染色によって間接的に運動性を検出することができる。また、懸濁液落下法や半固体穿刺法などの直接法による検出も可能です。 
細菌の運動性を検出するために学部微生物学研究室で一般的に使用される半固体穿刺法は、細菌の増殖の方向に応じて、0.4〜0.8%寒天を含む半固体寒天培地の穿刺に細菌を接種します。細菌が穿刺線に沿って増殖して周囲に広がると、雲のような(ブラシのような)成長痕跡が現れ、べん毛の存在、したがって運動性を示します。穿刺線の成長痕跡がない場合、細菌は鞭毛も運動性もありません。 
しかしながら、この方法はその欠点を有する:細菌は無色透明であり、べん毛活性は生きている細菌の生理学的特性および他の要因、ならびに寒天の濃度および試験管の小径によって影響を受ける。さらに、好気性細菌は寒天表面での増殖にのみ適しており、細菌の運動性の観察に影響を与えます。したがって、この実験を改善するために、2,3,5-トリフェニルテトラゾリウムクロリド(TTC)(無色)を培地に添加して、TTC 7,8,9,10の赤色生成物の形成を触媒する細胞内デヒドロゲナーゼを使用する現在の直接穿刺法よりも、細菌の運動性を決定するより信頼性が高く直感的な方法を確立しました。

<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

細菌の運動性は、細菌の病原性、バイオフィルム形成、および薬剤耐性にとって非常に重要です。細菌の運動性は、多くの病原性種の侵入および/または拡散にとって非常に重要です。したがって、細菌の運動性を検出することが重要です。酸素、pH、温度などの細菌の増殖条件は、細菌の増殖と細菌べん毛の発現に影響を与える可能性があります。これは、運動性の低下または運動性の喪失につながり、細菌の運動性の不正確な評価をもたらす可能性があります。生細菌の細胞内デヒドロゲナーゼによる2,3,5-トリフェニルテトラゾリウムクロリド(TTC)の呈色反応に基づいて、TTCを細菌の運動性検出のために従来の半固体寒天に添加しました。その結果、このTTC半固体寒天法は、細菌の運動性を検出するための簡便で操作が容易であり、大型で高価な機器を必要としないことが示されました。結果はまた、0.3%寒天で調製された半固体培地で最も高い運動が観察されることを示した。従来の半固体培地と比較して、結果は評価が容易で正確です。

運動性検出のために標準株と単離株の両方を比較し、結果を表1に示した。べん毛がないため、黄色ブドウ球菌と肺炎桿菌は、従来の培地とTTC半固体培地の両方で接種された線に沿ってのみ成長しました。一方,緑膿菌,大腸菌,ネズミチフス菌はTTC半固体培地で24時間培養した後,接種ライン付近で全方向に増殖を示した。これは、48時間培養後にさらに明らかになりました(図1)。従来の半固体培地では細菌が四方八方に増殖したが、接種ラインの外側の細菌数が少ないため、TTC培地よりも可視化がはるかに困難であった。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63706/63706fig01.jpg"> 図1:TTC半固体培地を用いた運動性試験結果。 左側が黄色ブドウ球菌、右側が大腸菌。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63706/63706fig01large.jpg" target="_blank">この図の拡大版を表示するには、ここをクリックしてください。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2" rowspan="2">濾す</td>
			<td colspan="2">0.4%寒天を含む半固体培地</td>
			<td colspan="2">0.4%寒天および0.005%TTCを含む半固体培地</td>
		</tr><tr><td>24時間</td>
			<td>48時間</td>
			<td>24時間</td>
			<td>48時間</td>
		</tr><tr><td colspan="2">緑膿菌 ATCC27853</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr><tr><td colspan="2">大腸菌 ATCC25922</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr><tr><td colspan="2">S.チフィムリウム ATCC14028</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr><tr><td colspan="2">黄色ブドウ球菌 ATCC25923</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr><tr><td colspan="2">大腸菌 (15)</td>
			<td>12</td>
			<td>14</td>
			<td>13</td>
			<td>14</td>
		</tr><tr><td colspan="2">サルモネラ属菌 (8)</td>
			<td>7</td>
			<td>8</td>
			<td>8</td>
			<td>8</td>
		</tr><tr><td colspan="2">A.親水性 (20)</td>
			<td>18</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
		</tr><tr><td colspan="2">ビブリオ 属 (8)</td>
			<td>7</td>
			<td>8</td>
			<td>8</td>
			<td>8</td>
		</tr><tr><td colspan="2">緑膿菌 (24)</td>
			<td>18</td>
			<td>20</td>
			<td>22</td>
			<td>23</td>
		</tr><tr><td colspan="2">肺炎桿菌 (5)</td>
			<td>-5</td>
			<td>-5</td>
			<td>-5</td>
			<td>-5</td>
		</tr><tr><td colspan="6">数字は、陽性の菌株(+)と負の染色(-)の数を示します。</td>
		</tr></tbody></table>表1:細菌の運動性の比較。 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63706/63706fig02.jpg"> 図2:異なる寒天濃度での 大腸菌 の運動活性の観察。 左から右へ:0.3%、0.5%、0.8%寒天。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63706/63706fig02large.jpg" target="_blank">この図の拡大版を表示するには、ここをクリックしてください。</a>
細菌の運動性に対する寒天濃度の影響を 図2に示します。我々は、0.3%寒天で調製した半固体培地で最も高い運動が観察されることを見出した。チューブ内のミディアムカラーはほぼ完全に赤に変わりました。一方,赤色拡散面積は減少し,寒天濃度の増加とともに拡散は長引いた。

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

ここでは、呈色反応に基づいて細菌の運動性を検出するためのプロトコルを提示します。この方法の主な利点は、評価が容易で精度が高く、特殊な機器を必要としないことです。

呈色反応に基づく細菌の運動性の可視化

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

Universidad San Sebastian

Research

JoVE Journal

Biology

3.8K ビュー.  China Pharmaceutical University. ここでは、呈色反応に基づいて細菌の運動性を検出するためのプロトコルを提示します。この方法の主な利点は、評価が容易で精度が高く、特殊な機器を必要としないことです。

ビデオ: 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.

安定した濃度勾配で細菌走化性を定量化するためのマイクロ流体デバイス

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

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.

インデューサーグラジエントプレート上の細菌表面群動運動性の定量化

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

生化学

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.

S. cerevisiae細胞から精製された双方向キネシン-5 Cin8の一分子およびクラスターの運動性

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.

微生物の挙動を研究するための制御された動的化学景観の生成

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

生物工学

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.

蛍光系抗生物質プローブを用いた細菌耐性の可視化

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

免疫・感染学

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.

単細胞間細菌相互作用の運動的可視化

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.

準備、イメージング、および細菌表面運動性アッセイの定量化

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.

べん毛モーターの機能の生物物理学的特性評価

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.

シリーズで3つの確立された技術を適用することにより 、大腸菌 におけるフラゲラ駆動運動性の調査

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.bdigitaluss.remotexs.co/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.

呈色反応に基づく細菌の運動性の可視化

要約

さらに動画を探す

この動画の章

安定した濃度勾配で細菌走化性を定量化するためのマイクロ流体デバイス

インデューサーグラジエントプレート上の細菌表面群動運動性の定量化

S. cerevisiae細胞から精製された双方向キネシン-5 Cin8の一分子およびクラスターの運動性

微生物の挙動を研究するための制御された動的化学景観の生成

蛍光系抗生物質プローブを用いた細菌耐性の可視化

単細胞間細菌相互作用の運動的可視化

準備、イメージング、および細菌表面運動性アッセイの定量化

べん毛モーターの機能の生物物理学的特性評価

シリーズで3つの確立された技術を適用することにより、大腸菌におけるフラゲラ駆動運動性の調査

Bacterial Co-Incubation Assay:単一細胞レベルでの種内細菌の競合を可視化するための蛍光顕微鏡ベースの技術

呈色反応に基づく細菌の運動性の可視化

要約

さらに動画を探す

この動画の章

安定した濃度勾配で細菌走化性を定量化するためのマイクロ流体デバイス

インデューサーグラジエントプレート上の細菌表面群動運動性の定量化

S. cerevisiae細胞から精製された双方向キネシン-5 Cin8の一分子およびクラスターの運動性

微生物の挙動を研究するための制御された動的化学景観の生成

蛍光系抗生物質プローブを用いた細菌耐性の可視化

単細胞間細菌相互作用の運動的可視化

準備、イメージング、および細菌表面運動性アッセイの定量化

べん毛モーターの機能の生物物理学的特性評価

シリーズで3つの確立された技術を適用することにより 、大腸菌 におけるフラゲラ駆動運動性の調査

Bacterial Co-Incubation Assay:単一細胞レベルでの種内細菌の競合を可視化するための蛍光顕微鏡ベースの技術

シリーズで3つの確立された技術を適用することにより、大腸菌におけるフラゲラ駆動運動性の調査