Este estudio fue apoyado por el Desarrollo del Programa Académico Prioritario de las Instituciones de Educación Superior de Jiangsu (PAPD) y el Proyecto de Investigación de Reforma Docente de la Universidad Farmacéutica de China (2019XJYB18).

1. Preparación del medio semisólido
<ol><li>Agar semisólido tradicional
	<ol><li>Prepare el agar semisólido tradicional de acuerdo con la receta del medio de prueba de motilidad bacteriana utilizando los ingredientes básicos11. Disuelva 10 g de triptosa, 15 g de NaCl, 4 g de agar en suficiente agua destilada, ajuste el pH a 7.2 ± 0.2 y enrasar el volumen final a 1,000 mL.</li>
		<li>Autoclave el agar a 121 °C durante 20 min, y dispense en tubos de ensayo de 10 ml como un medio semisólido de 3 cm de altura.</li>
	</ol></li>
	<li>Agar semisólido tradicional con TTC
	<ol><li>Después de esterilizar en autoclave el medio semisólido convencional, enfriarlo a 50 °C, agregar 5 ml de solución estéril de TTC al 100 ml de medio, mezclar y dispensar en tubos de ensayo de 10 ml para formar un medio semisólido de 3 cm de altura.</li>
	</ol></li>
</ol>2. Cepas bacterianas
NOTA: Se aislaron ochenta cepas del medio acuático y se identificaron utilizando un instrumento automatizado de identificación de bacterias (ver la Tabla de materiales), incluyendo Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., Vibrio spp., Klebsiella pneumoniae y Aeromonas hydrophila (Tabla 1). Staphylococcus aureus (ver la Tabla de materiales) se utilizó como un control negativo inmóvil; Escherichia coli, Pseudomonas aeruginosa y Salmonella typhimurium (ver la Tabla de materiales) se utilizaron como cepas de control positivo.
<ol><li>Identificar las cepas bacterianas de prueba que se utilizarán para el análisis de la motilidad.</li>
	<li>Incluye controles negativos inmóviles y tensiones móviles de control positivo.</li>
</ol>3. Observación de la motilidad bacteriana mejorada por TTC
<ol><li>Recoger colonias individuales de bacterias problema de las placas de agar e inocularlas en los dos medios semisólidos anteriores (pasos 1.1.2 y 1.2.1) mediante punción con agujas inoculantes.</li>
	<li>Cultivar los tubos a 37 °C en la incubadora durante 24-48 h para observar los resultados.</li>
	<li>Observe el estado de crecimiento: caracterice las bacterias como inmóviles (-) si solo la línea de punción es roja. Caracterizar las bacterias como móviles (+) si el color rojo se extiende ligeramente hacia afuera a lo largo de la línea de punción12.</li>
</ol>4. Efecto de diferentes concentraciones en agar sobre la motilidad bacteriana
<ol><li>Prepare medios semisólidos que contengan agar 0.3%, 0.5% y 0.8% e inoculos por punción, como se describió anteriormente. Observe los resultados después de 24-48 h de incubación.</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>

Los autores no tienen conflictos de intereses que revelar.

La detección de la motilidad bacteriana por el método del medio semisólido se ve afectada por muchos factores13,14. Las condiciones de crecimiento bacteriano, como el oxígeno (aeróbico en la superficie del agar, no aeróbico en la parte inferior del tubo con el medio semisólido), el pH y la temperatura, pueden afectar la viabilidad de los flagelos bacterianos, lo que puede conducir a una motilidad reducida o incluso a la pérdida de la motilidad<sup class="...

La motilidad bacteriana juega un papel crítico en la patogenicidad bacteriana, la formación de biopelículas y la resistencia a los medicamentos1. La motilidad bacteriana está estrechamente asociada con la patogenicidad y es necesaria para la colonización bacteriana durante la infección temprana de las células huésped2. La formación de biopelículas está estrechamente relacionada con la motilidad bacteriana, donde las bacterias se adhieren a la superficie de los medios sólidos a través de la motilidad. Durante mucho tiempo se ha considerado que la motilidad bacteriana se correlaciona positivamente con la formación de biopelículas. Un alto grado de resistencia bacteriana a los medicamentos debido al biofilm puede conducir a infecciones persistentes que son una amenaza para la salud humana 3,4,5. Por lo tanto, es importante detectar la motilidad bacteriana. La prueba de motilidad bacteriana se utiliza principalmente para examinar la motilidad de diferentes formas de bacterias en el estado vivo, lo que puede determinar indirectamente la presencia o ausencia de flagelos y, por lo tanto, tiene un papel importante en la identificación de bacterias.
Existen métodos directos e indirectos para detectar la motilidad bacteriana6. Como las bacterias con flagelos muestran motilidad, es posible detectar si las bacterias son móviles indirectamente detectando la presencia o ausencia de flagelos. Por ejemplo, es posible detectar la motilidad indirectamente mediante microscopía electrónica y tinción flagelar para indicar que las bacterias son móviles. También es posible detectar por métodos directos, como la caída de suspensión y los métodos de punción semisólida. 
El método de punción semisólida comúnmente utilizado en los laboratorios de microbiología de pregrado para detectar la motilidad bacteriana inocula a las bacterias en la punción en el medio de agar semisólido que contiene 0.4-0.8% de agar, de acuerdo con la dirección del crecimiento bacteriano. Si las bacterias crecen a lo largo de la línea de punción para extenderse, aparecen rastros de crecimiento en forma de nube (en forma de cepillo), lo que indica la presencia de flagelos y, por lo tanto, motilidad. Si no hay rastros de crecimiento de la línea de perforación, la bacteria no es flagelada ni móvil. 
Sin embargo, este método tiene sus inconvenientes: las bacterias son incoloras y transparentes, la actividad flagelar se ve afectada por las características fisiológicas de las bacterias vivas y otros factores, y la concentración de agar y el pequeño diámetro del tubo de ensayo. Además, las bacterias aeróbicas solo son adecuadas para el crecimiento en la superficie del agar, lo que afecta la observación de la motilidad bacteriana. Por lo tanto, para mejorar este experimento, se agregó cloruro de 2,3,5-trifeniltetrazolio (TTC) (incoloro) al medio para establecer un método más confiable e intuitivo para determinar la motilidad bacteriana que el método actual de punción directa utilizando deshidrogenasas intracelulares para catalizar la formación de un producto rojo de TTC 7,8,9,10.

<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

La motilidad bacteriana es crucial para la patogenicidad bacteriana, la formación de biopelículas y la resistencia a los medicamentos. La motilidad bacteriana es crucial para la invasión y/o diseminación de muchas especies patógenas. Por lo tanto, es importante detectar la motilidad bacteriana. Las condiciones de crecimiento bacteriano, como el oxígeno, el pH y la temperatura, pueden afectar el crecimiento bacteriano y la expresión de flagelos bacterianos. Esto puede conducir a una motilidad reducida o incluso a la pérdida de motilidad, lo que resulta en la evaluación inexacta de la motilidad bacteriana. Basado en la reacción de color del cloruro de 2,3,5-trifenil tetrazolio (TTC) por deshidrogenasas intracelulares de bacterias vivas, TTC se agregó al agar semisólido tradicional para la detección de la motilidad bacteriana. Los resultados mostraron que este método de agar semisólido TTC para la detección de la motilidad bacteriana es simple, fácil de operar y no involucra instrumentos grandes y costosos. Los resultados también mostraron que la mayor motilidad se observó en medio semisólido preparado con agar 0,3%. En comparación con el medio semisólido tradicional, los resultados son más fáciles de evaluar y más precisos.

Tanto las cepas estándar como las cepas aisladas se compararon para la detección de la motilidad, y los resultados se muestran en la Tabla 1. Debido a la ausencia de flagelos, Staphylococcus aureus y Klebsiella pneumoniae solo crecieron a lo largo de la línea inoculada en medios semisólidos tradicionales y TTC. En contraste, Pseudomonas aeruginosa, Escherichia coli y Salmonella typhimurium mostraron crecimiento en todas las direcciones alrededor de la lín...

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

Aquí, presentamos un protocolo para detectar la motilidad bacteriana basada en una reacción de color. Las ventajas clave de este método son que es fácil de evaluar y más preciso, y no requiere equipo especializado.

Visualización de la motilidad bacteriana basada en una reacción de color

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

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

Research

Education

JoVE Core

JoVE Journal

Microbiology

Biology

Unit

Microbial Cell Structure and Function        

SCIENTISTS IN ACTION

3.9K vistas.  China Pharmaceutical University. Aquí, presentamos un protocolo para detectar la motilidad bacteriana basada en una reacción de color. Las ventajas clave de este método son que es fácil de evaluar y más preciso, y no requiere equipo especializado.

Video: 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.

Visualizar solo los complejos moleculares En Vivo Uso de microscopía de fluorescencia avanzada

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 ...

Investigación

Biología

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.

Un dispositivo de microfluidos para la cuantificación de la quimiotaxis bacteriana en gradientes de concentración estable

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.

Motilidad de moléculas individuales y grupos de cinesina-5 cin8 bidireccional purificada a partir de células de S. cerevisiae

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

Bioquímica

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.

Visualización de la resistencia bacteriana mediante sondas fluorescentes antibióticas

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

Inmunología e Infección

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.

Visualización cinética de interacciones bacterianas entre especies de una sola célula

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.

Preparación, Imaging, y cuantificación de la superficie bacteriana Motilidad ensayos

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.

Caracterización biofísica de las funciones de motor flagelar

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.

Investigación De La Motilidad Impulsada Por Flagelos En Escherichia coli mediante la Aplicación De Tres Técnicas Establecidas En Una 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.

Visualización de bacterias anaeróbicas internalizadas dentro de las células endoteliales del huésped

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.

Ensayo de co-incubación bacteriana: una técnica basada en microscopía de fluorescencia para visualizar la competencia bacteriana intraespecífica a nivel de una sola célula