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

Bu çalışma, Jiangsu Yüksek Öğretim Kurumlarının Öncelikli Akademik Program Geliştirme (PAPD) ve Çin İlaç Üniversitesi Öğretim Reformu Araştırma Projesi (2019XJYB18) tarafından desteklenmiştir.

1. Yarı katı ortamın hazırlanması
<ol><li>Geleneksel yarı katı agar
	<ol><li>Geleneksel yarı katı agarı, temel bileşenleri kullanarak bakteriyel motilite, test ortamı tarifine göre hazırlayın11. 10 g Triptoz, 15 g NaCl, 4 g agar'ı yeterince damıtılmış suda çözün, pH'ı 7.2 ± 0.2'ye ayarlayın ve nihai hacmi 1.000 mL'ye ayarlayın.</li>
		<li>Agar'ı 121 ° C'de 20 dakika boyunca otoklavlayın ve 3 cm yüksekliğinde yarı katı bir ortam olarak 10 mL test tüplerine dağıtın.</li>
	</ol></li>
	<li>TTC'li geleneksel yarı katı agar
	<ol><li>Geleneksel yarı katı ortamı otoklavladıktan sonra, 50 ° C'ye soğutun, 100 mL ortama 5 mL steril% 1 TTC çözeltisi ekleyin, karıştırın ve 3 cm yüksekliğinde bir yarı katı ortam oluşturmak için 10 mL test tüplerine dağıtın.</li>
	</ol></li>
</ol>2. Bakteri suşları
NOT: Seksen suş su ortamından izole edilmiş ve Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., Vibrio spp., Klebsiella pneumoniae ve Aeromonas hydrophila dahil olmak üzere otomatik bir bakteri tanımlama cihazı (Malzeme Tablosuna bakınız) kullanılarak tanımlanmıştır (Tablo 1). Staphylococcus aureus (Malzeme Tablosuna bakınız) negatif hareketsiz bir kontrol olarak kullanılmıştır; Escherichia coli, Pseudomonas aeruginosa ve Salmonella typhimurium (Malzeme Tablosuna bakınız) pozitif kontrol suşları olarak kullanılmıştır.
<ol><li>Motilitenin analizi için kullanılacak test bakteri suşlarını tanımlayın.</li>
	<li>Negatif hareketsiz kontrolleri ve hareketli pozitif kontrol suşlarını dahil edin.</li>
</ol>3. TTC ile güçlendirilmiş bakteriyel motilitenin gözlemlenmesi
<ol><li>Agar plakalarından tek test bakteri kolonilerini seçin ve aşılama iğneleri kullanarak delinerek yukarıdaki iki yarı katı ortama (adım 1.1.2 ve 1.2.1) aşılayın.</li>
	<li>Sonuçları gözlemlemek için tüpleri inkübatörde 37 ° C'de 24-48 saat boyunca kültürleyin.</li>
	<li>Büyüme durumunu gözlemleyin: sadece delinme çizgisi kırmızıysa, bakterileri hareketsiz (-) olarak karakterize edin. Kırmızı renk delinme çizgisi12 boyunca hafifçe dışa doğru yayılırsa, bakterileri hareketli (+) olarak karakterize edin.</li>
</ol>4. Farklı agar konsantrasyonlarının bakteriyel motiliteye etkisi
<ol><li>% 0.3,% 0.5 ve% 0.8 agar içeren yarı katı ortamlar hazırlayın ve yukarıda açıklandığı gibi delinerek aşılayın. 24-48 saatlik inkübasyondan sonra sonuçları gözlemleyin.</li>
</ol>

<ol>
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Y., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explore+the+influencing+factors+of+bacterial+motility.">Explore the influencing factors of bacterial motility.</a> Health Care Today. 6, 50-51 (2018).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+Phytophthora+pathogens+deploy+effectors+to+interfere+with+bacterial+growth+and+motility.">Filamentous Phytophthora pathogens deploy effectors to interfere with bacterial growth and motility.</a> Frontiers in Microbiology. 11, 581511 (2020).</li><li>K&#252;hn, M. 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Yazarların açıklayacak çıkar çatışmaları yoktur.

Bakteriyel motilitenin semisolid medium yöntemi ile saptanması birçok faktörden etkilenmektedir13,14. Oksijen (agar yüzeyinde aerobik, yarı katı ortam ile tüpün dibinde aerobik olmayan), pH ve sıcaklık gibi bakteriyel büyüme koşulları, bakteriyel flagella'nın canlılığını etkileyebilir, bu da hareketliliğin azalmasına ve hatta hareketlilik kaybına neden olabilir15. Ek olarak, hareketlilikleri olarak bazı mukus tipi bakteriler, podokonjugatların üretiminden etkilenebilir.
TTC'nin yarı katı ortama eklenmesi, hareketliliğin gözlemlenmesine yardımcı olur. Yönlendirici güce sahip fermentatif bakteriler, inkübasyondan sonra delinme çizgisi boyunca her yönde büyüyebilir. Bu nedenle, delinme çizgisinin etrafındaki ortam kırmızı olur. Yönlendirici güce sahip fermentatif olmayan bakteriler, ortamın alt kısmında düşük oksijen içeriğine sahiptir. Bu nedenle, bu bakteriler zayıf büyür, böylece ortamın sadece üst tabakası kırmızıdır. İtici gücü olmayan bakteriler sadece aşılama hattında büyüyebilir ve sadece delinme çizgisi kırmızı görünür. 
TTC-yarı katı ortam ile bakteriyel motiliteyi tespit ederken, kültür süresi ne kadar uzun olursa, özellikle düşük agar konsantrasyonlarında sonuçlar o kadar belirgin olur. Sonucun yorumlanması zorsa, kültür süresi uygun şekilde uzatılmalıdır. Bu, yarı katı ortamın agar konsantrasyonu, bakteri sayısı ve hareketlilikleri16 ile ilgili olabilir. Ek olarak, bu yöntem, E. coli ve P. aeruginosa'nın bazı suşlarının geleneksel yarı katı agar ortamında büyüyemediğini ve TTC yarı katı agar ortamında ortamın yüzeyinde sadece soluk kırmızı bir renk gösterdiğini ortaya koymuştur. Bu, bu tür suşlar tarafından kapsüllerin üretilmesinden kaynaklanabilir ve bu da hareketliliklerini etkiler17. Bu fenomen ayrıca kapsülü nedeniyle Neisseria meningitidis18'de de görülür. Sonuç olarak, TTC içeren kromojenik bir yarı katı ortam kullanılarak bakteriyel motilitenin tespiti, bakteriyel faktörlerin test sonuçları üzerindeki etkisini azaltır ve sonuçların çıplak gözle gözlemlenmesini kolaylaştırır. Yüksek algılama oranının avantajı, bunu bakteriyel hareketliliği tespit etmek için geleneksel yarı katı ortamın yerini alabilecek etkili bir yöntem haline getirir.

Bakteriyel motilite, bakteriyel patojenite, biyofilm oluşumu ve ilaç direncinde kritik bir rol oynar1. Bakteriyel motilite, patojenite ile yakından ilişkilidir ve konakçı hücrelerin erken enfeksiyonu sırasında bakteriyel kolonizasyon için gereklidir2. Biyofilm oluşumu, bakterilerin katı ortamın yüzeyine motiliteyle yapıştığı bakteriyel motiliteyle yakından ilişkilidir. Bakteriyel motilitenin uzun zamandır biyofilm oluşumu ile pozitif ilişkili olduğu düşünülmektedir. Biyofilme bağlı yüksek derecede bakteriyel ilaç direnci, insan sağlığı için tehdit oluşturan kalıcı enfeksiyonlara yol açabilir 3,4,5. Bu nedenle, bakteriyel motiliteyi tespit etmek önemlidir. Bakteriyel motilite, esas olarak, canlı haldeki farklı bakteri formlarının hareketliliğini incelemek için kullanılır, bu da dolaylı olarak flagella'nın varlığını veya yokluğunu belirleyebilir ve bu nedenle bakterilerin tanımlanmasında önemli bir role sahiptir.
Bakteriyel motiliteyi tespit etmek için doğrudan ve dolaylı yöntemler vardır6. Flagellalı bakteriler motiliteyi gösterdiğinden, flagella'nın varlığını veya yokluğunu tespit ederek bakterilerin dolaylı olarak hareketli olup olmadığını tespit etmek mümkündür. Örneğin, bakterilerin hareketli olduğunu göstermek için elektron mikroskobu ve flagellar boyama ile hareketliliği dolaylı olarak tespit etmek mümkündür. Süspansiyon düşürme ve yarı katı delinme yöntemleri gibi doğrudan yöntemlerle tespit etmek de mümkündür. 
Lisans mikrobiyoloji laboratuvarlarında bakteriyel motiliteyi tespit etmek için yaygın olarak kullanılan yarı katı delinme yöntemi, bakterilerin üreme yönüne göre, bakterileri% 0.4-0.8 agar içeren yarı katı agar ortamındaki delinmeye aşılar. Bakteriler etrafa yayılmak için delinme çizgisi boyunca büyürse, flagella'nın varlığını ve dolayısıyla hareketliliği gösteren bulut benzeri (fırça benzeri) büyüme izleri ortaya çıkar. Delinme çizgisi büyüme izleri yoksa, bakteri ne kamçılanır ne de hareketlidir. 
Bununla birlikte, bu yöntemin dezavantajları vardır: bakteriler renksiz ve şeffaftır, kamçı aktivitesi canlı bakterilerin fizyolojik özelliklerinden ve diğer faktörlerden ve agar konsantrasyonundan ve test tüpünün küçük çapından etkilenir. Dahası, aerobik bakteriler sadece agar yüzeyinde büyüme için uygundur ve bakteriyel hareketliliğin gözlemlenmesini etkiler. Bu nedenle, bu deneyi geliştirmek için, TTC7,8,9,10'un kırmızı bir ürününün oluşumunu katalize etmek için hücre içi dehidrojenazlar kullanan mevcut doğrudan delinme yönteminden daha güvenilir ve sezgisel bir bakteri hareketliliğini belirlemek için ortama 2,3,5-trifeniltetrazolyum klorür (TTC) (renksiz) eklenmiştir.

<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

Bakteriyel motilite, bakteriyel patojenite, biyofilm oluşumu ve ilaç direnci için çok önemlidir. Bakteriyel motilite, birçok patojenik türün istilası ve / veya yayılması için çok önemlidir. Bu nedenle, bakteriyel motiliteyi tespit etmek önemlidir. Oksijen, pH ve sıcaklık gibi bakteriyel büyüme koşulları, bakteriyel büyümeyi ve bakteriyel flagella ekspresyonunu etkileyebilir. Bu, hareketliliğin azalmasına ve hatta motilitenin kaybına neden olabilir, bu da bakteriyel motilitenin yanlış değerlendirilmesine neden olabilir. Canlı bakterilerin hücre içi dehidrojenazları ile 2,3,5-trifenil tetrazolyum klorürün (TTC) renk reaksiyonuna dayanarak, TTC, bakteriyel motilitenin tespiti için geleneksel yarı katı agar'a eklenmiştir. Sonuçlar, bakteriyel hareketliliğin tespiti için bu TTC yarı katı agar yönteminin basit, kullanımı kolay ve büyük ve pahalı aletler içermediğini göstermiştir. Sonuçlar ayrıca en yüksek hareketliliğin% 0.3 agar ile hazırlanan yarı katı ortamda gözlendiğini göstermiştir. Geleneksel yarı katı ortam ile karşılaştırıldığında, sonuçların değerlendirilmesi daha kolay ve daha doğrudur.

Motilitenin tespiti için hem standart suşlar hem de izole suşlar karşılaştırılmış ve sonuçlar Tablo 1'de gösterilmiştir. Flagella'nın yokluğu nedeniyle, Staphylococcus aureus ve Klebsiella pneumoniae , hem geleneksel hem de TTC yarı katı ortamlarda aşılanmış çizgi boyunca büyüdü. Buna karşılık, Pseudomonas aeruginosa, Escherichia coli ve Salmonella typhimurium , TTC yarı katı ortamda 24 saat boyunca kültürlendikten sonra aşılanmış çizginin etrafında her yönde büyüme gösterdi. Bu, 48 saatlik kültürden sonra daha da belirgindi (Şekil 1). Bakteriler geleneksel yarı katı ortamda her yönde büyümüş olsalar da, aşılanmış çizginin dış tarafındaki az sayıda bakteri nedeniyle görselleştirmek TTC ortamından çok daha zordu.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63706/63706fig01.jpg"> Şekil 1: TTC yarı katı ortam kullanılarak yapılan motilite testi sonuçları. Solda Staphylococcus aureus, sağda Escherichia coli. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63706/63706fig01large.jpg" target="_blank">Bu şeklin daha büyük bir versiyonunu görmek için lütfen buraya tıklayın.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2" rowspan="2">Soy</td>
			<td colspan="2">% 0.4 agar ile yarı katı ortam</td>
			<td colspan="2">% 0.4 agar ve% 0.005 TTC ile yarı katı ortam</td>
		</tr><tr><td>24 saat</td>
			<td>48 saat</td>
			<td>24 saat</td>
			<td>48 saat</td>
		</tr><tr><td colspan="2">P. aeruginosa ATCC27853</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr><tr><td colspan="2">E. coli ATCC25922</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr><tr><td colspan="2">S. typhimurium ATCC14028</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr><tr><td colspan="2">S. aureus · ATCC25923</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr><tr><td colspan="2">E. coli (15)</td>
			<td>12</td>
			<td>14</td>
			<td>13</td>
			<td>14</td>
		</tr><tr><td colspan="2">Salmonella spp. (8)</td>
			<td>7</td>
			<td>8</td>
			<td>8</td>
			<td>8</td>
		</tr><tr><td colspan="2">A. hidrofila (20)</td>
			<td>18</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
		</tr><tr><td colspan="2">Vibrio sayfa (8)</td>
			<td>7</td>
			<td>8</td>
			<td>8</td>
			<td>8</td>
		</tr><tr><td colspan="2">P. aeruginosa (24)</td>
			<td>18</td>
			<td>20</td>
			<td>22</td>
			<td>23</td>
		</tr><tr><td colspan="2">K. pneumoniae (5)</td>
			<td>-5</td>
			<td>-5</td>
			<td>-5</td>
			<td>-5</td>
		</tr><tr><td colspan="6">Sayılar, pozitif suşların (+) ve negatif lekelerin (-) sayılarını gösterir.</td>
		</tr></tbody></table>Tablo 1: Bakteriyel motilitenin karşılaştırılması. 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63706/63706fig02.jpg"> Şekil 2: Farklı agar konsantrasyonlarında Escherichia coli motilitesinin gözlenmesi. Soldan sağa: %0,3, %0,5, %0,8 agar. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63706/63706fig02large.jpg" target="_blank">Bu şeklin daha büyük bir versiyonunu görmek için lütfen buraya tıklayın.</a>
Agar konsantrasyonunun bakteriyel motiliteye etkisi Şekil 2'de gösterilmiştir. En yüksek motilitenin %0.3 agar ile hazırlanan yarı katı ortamda gözlendiğini bulduk. Tüpteki orta renk neredeyse tamamen kırmızıya döndü. Buna karşılık, kırmızı difüzyon alanı azaldı ve difüzyon artan agar konsantrasyonu ile uzadı.

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

Burada, bir renk reaksiyonuna dayalı bakteriyel motiliteyi tespit etmek için bir protokol sunuyoruz. Bu yöntemin temel avantajları, değerlendirilmesi kolay ve daha doğru olması ve özel ekipman gerektirmemesidir.

Bir renk reaksiyonuna dayalı bakteriyel hareketliliğin görselleştirilmesi

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 Görüntüleme.  China Pharmaceutical University. Burada, bir renk reaksiyonuna dayalı bakteriyel motiliteyi tespit etmek için bir protokol sunuyoruz. Bu yöntemin temel avantajları, değerlendirilmesi kolay ve daha doğru olması ve özel ekipman gerektirmemesidir.

Video: 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.

Kararlı Konsantrasyon Degradeler Bakteriyel Kemotaksis miktarının mikroakışkan Aygıt

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

Biyoloji

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.

İndükleyici Gradyan Plakaları Üzerindeki Bakteriyel Yüzey Kaynatma Motilitesinin Ölçülmesi

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

Biyokimya

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.

Tek Moleküllerin Motilitesi ve S. cerevisiae Hücrelerinden Saflaştırılmış Çift Yönlü Kinesin-5 Cin8 Kümeleri

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.

Mikrobiyal Davranışı İncelemek Için Kontrollü, Dinamik Kimyasal Manzaralar Üretmek

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

Biyomühendislik

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.

Floresan Antibiyotik Probları Kullanılarak Bakteriyel Direncin Görüntülenmesi

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

İmmünoloji ve Enfeksiyon

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.

Tek Hücreli Türler Arası Bakteri Etkileşimlerinin Kinetik Görselleştirilmesi

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.

Bakteriyel Yüzey Motilite Tahliller hazırlanması, Görüntüleme ve miktarının belirlenmesi

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.

Kuyruktaki Motor fonksiyonların Biyofiziksel Karakterizasyonu

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.

Bir Seride Üç Yerleşik Teknik Uygulayarak Escherichia coli'de Flagella Odaklı Hareketliliği Araştırmak

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.