Name: High-throughput methode voor het observeren van motiliteitsfenotypes in Pseudomonas aeruginosa (Video) | JoVE Journal
Uploaded: 2025-06-20T12:30:00.000+00:00
Duration: 7 min 23 s
Description: Leer een high-throughput methode voor het analyseren van motiliteit in Pseudomonas aeruginosa. Dit protocol identificeert genetische factoren die zwerm- en twitchinggedrag beïnvloeden voor microbiologisch onderzoek.

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</ol>

<table><tbody><tr><td>96 well-plates&nbsp;&nbsp;</td><td>Corning&nbsp;&nbsp;</td><td>3701&nbsp;</td><td></td></tr><tr><td>Agar&nbsp;</td><td>Fisher Scientific&nbsp;&nbsp;</td><td>BP1423-2&nbsp;</td><td>Finale concentratie van 0,5% voor swarming motiliteit of 1% voor twitching motiliteit respectievelijk.&nbsp;</td></tr><tr><td>Casamino Acids&nbsp;&nbsp;</td><td>Fisher Scientific&nbsp;&nbsp;</td><td>223050&nbsp;</td><td>Bereid een 20% voorraad in water. Filter steriliseert met een 0,22 μm filter en bewaar bij 4 °C. Finale concentratie 0,5% in M9 minimum medium.&nbsp;</td></tr><tr><td>Kristalviolet&nbsp;</td><td>Bio Basic&nbsp;&nbsp;</td><td>CB0331</td><td>Bereid een 1% oplossing in water.&nbsp;</td></tr><tr><td>D-Glucose&nbsp;&nbsp;</td><td>Bio Basic&nbsp;&nbsp;</td><td>GB0219&nbsp;</td><td>Bereid een 20% voorraad (1,1 M) in water en steriliseer door autoclaving. Finale concentratie 0,2% in M9 minimum medium.&nbsp;</td></tr><tr><td>Filtropur S 0.2&nbsp;</td><td>Sarstedt&nbsp;</td><td>83.1826.001</td><td></td></tr><tr><td>Gentamycin sulfaat&nbsp;&nbsp;</td><td>Bio Basic&nbsp;&nbsp;</td><td>GB0217&nbsp;</td><td>Bereid een 30 mg/ml voorraad in water. Filter steriliseert met een 0,22 μm filter en bewaar bij 4 °C – finale concentratie in platen 15 µg/mL..&nbsp;&nbsp;</td></tr><tr><td>Inoculatieslinger en naald&nbsp;</td><td>Fisher Scientific&nbsp;</td><td>22363597</td><td></td></tr><tr><td>M9 Minimal Salts, 5X&nbsp;</td><td>Fisher Scientific&nbsp;</td><td>248510</td><td>Bereid een 1M voorraad in water en steriliseer door autoclaaf. Finale concentratie 1 mM in M9 minimum medium.&nbsp;&nbsp;</td></tr><tr><td>Magnesium Sulfate (MgSO4)&nbsp;&nbsp;</td><td>Fisher Scientific&nbsp;</td><td>M63-500</td><td>&nbsp;Bereid een 1 M MgSO4 voorraad in water. Filter steriliseert met een 0,22 μm filter en bewaar bij kamertemperatuur. Finale concentratie&nbsp;</td></tr><tr><td>PA14 Transposon Insertie Mutanten Bibliotheek&nbsp;&nbsp;</td><td></td><td>Liberati et al., 2006 (Zie referentie 29)</td><td>59 96-well platen voor de volledige collectie. Elke put bevat een enkele mutant in LB met 25% glycerol.&nbsp;</td></tr><tr><td>Petrischaal 92x16mm&nbsp;</td><td>Sarstedt&nbsp;</td><td>82.1473.001</td><td></td></tr><tr><td>PhenoBooth+&nbsp;</td><td>Singer Instruments&nbsp;</td><td>https://www.singerinstruments.com/solution/phenobooth/specification/&nbsp;</td><td>Los 11,3 g van het poeder in 200 mL gezuiverd water. Autoclaveer bij 121 °C gedurende 15 min.&nbsp;</td></tr><tr><td>Kunststof spuit 10 mL&nbsp;</td><td>Fisher Scientific&nbsp;</td><td>14955459</td><td></td></tr><tr><td>Rotor+&nbsp;&nbsp;&nbsp;</td><td>Singer Instruments&nbsp;</td><td>https://www.singerinstruments.com/resource/rotor-hda/&nbsp;</td><td></td></tr><tr><td>Short pin RePads 384 dichtheden&nbsp;</td><td>Singer Instruments&nbsp;&nbsp;</td><td>REP-004&nbsp;</td><td></td></tr><tr><td>Short pin RePads 96 dichtheden&nbsp;&nbsp;</td><td>Singer Instruments&nbsp;&nbsp;</td><td>REP-002&nbsp;</td><td></td></tr><tr><td>Natriumchloride (NaCl)&nbsp;&nbsp;</td><td>Fisher Scientific&nbsp;</td><td>BP358-212&nbsp;</td><td></td></tr><tr><td>Veerlading 96-pin replicator</td><td>EnzyScreen&nbsp;&nbsp;</td><td>CR1000&nbsp;</td><td></td></tr><tr><td>Chirurgische kling roestvrij No. 25&nbsp;</td><td>Fisher Scientific&nbsp;</td><td>08-918-5F</td><td></td></tr><tr><td>Tryptone&nbsp;</td><td>Oxoid&nbsp;&nbsp;</td><td>LP0042B&nbsp;</td><td></td></tr><tr><td>Uniwell platen</td><td>Singer Instruments&nbsp;&nbsp;</td><td>PlusPlates: PLU-003&nbsp;&nbsp;</td><td>Platen afmeting: 54 × 34 × 38 cm - zonder binnenwanden, die grotere interne afmetingen bieden.&nbsp;</td></tr><tr><td>Uniwell platen</td><td>VWR</td><td>Single well weefselkweek platen: 75780-348</td><td>Zelfde afmetingen als de PlusPlates.</td></tr><tr><td>Gist Extract&nbsp;</td><td>Fisher Scientific&nbsp;</td><td>248510&nbsp;</td><td></td></tr></tbody></table>

high-throughput methode voor het observeren van motiliteitsfenotypes in pseudomonas aeruginosa

High-throughput Method for Observing Motility Phenotypes in Pseudomonas aeruginosa

High-throughput methode voor het observeren van motiliteitsfenotypes in Pseudomonas aeruginosa

Motility behaviors often play a significant role in the ability of a bacterium to exploit the resources available in its environment. This is particularly ...

high-throughput-method-for-observing-motility-phenotypes-pseudomonas

Research

JoVE Journal

Immunology and Infection

Immunologie en Infectie

Dit protocol beschrijft de ontwikkeling van een microfluïdische apparaat voor het onderzoeken van bacteriële chemotaxis in stabiele concentratiegradiënten van chemoeffectors.

Een microfluïdische apparaat voor het kwantificeren van Bacteriële Chemotaxis in stabiele concentratiegradiënten

Source: Morales-Soto, N. et. al., Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays. J. Vis. Exp. (2015)This video demonstrates a swarming assay using bacteria that express green fluorescent protein to observe surface motility on soft agar. Time-lapse imaging captures coordinated expansion and tendril formation, highlighting bacterial surface motility.

Swarming Assay for Visualizing Bacterial Surface Motility

Source: Chu, W., and Zhuang, X. Visualizing Bacterial Motility Based on a Color Reaction. J. Vis. Exp. (2022)This video demonstrates a redox dye-based semisolid agar assay for assessing bacterial motility by visually distinguishing motile and non-motile strains.

Visualization of Bacterial Motility Using Redox Dye–Supplemented Semisolid Medium

Source: Scheidweiler, D., et al. Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales. J. Vis. Exp. (2020).This video demonstrates the use of a microfluidic device to quantify bacterial motility through a porous matrix composed of randomly arranged pillars. Fluorescently labeled bacteria are introduced into the device, imaged via microscopy as they move through the porous matrix into the observation channel, and analyzed...

Assessing Bacterial Motility Using a Microfluidic Device

Zwermen motiliteit wordt beïnvloed door fysische en omgevingsfactoren. We beschrijven een tweefasenprotocol en richtlijnen om de uitdagingen die gewoonlijk worden geassocieerd met zwermtestvoorbereiding en gegevensverzameling te omzeilen. Een macroscopische beeldtechniek wordt gebruikt om gedetailleerde informatie over zwermgedrag te verkrijgen die niet wordt geleverd door huidige analysetechnieken.

Voorbereiding, Imaging, en kwantificeren van Bacteriële Surface Motility Testen

In deze studie werd een nano-microfluïdische stroom kamer gebruikt om te visualiseren en functioneel karakteriseren het trillen beweeglijkheid van Xylella fastidiosa, een bacterie die de ziekte van Pierce veroorzaakt in Grapevine.

Visualisatie van Twitching Beweeglijkheid en karakterisering van de rol van de<em&gt; PilG</em&gt; in<em&gt; Xylella fastidiosa</em

Dit artikel beschrijft de high-throughput assay die met succes heeft bewezen grote bibliotheken van kleine moleculen te screenen op hun potentiële vermogen om cellulaire niveaus van cyclisch di-GMP manipuleren Pseudomonas aeruginosa, die een nieuw krachtig hulpmiddel voor antibacteriële geneesmiddelen en beproeving in.

Oprichting van een High-throughput Setup voor het screenen van kleine moleculen die Moduleer c-di-GMP Signalering in<em&gt; Pseudomonas aeruginosa</em

Veel bacteriën gebruiken flagella-gedreven beweeglijkheid om door hun omgeving te navigeren en gunstige omgevingen te koloniseren, zowel individueel als als collectief. Hier wordt het gebruik aangetoond van drie gevestigde methoden die motiliteit als selectietool gebruiken om componenten / paden te identificeren die bijdragen aan zwem- en zwermmotiliteit.

Onderzoek naar flagella-gedreven beweeglijkheid in Escherichia coli door drie gevestigde technieken in een serie toe te passen

Hier beschrijven we de constructie van nulmutanten van Aeromonas in specifieke glycosyltransferasen of regio's die glycosyltransferasen bevatten, de beweeglijkheidstests en flagellenzuivering die worden uitgevoerd om de betrokkenheid en functie van hun gecodeerde enzymen in de biosynthese van een glycaan vast te stellen, evenals de rol van deze glycaan in bacteriële pathogenese.

Generatie van null-mutanten om de rol van bacteriële glycosyltransferasen in bacteriële motiliteit op te helderen

Hier presenteren we een protocol om bacteriële motiliteit te detecteren op basis van een kleurreactie. De belangrijkste voordelen van deze methode zijn dat deze gemakkelijk te evalueren en nauwkeuriger is en geen gespecialiseerde apparatuur vereist.

Bacteriële beweeglijkheid visualiseren op basis van een kleurreactie

In dit manuscript bieden we een uitgebreid protocol voor het beoordelen van de antibioticaoverleving voor Pseudomonas aeruginosa en Staphylococcus aureus, waarbij plasmiden worden getransformeerd in P. aeruginosa en S. aureus om reporterstammen te creëren en fenotypische varianten, zoals persisters, te visualiseren door time-lapse epifluorescentiemicroscopie.

Time-Lapse Epifluorescentiemicroscopie Beeldvorming van Pseudomonas aeruginosa en Staphylococcus aureus Heterogene fenotypes

Leer een high-throughput methode voor het analyseren van motiliteit in Pseudomonas aeruginosa. Dit protocol identificeert genetische factoren die zwerm- en twitchinggedrag beïnvloeden voor microbiologisch onderzoek.

High-throughput methode voor het observeren van motiliteitsfenotypes in Pseudomonas aeruginosa

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