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

Este estudo foi apoiado pelo Desenvolvimento do Programa Acadêmico Prioritário das Instituições de Ensino Superior de Jiangsu (PAPD) e pelo Projeto de Pesquisa de Reforma do Ensino da Universidade Farmacêutica da China (2019XJYB18).

1. Preparo do meio semissólido
<ol><li>Ágar semissólido tradicional
	<ol><li>Preparar o ágar semissólido tradicional de acordo com a receita do meio teste de motilidade bacteriana utilizando os ingredientes básicos11. Dissolver 10 g de triptose, 15 g de NaCl, 4 g de ágar em água destilada suficiente, ajustar o pH para 7,2 ± 0,2 e perfazer o volume final para 1.000 mL.</li>
		<li>Autoclavar o ágar a 121 °C por 20 min e distribuí-lo em tubos de ensaio de 10 mL como meio semissólido de 3 cm de altura.</li>
	</ol></li>
	<li>Ágar semissólido tradicional com TTC
	<ol><li>Após a autoclavagem do meio semissólido convencional, resfriá-lo a 50 °C, adicionar 5 mL de solução TTC 1% estéril a 100 mL de meio, misturar e dispensá-lo em tubos de ensaio de 10 mL para formar um meio semissólido de 3 cm de altura.</li>
	</ol></li>
</ol>2. Cepas bacterianas
NOTA: Oitenta cepas foram isoladas do ambiente aquático e identificadas usando um instrumento automatizado de identificação bacteriana (ver Tabela de Materiais), incluindo Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., Vibrio spp. , Klebsiella pneumoniae e Aeromonas hydrophila (Tabela 1). Staphylococcus aureus (ver Tabela de Materiais) foi utilizado como controle negativo não móvel; Escherichia coli, Pseudomonas aeruginosa e Salmonella typhimurium (ver Tabela de Materiais) foram utilizadas como cepas controle positivas.
<ol><li>Identificar cepas bacterianas de teste a serem usadas para análise de motilidade.</li>
	<li>Inclua controles negativos não móveis e cepas de controle positivo móveis.</li>
</ol>3. Observação da motilidade bacteriana reforçada pelo TTC
<ol><li>Seleccionar colónias únicas de bactérias de ensaio a partir de placas de ágar e inocular-as nos dois meios semissólidos acima referidos (passos 1.1.2 e 1.2.1) por punção utilizando agulhas inoculantes.</li>
	<li>Cultivar os tubos a 37 °C na incubadora por 24-48 h para observar os resultados.</li>
	<li>Observe o estado de crescimento: caracterize as bactérias como não móveis (-) se apenas a linha de punção for vermelha. Caracterizar as bactérias como móveis (+) se a cor vermelha se espalhar levemente para fora ao longo da linha de punção12.</li>
</ol>4. Efeito de diferentes concentrações de ágar sobre a motilidade bacteriana
<ol><li>Preparar meios semissólidos contendo 0,3%, 0,5% e 0,8% de ágar e inocular-os por punção, conforme descrito acima. Observe os resultados após 24-48 h de incubação.</li>
</ol>

<ol>
	<li>Jordan, E. O., Caldwell, M. E., Reiter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+motility.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+motility+and+its+role+during+initial+stage+of+pathogenesis.">Bacterial motility and its role during initial stage of pathogenesis.</a> Journal of Microbiology. 26 (5), 68-70 (2006).</li><li>Ding, S. S., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+flagella-dependent+motility+and+biofilm+in+bacteria+-+A+review.">Relationship between flagella-dependent motility and biofilm in bacteria - A review.</a> Acta Microbiologica Sinica. 49 (4), 417-422 (2009).</li><li>Zeng, J., Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+mechanism+of+bacterial+resistance+and+tolerance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+mechanism+of+bacterial+flagellum+motility,+adhesion+and+immune+escape.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microbiology: laboratory theory and application. Third edition. , (2015).</li><li>Ball, R. J., Sellers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+motility+medium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Pseudomonas+aeruginosa+biofilm+dispersal+by+a+cyclic-di-GMP+phosphodiesterase+with+a+putative+hypoxia-sensing+domain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+site-directed+mutagenesis+at+the+GGEEF+domain+of+the+biofilm+forming+GGEEF+protein+from+Vibrio+cholerae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+colorimetric+microplate+test+of+phage+lysis+in+Salmonella+enterica.">Simple colorimetric microplate test of phage lysis in Salmonella enterica.</a> Journal of Microbiological Methods. 69 (2), 394-398 (2007).</li><li>Difco Laboratories. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difco+manual:+Dehydrated+culture+media+and+reagents+for+microbiology.">Difco manual: Dehydrated culture media and reagents for microbiology.</a> Difco Laboratories. , (1984).</li><li>Tittsler, R. P., Sandholzer, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+semi-solid+agar+for+the+detection+of+bacterial+motility.">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/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. 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=Spatial+arrangement+of+several+flagellins+within+bacterial+flagella+improves+motility+in+different+environments.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+agar+concentration+on+the+growth+and+morphology+of+submerged+colonies+of+motile+and+non-motile+bacteria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+mechanism+for+how+sigma+factor+AlgT+and+transcriptional+regulator+AmrZ+inhibit+twitching+motility+in+Pseudomonas+aeruginosa.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+and+invasion+of+Neisseria+meningitidis+to+host+cells+is+related+to+surface+hydrophobicity,+bacterial+cell+size+and+capsule.">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>

Os autores não têm conflitos de interesse a declarar.

A detecção da motilidade bacteriana pelo método do meio semissólido é afetada por váriosfatores13,14. Condições de crescimento bacteriano, como oxigênio (aeróbio na superfície do ágar, não aeróbio no fundo do tubo com o meio semissólido), pH e temperatura, podem afetar a viabilidade dos flagelos bacterianos, o que pode levar à redução da motilidade ou mesmo à perda da motilidade15. Além disso, algumas bactérias do tipo muco como sua motilidade podem ser afetadas pela produção de podoconjugados.
A adição de TTC ao meio semissólido auxilia na observação da motilidade. As bactérias fermentativas com poder motriz podem crescer em todas as direções ao longo da linha de punção após a incubação. Assim, o meio ao redor da linha de punção torna-se vermelho. As bactérias não fermentativas com poder motriz apresentam baixo teor de oxigênio na parte inferior do meio. Assim, essas bactérias crescem mal, de modo que apenas a camada superior do meio é vermelha. Bactérias sem força motriz só podem crescer na linha de inoculação, e apenas a linha de punção aparece vermelha. 
Ao detectar a motilidade bacteriana com meio TTC-semissólido, quanto maior o tempo de cultivo, mais evidentes são os resultados, principalmente com menores concentrações de ágar. Se o resultado for difícil de interpretar, o tempo de cultura deve ser estendido adequadamente. Isso pode estar relacionado à concentração de ágar do meio semissólido, ao número de bactérias e à sua motilidade16. Além disso, este método revelou que algumas cepas de E. coli e P. aeruginosa não puderam crescer em meio ágar semissólido convencional e apresentaram apenas uma leve coloração vermelha na superfície do meio em meio ágar semissólido TTC. Isso pode ser devido à produção de cápsulas por essas cepas, o que afeta sua motilidade17. Esse fenômeno também ocorre em Neisseria meningitidis18 devido à sua cápsula. Em conclusão, a detecção da motilidade bacteriana utilizando um meio semissólido cromogênico contendo TTC reduz a influência de fatores bacterianos nos resultados dos testes e torna os resultados mais fáceis de serem observados a olho nu. A vantagem de uma alta taxa de detecção torna este um método eficaz que pode substituir o meio semissólido tradicional para detectar a motilidade bacteriana.

A motilidade bacteriana desempenha um papel crítico na patogenicidade bacteriana, na formação de biofilme e na resistência a drogas1. A motilidade bacteriana está intimamente associada à patogenicidade e é necessária para a colonização bacteriana durante a infecção precoce das células dohospedeiro2. A formação de biofilme está intimamente relacionada à motilidade bacteriana, onde as bactérias aderem à superfície do meio sólido através da motilidade. A motilidade bacteriana tem sido considerada positivamente correlacionada com a formação de biofilme. Um alto grau de resistência bacteriana a drogas devido ao biofilme pode levar a infecções persistentes que são uma ameaça à saúde humana 3,4,5. Portanto, é importante detectar a motilidade bacteriana. O teste de motilidade bacteriana é usado principalmente para examinar a motilidade de diferentes formas de bactérias no estado vivo, que podem determinar indiretamente a presença ou ausência de flagelos e, portanto, tem um papel importante na identificação de bactérias.
Existem métodos diretos e indiretos para detectar a motilidade bacteriana6. Como as bactérias com flagelos apresentam motilidade, é possível detectar se as bactérias são móveis indiretamente, detectando a presença ou ausência de flagelos. Por exemplo, é possível detectar a motilidade indiretamente por microscopia eletrônica e coloração flagelar para indicar que as bactérias são móveis. Também é possível detectar por métodos diretos, como queda de suspensão e punção semissólida. 
O método de punção semissólido comumente utilizado em laboratórios de microbiologia de graduação para detectar motilidade bacteriana inocula a bactéria na punção em meio ágar semissólido contendo 0,4-0,8% de ágar, de acordo com a direção do crescimento bacteriano. Se a bactéria crescer ao longo da linha de punção para se espalhar, aparecem vestígios de crescimento semelhantes a nuvens (semelhantes a escovas), indicando a presença de flagelos e, portanto, motilidade. Se não houver vestígios de crescimento na linha de punção, a bactéria não é flagelada nem móvel. 
No entanto, este método tem suas desvantagens: as bactérias são incolores e transparentes, a atividade flagelar é afetada pelas características fisiológicas das bactérias vivas e outros fatores, e a concentração de ágar e o pequeno diâmetro do tubo de ensaio. Além disso, bactérias aeróbias são adequadas apenas para o crescimento na superfície do ágar, afetando a observação da motilidade bacteriana. Assim, para aprimorar este experimento, o cloreto de 2,3,5-trifeniltetrazólio (TTC) (incolor) foi adicionado ao meio para estabelecer um método mais confiável e intuitivo de determinação da motilidade bacteriana do que o método atual de punção direta, utilizando desidrogenases intracelulares para catalisar a formação de um produto vermelho do 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

A motilidade bacteriana é crucial para a patogenicidade bacteriana, formação de biofilme e resistência a drogas. A motilidade bacteriana é crucial para a invasão e/ou disseminação de muitas espécies patogênicas. Portanto, é importante detectar a motilidade bacteriana. Condições de crescimento bacteriano, como oxigênio, pH e temperatura, podem afetar o crescimento bacteriano e a expressão de flagelos bacterianos. Isso pode levar à redução da motilidade ou mesmo à perda da motilidade, resultando na avaliação imprecisa da motilidade bacteriana. Com base na reação de cor do cloreto de 2,3,5-trifenil tetrazólio (TTC) por desidrogenases intracelulares de bactérias vivas, o TTC foi adicionado ao ágar semissólido tradicional para detecção da motilidade bacteriana. Os resultados mostraram que este método de ágar semissólido TTC para detecção de motilidade bacteriana é simples, fácil de operar e não envolve instrumentos grandes e caros. Os resultados também mostraram que a maior motilidade foi observada em meio semissólido preparado com 0,3% de ágar. Em comparação com o meio semissólido tradicional, os resultados são mais fáceis de avaliar e mais precisos.

Tanto as cepas padrão quanto as isoladas foram comparadas para detecção da motilidade, e os resultados são mostrados na Tabela 1. Devido à ausência de flagelos, Staphylococcus aureus e Klebsiella pneumoniae cresceram apenas ao longo da linha inoculada em ambos os meios semissólido tradicional e TTC. Em contraste, Pseudomonas aeruginosa, Escherichia coli e Salmonella typhimurium apresentaram crescimento em todas as direções ao redor da linhagem inoculada após cultivo por 24 h em meio TTC semissólido. Isso ficou ainda mais evidente após 48 h de cultura (Figura 1). Embora as bactérias tenham crescido em todas as direções no meio semissólido tradicional, foi muito mais difícil de visualizar do que no meio TTC devido ao pequeno número de bactérias no lado externo da linha inoculada.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63706/63706fig01.jpg"> Figura 1: Resultados do teste de motilidade utilizando meio semissólido TTC. Staphylococcus aureus à esquerda, Escherichia coli à direita. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63706/63706fig01large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2" rowspan="2">Coar</td>
			<td colspan="2">Meio semissólido com 0,4% de ágar</td>
			<td colspan="2">Meio semissólido com 0,4% ágar e 0,005% TTC</td>
		</tr><tr><td>24 horas</td>
			<td>48 horas</td>
			<td>24 horas</td>
			<td>48 horas</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">(8)</td>
			<td>7</td>
			<td>8</td>
			<td>8</td>
			<td>8</td>
		</tr><tr><td colspan="2">A. hidrophila (20)</td>
			<td>18</td>
			<td>20</td>
			<td>20</td>
			<td>20</td>
		</tr><tr><td colspan="2">( 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">Os números indicam os números de cepas positivas (+) e negativas (-).</td>
		</tr></tbody></table>Tabela 1: Comparação da motilidade bacteriana. 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63706/63706fig02.jpg"> Figura 2: Observação da atividade da motilidade de Escherichia coli em diferentes concentrações de ágar. Da esquerda para a direita: 0,3%, 0,5%, 0,8% ágar. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63706/63706fig02large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
A influência da concentração de ágar na motilidade bacteriana é mostrada na Figura 2. Verificou-se que a maior motilidade foi observada em meio semissólido preparado com 0,3% de ágar. A cor média no tubo ficou quase inteiramente vermelha. Em contraste, a área de difusão vermelha diminuiu, e a difusão foi prolongada com o aumento da concentração de ágar.

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

Aqui, apresentamos um protocolo para detectar motilidade bacteriana baseado em uma reação de cor. As principais vantagens deste método são que é fácil de avaliar e mais preciso, e não requer equipamentos especializados.

Visualizando a motilidade bacteriana com base em uma reação de cor

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

Nanyang Technological University

Research

JoVE Journal

Biology

3.8K visualizações.  China Pharmaceutical University. Aqui, apresentamos um protocolo para detectar motilidade bacteriana baseado em uma reação de cor. As principais vantagens deste método são que é fácil de avaliar e mais preciso, e não requer equipamentos especializados.

Vídeo: 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.

Um dispositivo micro para Quantificar Quimiotaxia bacteriana em gradientes de concentração estável

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

Biologia

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.

Quantificando a motilidade de enxame de superfície bacteriana em placas de gradiente indutor

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

Bioquímica

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.

Motilidade de Moléculas Únicas e Aglomerados de Kinesin-5 Cin8 Bidiso

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.

Gerando paisagens químicas controladas e dinâmicas para estudar comportamento microbiano

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

Bioengenharia

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.

Visualização da resistência bacteriana usando sondas de antibióticos fluorescentes

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

Imunologia e Infecção

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.

Visualização cinética de interespécies interespécies unicelulares interespécies

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.

Preparação, Imaging, e quantificação de Motilidade Ensaios superfície da bactéria

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.

Caracterização biofísica das funções Flagellar motor

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.

Investigando a Motilidade Impulsionada por Flagella em Escherichia coli aplicando três técnicas estabelecidas em uma série

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.remotexs.ntu.edu.sg/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.

Ensaio de co-incubação bacteriana: uma técnica baseada em microscopia de fluorescência para visualizar a competição bacteriana intraespecífica no nível de célula única