Diese Studie wurde durch das Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) und das Teaching Reform Research Project der China Pharmaceutical University (2019XJYB18) unterstützt.

1. Aufbereitung des halbfesten Mediums
<ol><li>Traditioneller halbfester Agar
	<ol><li>Bereiten Sie den traditionellen halbfesten Agar gemäß der Rezeptur des bakteriellen Motilitätstestmediums unter Verwendung der Grundzutaten11 zu. 10 g Tryptose, 15 g NaCl, 4 g Agar in ausreichend destilliertem Wasser auflösen, den pH-Wert auf 7,2 ± 0,2 einstellen und das Endvolumen auf 1.000 ml auffüllen.</li>
		<li>Autoklavieren Sie den Agar bei 121 °C für 20 min und dosieren Sie ihn als 3 cm hohes halbfestes Medium in 10-ml-Reagenzgläser.</li>
	</ol></li>
	<li>Traditioneller halbfester Agar mit TTC
	<ol><li>Nach dem Autoklavieren des herkömmlichen halbfesten Mediums wird es auf 50 °C abgekühlt, 5 ml sterile 1%ige TTC-Lösung zu 100 ml Medium gegeben, gemischt und in 10 ml-Reagenzgläser dosiert, um ein 3 cm hohes halbfestes Medium zu bilden.</li>
	</ol></li>
</ol>2. Bakterienstämme
ANMERKUNG: Achtzig Stämme wurden aus der aquatischen Umwelt isoliert und mit einem automatisierten Bakterienidentifikationsinstrument identifiziert (siehe Materialtabelle), darunter Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., Vibrio spp., Klebsiella pneumoniae und Aeromonas hydrophila (Tabelle 1). Staphylococcus aureus (siehe Materialtabelle) wurde als negative unbewegliche Kontrolle verwendet; Als Positivkontrollstämme wurden Escherichia coli, Pseudomonas aeruginosa und Salmonella typhimurium ( siehe Materialtabelle) verwendet.
<ol><li>Identifizieren Sie Testbakterienstämme, die für die Motilitätsanalyse verwendet werden sollen.</li>
	<li>Negative unbewegliche Kontrollen und bewegliche Positivkontrollstämme einschließen.</li>
</ol>3. TTC-verstärkte Beobachtung der bakteriellen Motilität
<ol><li>Einzelne Kolonien von Testbakterien werden von Agarplatten entnommen und mit Impfnadeln in die beiden oben genannten halbfesten Medien (Schritte 1.1.2 und 1.2.1) geimpft.</li>
	<li>Kultivieren Sie die Röhrchen bei 37 °C im Inkubator für 24-48 h, um die Ergebnisse zu beobachten.</li>
	<li>Beobachten Sie den Wachstumszustand: Charakterisieren Sie die Bakterien als unbeweglich (-), wenn nur die Einstichlinie rot ist. Charakterisieren Sie die Bakterien als beweglich (+), wenn sich die rote Farbe entlang der Einstichlinie12 leicht nach außen ausbreitet.</li>
</ol>4. Einfluss unterschiedlicher Agarkonzentrationen auf die bakterielle Motilität
<ol><li>Bereiten Sie halbfeste Medien mit 0,3 %, 0,5 % und 0,8 % Agar vor und beimpfen Sie sie wie oben beschrieben durch Punktion. Beobachten Sie die Ergebnisse nach 24-48 h Inkubation.</li>
</ol>

<ol><li>Jordan, E. O., Caldwell, M. E., Reiter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+motility%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">Bacterial motility.</a> Journal of Bacteriology. 27 (2), 165(1934).</li>
<li>Lai, S. L., Hou, H., Jiang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+motility+and+its+role+during+initial+stage+of+pathogenesis%5BTitle%5D)+AND+%22Journal+of+Microbiology%22%5BJournal%5D)">Bacterial motility and its role during initial stage of pathogenesis.</a> Journal of Microbiology. 26 (5), in Chinese 68-70 (2006).</li>
<li>Ding, S. S., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+between+flagella-dependent+motility+and+biofilm+in+bacteria+-+A+review%5BTitle%5D)+AND+%22Acta+Microbiologica+Sinica%22%5BJournal%5D)">Relationship between flagella-dependent motility and biofilm in bacteria - A review.</a> Acta Microbiologica Sinica. 49 (4), in Chinese 417-422 (2009).</li>
<li>Zeng, J., Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+the+mechanism+of+bacterial+resistance+and+tolerance%5BTitle%5D)+AND+%22Chinese+Journal+of+Antibiotics%22%5BJournal%5D)">Recent advances in the mechanism of bacterial resistance and tolerance.</a> Chinese Journal of Antibiotics. 45 (2), 113-121 (2020).</li>
<li>Xu, M., Zhou, M. X., Zhu, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progress+in+the+mechanism+of+bacterial+flagellum+motility%2C+adhesion+and+immune+escape%5BTitle%5D)+AND+%22Chinese+Journal+of+Veterinary+Science%22%5BJournal%5D)">Progress in the mechanism of bacterial flagellum motility, adhesion and immune escape.</a> Chinese Journal of Veterinary Science. 37 (2), 369-375 (2017).</li>
<li>Leboffe, M. J., Pierce, B. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLeboffe%2C+MJ+%22Microbiology%3A+laboratory+theory+and+application.+Third+edition%22">Microbiology: laboratory theory and application. Third edition</a>. , Morton Publishing Company. Colorado. (2015).</li>
<li>Ball, R. J., Sellers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+motility+medium%5BTitle%5D)+AND+%22Applied+Microbiology%22%5BJournal%5D)">Improved motility medium.</a> Applied Microbiology. 14, 670-673 (1966).</li>
<li>An, S., Wu, J., Zhang, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+Pseudomonas+aeruginosa+biofilm+dispersal+by+a+cyclic-di-GMP+phosphodiesterase+with+a+putative+hypoxia-sensing+domain%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">Modulation of Pseudomonas aeruginosa biofilm dispersal by a cyclic-di-GMP phosphodiesterase with a putative hypoxia-sensing domain.</a> Applied and Environmental Microbiology. 76 (24), 8160-8173 (2010).</li>
<li>Chouhan, O. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+site-directed+mutagenesis+at+the+GGEEF+domain+of+the+biofilm+forming+GGEEF+protein+from+Vibrio+cholerae%5BTitle%5D)+AND+%22AMB+Express%22%5BJournal%5D)">Effect of site-directed mutagenesis at the GGEEF domain of the biofilm forming GGEEF protein from Vibrio cholerae.</a> AMB Express. 6 (1), 2(2016).</li>
<li>McLaughlin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+colorimetric+microplate+test+of+phage+lysis+in+Salmonella+enterica%5BTitle%5D)+AND+%22Journal+of+Microbiological+Methods%22%5BJournal%5D)">Simple colorimetric microplate test of phage lysis in Salmonella enterica.</a> Journal of Microbiological Methods. 69 (2), 394-398 (2007).</li>
<li>Difco Laboratories. Difco manual: Dehydrated culture media and reagents for microbiology. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Difco+Laboratories%22">Difco Laboratories</a>. , Detroit. (1984).</li>
<li>Tittsler, R. P., Sandholzer, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+semi-solid+agar+for+the+detection+of+bacterial+motility%5BTitle%5D)+AND+%22Journal+of+Bacteriology%22%5BJournal%5D)">The use of semi-solid agar for the detection of bacterial motility.</a> Journal of Bacteriology. 31 (6), 575(1936).</li>
<li>Qian, Y., Tian, X. Y., Zhang, S. Y., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Explore+the+influencing+factors+of+bacterial+motility%5BTitle%5D)+AND+%22Health+Care+Today%22%5BJournal%5D)">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/pubmed?term=((Filamentous+Phytophthora+pathogens+deploy+effectors+to+interfere+with+bacterial+growth+and+motility%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Filamentous Phytophthora pathogens deploy effectors to interfere with bacterial growth and motility.</a> Frontiers in Microbiology. 11, 581511(2020).</li>
<li>Kühn, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+arrangement+of+several+flagellins+within+bacterial+flagella+improves+motility+in+different+environments%5BTitle%5D)+AND+%22Nature+Communication%22%5BJournal%5D)">Spatial arrangement of several flagellins within bacterial flagella improves motility in different environments.</a> Nature Communication. 9 (1), 5369(2018).</li>
<li>Mitchell, A. J., Wimpenny, J. W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effects+of+agar+concentration+on+the+growth+and+morphology+of+submerged+colonies+of+motile+and+non-motile+bacteria%5BTitle%5D)+AND+%22Journal+of+Applied+Microbiology%22%5BJournal%5D)">The effects of agar concentration on the growth and morphology of submerged colonies of motile and non-motile bacteria.</a> Journal of Applied Microbiology. 83 (1), 76-84 (2010).</li>
<li>Xu, A., Zhang, M., Du, W., Wang, D., Ma, L. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+molecular+mechanism+for+how+sigma+factor+AlgT+and+transcriptional+regulator+AmrZ+inhibit+twitching+motility+in+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Environmental+Microbiology%22%5BJournal%5D)">A molecular mechanism for how sigma factor AlgT and transcriptional regulator AmrZ inhibit twitching motility in Pseudomonas aeruginosa.</a> Environmental Microbiology. 23 (2), 572-587 (2021).</li>
<li>Bartley, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Attachment+and+invasion+of+Neisseria+meningitidis+to+host+cells+is+related+to+surface+hydrophobicity%2C+bacterial+cell+size+and+capsule%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Attachment and invasion of Neisseria meningitidis to host cells is related to surface hydrophobicity, bacterial cell size and capsule.</a> PLoS One. 8, 55798(2013).</li>
</ol>

Die Autoren haben keine Interessenkonflikte offenzulegen.

Der Nachweis der bakteriellen Motilität durch die Methode des halbfesten Mediums wird von vielen Faktoren beeinflusst13,14. Bakterielle Wachstumsbedingungen, wie z. B. Sauerstoff (aerob auf der Agaroberfläche, nicht aerob am Boden des Röhrchens mit dem halbfesten Medium), pH-Wert und Temperatur, können die Lebensfähigkeit bakterieller Flagellen beeinträchtigen, was zu einer verminderten Motilität oder sogar zu einem Verlust der Motilität führen kann<sup ...

Die bakterielle Motilität spielt eine entscheidende Rolle bei der bakteriellen Pathogenität, der Biofilmbildung und der Arzneimittelresistenz1. Die bakterielle Motilität ist eng mit der Pathogenität verbunden und ist für die bakterielle Besiedlung während der frühen Infektion von Wirtszellen notwendig2. Die Bildung von Biofilmen steht in engem Zusammenhang mit der bakteriellen Motilität, bei der Bakterien durch Motilität an der Oberfläche fester Medien haften. Es wurde lange angenommen, dass die bakterielle Motilität positiv mit der Biofilmbildung korreliert. Ein hohes Maß an bakterieller Arzneimittelresistenz aufgrund von Biofilmen kann zu anhaltenden Infektionen führen, die eine Bedrohung für die menschliche Gesundheit darstellen 3,4,5. Daher ist es wichtig, die bakterielle Motilität nachzuweisen. Der bakterielle Motilitätstest wird hauptsächlich verwendet, um die Motilität verschiedener Bakterienformen im lebenden Zustand zu untersuchen, die indirekt das Vorhandensein oder Fehlen von Flagellen bestimmen können und somit eine wichtige Rolle bei der Identifizierung von Bakterien spielen.
Es gibt direkte und indirekte Methoden zum Nachweis der bakteriellen Motilität6. Da Bakterien mit Geißeln Motilität aufweisen, ist es möglich, indirekt festzustellen, ob Bakterien beweglich sind, indem das Vorhandensein oder Fehlen von Geißeln nachgewiesen wird. So ist es beispielsweise möglich, die Motilität indirekt durch Elektronenmikroskopie und Flagellenfärbung nachzuweisen, um anzuzeigen, dass Bakterien beweglich sind. Es ist auch möglich, durch direkte Methoden zu detektieren, wie z. B. Suspensionstropfen und halbfeste Punktionsmethoden. 
Die halbfeste Punktionsmethode, die üblicherweise in mikrobiologischen Labors verwendet wird, um die bakterielle Motilität nachzuweisen, impft die Bakterien in das Einstichmedium des halbfesten Agars, das 0,4-0,8 % Agar enthält, je nach Richtung des Bakterienwachstums. Wenn die Bakterien entlang der Einstichlinie wachsen, um sich auszubreiten, erscheinen wolkenartige (bürstenartige) Wachstumsspuren, die auf das Vorhandensein von Geißeln und damit auf Motilität hinweisen. Wenn keine Wachstumsspuren an der Einstichlinie vorhanden sind, ist das Bakterium weder gegeißelt noch beweglich. 
Diese Methode hat jedoch ihre Nachteile: Die Bakterien sind farblos und durchsichtig, die Flagellenaktivität wird durch die physiologischen Eigenschaften der lebenden Bakterien und andere Faktoren sowie die Konzentration des Agars und den geringen Durchmesser des Reagenzglases beeinflusst. Darüber hinaus sind aerobe Bakterien nur für das Wachstum auf der Agaroberfläche geeignet, was die Beobachtung der bakteriellen Motilität beeinträchtigt. Um dieses Experiment zu verbessern, wurde dem Medium daher 2,3,5-Triphenyltetrazoliumchlorid (TTC) (farblos) zugesetzt, um eine zuverlässigere und intuitivere Methode zur Bestimmung der bakteriellen Motilität zu etablieren als die derzeitige direkte Punktionsmethode, bei der intrazelluläre Dehydrogenasen verwendet werden, um die Bildung eines roten Produkts von TTC 7,8,9,10 zu katalysieren.

<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

Die bakterielle Motilität ist entscheidend für die bakterielle Pathogenität, die Biofilmbildung und die Arzneimittelresistenz. Die bakterielle Motilität ist entscheidend für die Invasion und/oder Verbreitung vieler pathogener Spezies. Daher ist es wichtig, die bakterielle Motilität nachzuweisen. Bakterienwachstumsbedingungen wie Sauerstoff, pH-Wert und Temperatur können das Bakterienwachstum und die Expression von bakteriellen Flagellen beeinflussen. Dies kann zu einer verminderten Motilität oder sogar zu einem Verlust der Motilität führen, was zu einer ungenauen Bewertung der bakteriellen Motilität führt. Basierend auf der Farbreaktion von 2,3,5-Triphenyltetrazoliumchlorid (TTC) durch intrazelluläre Dehydrogenasen lebender Bakterien wurde TTC zu herkömmlichem halbfestem Agar zum Nachweis der bakteriellen Motilität hinzugefügt. Die Ergebnisse zeigten, dass diese TTC-Methode mit halbfestem Agar zum Nachweis der bakteriellen Motilität einfach und leicht zu bedienen ist und keine großen und teuren Instrumente erfordert. Die Ergebnisse zeigten auch, dass die höchste Motilität in halbfestem Medium beobachtet wurde, das mit 0,3%igem Agar hergestellt wurde. Im Vergleich zum herkömmlichen halbfesten Medium sind die Ergebnisse einfacher auszuwerten und genauer.

Sowohl Standardstämme als auch isolierte Stämme wurden zur Motilitätsdetektion verglichen, und die Ergebnisse sind in Tabelle 1 dargestellt. Aufgrund des Fehlens von Flagellen wuchsen Staphylococcus aureus und Klebsiella pneumoniae nur entlang der inokulierten Linie sowohl auf traditionellen als auch auf halbfesten TTC-Medien. Im Gegensatz dazu zeigten Pseudomonas aeruginosa, Escherichia coli und Salmonella typhimurium nach 24-stündiger Kultivierung auf ha...

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

Hier stellen wir ein Protokoll zum Nachweis der bakteriellen Motilität anhand einer Farbreaktion vor. Die Hauptvorteile dieser Methode sind, dass sie einfacher auszuwerten und genauer ist und keine spezielle Ausrüstung erfordert.

Visualisierung der bakteriellen Motilität anhand einer Farbreaktion

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

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3.9K Aufrufe.  China Pharmaceutical University. Hier stellen wir ein Protokoll zum Nachweis der bakteriellen Motilität anhand einer Farbreaktion vor. Die Hauptvorteile dieser Methode sind, dass sie einfacher auszuwerten und genauer ist und keine spezielle Ausrüstung erfordert.

Serie: Visualizing Bacterial Motility Based on a Color Reaction

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Visualizing Einzelne molekulare Komplexe In Vivo Verwenden der erweiterten Fluoreszenzmikroskopie

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Forschung

Biologie

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.

Einer mikrofluidischen Vorrichtung zur Quantifizierung von bakteriellen Chemotaxis in Stable Konzentrationsgradienten

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

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.

Motilität einzelner Moleküle und Cluster von bidirektionalem Kinesin-5 Cin8, gereinigt aus S. cerevisiae-Zellen

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

Biochemie

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.

Visualisierung der bakteriellen Resistenz mit fluoreszierenden Antibiotikasonden

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

Immunologie und Infektion

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.

Kinetische Visualisierung von einzelzelligen Interspezies-Bakterien-Wechselwirkungen

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.

Vorbereitung, Imaging und Quantifizierung der Bakterienoberfläche Motilität Tests

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.

Biophysikalische Charakterisierung von Flagellenmotors Funktionen

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.

Untersuchung der Flagellen-getriebenen Motilität in Escherichia coli durch Anwendung von drei etablierten Techniken in einer Serie

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

Visualization of Internalized Anaerobic Bacteria within Host Endothelial Cells

Source: Wunsch, C. M. et. al., <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
In this video, we demonstrate a procedure to visualize internalized Porphyromonas gingivalis, an anaerobic pathogenic bacterium, within human endothelial cells using confocal microscopy. The bacteria are stained with a fluorescent dye while the host endothelial cell actin cytoskeleton is labeled with a different fluorescent dye. The internalized Porphyromonas gingivalis inside endothelial cells are visualized using confocal microscopy.

Visualisierung von internalisierten anaeroben Bakterien in Wirtsendothelzellen

1. Internalization of Bacteria into Host Cells (Fluorescent Microscopy)
Note: P. gingivalis is labeled with ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

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.

Bakterieller Co-Inkubations-Assay: Eine auf Fluoreszenzmikroskopie basierende Technik zur Visualisierung der intraspezifischen bakteriellen Konkurrenz auf Einzelzellebene