Name: protocol for biofilm streamer formation in a microfluidic device with micro-pillars
Uploaded: 2014-08-20T14:00:00.000+00:00
Duration: 7 min 19 s
Description: Watch this Scientific Journal Video about Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars at JoVE.com

The authors would like to thank Professor Howard Ceri from the Biological Sciences Department of the University of Calgary for providing bacterial strains. A. Kumar acknowledges support from NSERC. T. Thundat acknowledges financial support from the Canada Excellence Research Chair (CERC) program. The authors would also like to acknowledge help from Ms. Zahra Nikakhtari for help with videography.

Executar os protocolos experimentais aqui na ordem descrita abaixo. Protocolos de microfabricação para criar a plataforma de microfluidos são discutidos no Passo 1 Passo 2 descreve o protocolo de cultura de bactérias (Figura 2), e Passo 3 refere-se a montagem da instalação experimental (Figura 3). Finalmente, o passo real experimental é descrito no Passo 4. 1 Chip Fabricação Procedimento NOTA: Proper procedimentos de segurança devem ser seguidas para os processos descritos abaixo. Consulte o oficial de segurança institucional para mais detalhes. <ol><li> Projete a máscara com um software apropriado (por exemplo, L-Edit). O desenho é constituído por um canal principal micro-canal de largura de 625 m. A região central do canal contém uma matriz de micro-postos 50 um de diâmetro, espaçados de 25 um (ver a ficheiros suplementar). </li><li> Imprima este projeto em vidro (5 &quot;x 5&quot; vidro soda cal), Que tem uma espessura de 0,09 &quot;e é revestido por cerca de 70 nm de espessura da camada de crómio (Cr), usando mascaramento de modo a preparar uma máscara de foto. Utilização AZ400K programador durante 1 min. Seguida, gravar a camada de Cr Cr usando cáustico durante cerca de 1 min a utilização de acetona para retirar a resistir e limpá-lo com uma solução fria de piranha. (H 2 SO 4 e H 2 O 2 em uma proporção de 3: 1). </li><li> Fotolitografia <ol><li> Limpe uma bolacha 4 &quot;de silício padrão quimicamente com solução piranha para 20 min. </li><li> Lavar o wafer com água DI e secá-la. </li><li> Aquece-se a bolacha em uma placa quente (200 ° C durante 15 min). </li><li> Cubra o wafer de silício com photoresist. Aqui, o revestimento fotoprotector positivo AZ4620 foi revestido por centrifugação sobre uma bolacha de silício, a 2000 rpm durante 25 segundos para se obter uma camada de espessura de 12,5 | iM. </li><li> Remover todo o solvente por cozimento da bolacha mole em uma placa quente, flutuando a bolacha durante 90 segundos em fluxo de azoto a 100 ° C. Em seguida, mantê-lo no vácuo a tele mesma temperatura durante 60 seg. </li><li> Coloque a bolacha em uma caixa escura por 24 horas para a desidratação. </li><li> Expor a pastilha à luz ultravioleta, a fim de transferir o padrão concebido para o foto-resistente. </li><li> Mergulha-se o wafer foto-resistente na solução de revelação (AZ400K) para 240 seg. Em seguida, lavar a bolacha com álcool isopropílico e secá-lo, colocando em uma corrente de azoto gasoso. </li></ol></li><li> ICP-DRIE (plasma indutivamente acoplado - Deep Reactive Ion Etching) Processo <ol><li> Aplicar DRIE gravura. Escolha a profundidade etch adequado de acordo com a profundidade final necessária para o dispositivo (50 mm nesta investigação). Photoresist actua como uma camada de máscara, durante este processo. </li><li> Remover o fotorresistente remanescente com acetona e limpar a bolacha. </li></ol></li><li> PDMS (polidimetilsiloxano) Fundição <ol><li> Use triclorometilsilano (EMT) para silanização o molde mestre de silício. Despeje 2 ou 3 gotas de triclorometilsilano em um frasco e colocá-lo num exsicador junto aomolde mestre de silício. Permitir 2-3 horas para o processo de silanização para ser concluído. </li><li> Num recipiente separado, misturar a base de silicone Sylgard 184 com o agente de cura pela relação de peso de 10: 1 para preparar PDMS. Desgasificar o PDMS, submetendo-o a condições de vácuo (cerca de 2 h). </li><li> Coloque o molde mestre de silício em um suporte. Em seguida, despejar o PDMS sobre o molde mestre de silício para formar o selo de PDMS. Certifique-se de que não se formam bolhas de PDMS durante este processo. </li><li> Curar o PDMS durante 2 horas a 80 ° C. </li><li> Retire o selo PDMS do molde mestre. Em seguida, corte os PDMS carimbar em microchips separados. Finalmente, a utilização de um núcleo de corte de furos para a entrada (s) e saída (s). </li></ol></li><li> Colagem de PDMS para Vidro <ol><li> Expor a lamínula e PDMS selo de plasma de oxigênio por 30 seg. Vínculo PDMS selo à lamela. </li><li> Para obter uma vedação adequada entre o carimbo de PDMS e o deslizamento da tampa, o dispositivo de recozimento, colocando-o num forno a 70 ° C durante 10 min. </li&gt; </ol></li></ol> 2 Cultura bacteriana NOTA: protocolos adequados de biossegurança devem ser seguidas para os passos 2-4. Consulte o oficial de segurança institucional para mais detalhes. <ol><li> Prepare placas LB Agar <ol><li> Adicionar 20 g de meio de Luria-Bertani (LB) agar (Miller) pós e 500 ml de água ultra-pura a um balão de 1 L. Agita-se a dissolver o pó. </li><li> Esterilizar por autoclave a 15 psi, a 121 ° C durante 15 min. </li><li> Permitir que o balão arrefeça a 50-55 ° C num banco ou num banho de água na capa de biossegurança. </li><li> Adicionar o antibiótico tetraciclina para se conseguir uma concentração final de 50 ug / ml. Misture bem por agitação. </li><li> Despeje a mistura em placas. Encha cada prato até meia - 2/3 cheio. </li><li> Chama bolhas de ar brevemente para estourá-los se formar. Bolhas de ar solidificados são difíceis de espalhar a cultura bacteriana over. </li><li> Permitir que as placas de arrefecer à temperatura ambiente durante a noite. </li><li> Quando eles são legais,colocar placas de volta para a sua manga, selar o saco, etiqueta (antibiótico e data), e armazenar a 4 ° C. NOTA: Cubra o estoque de placas com papel de alumínio, como a luz desativa muitos antibióticos. </li></ol></li><li> Caldo LB Preparação <ol><li> Adicionar 20 g de meio de Luria-Bertani (LB) broth (Miller) pós e 1 L de água ultrapura com um balão. Agita-se a dissolver o pó. </li><li> Esterilizar por autoclave a 15 psi, a 121 ° C durante 15 min. </li><li> Permitir balão arrefecer até 50-55 ° C num banco ou num banho de água na capa de biossegurança. </li><li> Adicionar o antibiótico tetraciclina para se conseguir uma concentração final de 50 ug / ml. Misture bem por agitação. </li><li> Quando estiver frio, coloque a garrafa rotulada a 4 ° C. NOTA: Cubra a garrafa com papel de alumínio, como a luz desativa muitos antibióticos. </li></ol></li><li> Cultura das bactérias em uma placa de LB Agar (Este protocolo utiliza Pseudomonas fluorescens) <ol><li> Pegue o estoque de bactérias do freezer (-80 °; C) e coloque-o no gelo. </li><li> Coloque o ° C -80 estoque bacteriana e uma placa de ágar LB dentro de uma capa de biossegurança. </li><li> Streak a estirpe bacteriana em uma placa de ágar LB em ziguezague. Cubra a placa de ágar e incubar-lo a 30 ° C durante a noite. Finalmente, a placa de armazenar no frigorífico a 4 ° C. </li></ol></li><li> Preparar a solução bacteriana (S1) <ol><li> Despeje 50 ml de caldo LB mídia para um frasco autoclavado. Realize esta operação dentro de uma capa de biossegurança. </li><li> Transferir uma única colónia bacteriana da placa de agar LB para o balão. Esta operação deverá ser realizada dentro de um capuz de biossegurança. </li><li> Colocar o balão numa incubadora com agitação a 30 ° C e 150 rpm durante um tempo suficiente (4 h). </li></ol></li><li> Preparar a solução bacteriana diluída (S2) <ol><li> Despeje 5 ml de caldo de meio LB num tubo de plástico esterilizado. </li><li> Diluir S1 por mistura com meio LB. Então, Vórtice a solução. Dilui-se a solução atingir a desejada ópticaai densidade (OD medido a 600 nm = 0,1). NOTA: experimentos biofilme normalmente empregam valores de DO nesta vizinhança. </li></ol></li></ol> 3 Prepare o Setup Experimental <ol><li> Usando pinças ligar tubos de plástico flexíveis (0.20 &quot;ID) na entrada (s) e saída (s) de que o microchip. As entradas e saídas foram previamente perfurados na porção de PDMS do microchip (passo 1.5.5). Nesta investigação , o circuito integrado é composto de duas entradas e uma saída. </li><li> Encher a seringa (s) com a solução bacteriana (solução S2) e remover todas as bolhas na seringa (s). </li><li> Conecte a ponta da seringa (s) (30 G 0.5 &quot;agulha romba) no tubo (s) de entrada. Então, conecte tubo de saída (s) para contentor de resíduos. </li></ol> 4 Execute o Experimento <ol><li> Ligar a seringa (s) para a ponta (s) seringa. </li><li> Local e correção seringa (s) para a bomba de seringa. Em seguida, coloque o microchip sob um microscópio óptico com lentes objetivas de desired ampliação (por exemplo, 40X). Cobrir o microchip com um aparelho de câmara de células vivas para manter um ambiente de temperatura constante para o crescimento bacteriano (30 ° C por P. fluorescens). </li><li> A bomba para o nível de fluxo desejado (por exemplo 10 l / h) e iniciar o bombeamento de fluidos. </li><li> Uma vez que as bactérias são introduzidas na câmara, a formação de biofilme é também iniciada. A formação de biofilme e de maturação tipicamente ocorrem ao longo de um período de várias horas ou mesmo dias. Observar e tirar fotos de crescimento do biofilme através do microscópio. </li></ol>

<ol>
	<li>Valiei, A., Kumar, A., Mukherjee, P. P., Liu, Y., Thundat, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+web+of+streamers:+biofilm+formation+in+a+porous+microfluidic+device.">A web of streamers: biofilm formation in a porous microfluidic device.</a> Lab Chip. 12, 5133-5137 (2012).</li><li>Costerton, J. 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+Biofilms:+A+Common+Cause+of+Persistent+Infections.">Bacterial Biofilms: A Common Cause of Persistent Infections.</a> Science. 284, 1318-1322 (1999).</li><li>Flemming, H. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nat Rev Microbiol. 8, 623-633 (2010).</li><li>Wong, G. C. L., O&#8217;Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+together+now:+Integrating+biofilm+research+across+disciplines.">All together now: Integrating biofilm research across disciplines.</a> MRS Bulletin. 36, 339-342 (2011).</li><li>Yazdi, S., Ardekani, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+aggregation+and+biofilm+formation+in+a+vortical+flow.">Bacterial aggregation and biofilm formation in a vortical flow.</a> Biomicrofluidics. 6, 044114 (2012).</li><li>Rusconi, R., Lecuyer, S., Guglielmini, L., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminar+flow+around+corners+triggers+the+formation+of+biofilm+streamers.">Laminar flow around corners triggers the formation of biofilm streamers.</a> J R Soc Interface. 7, 1293-1299 (2010).</li><li>Drescher, K., Shen, Y., Bassler, B. L., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+streamers+cause+catastrophic+disruption+of+flow+with+consequences+for+environmental+and+medical+systems.">Biofilm streamers cause catastrophic disruption of flow with consequences for environmental and medical systems.</a> P Natl Acad Sci USA. 110, 4345-4350 (2013).</li><li>Marty, A., Roques, C., Causserand, C., Bacchin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+bacterial+streamers+during+filtration+in+microfluidic+systems.">Formation of bacterial streamers during filtration in microfluidic systems.</a> Biofouling. 28, 551-562 (2012).</li><li>Taherzadeh, D., 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=Computational+Study+of+the+Drag+and+Oscillatory+Movement+of+Biofilm+Streamers+in+Fast+Flows.">Computational Study of the Drag and Oscillatory Movement of Biofilm Streamers in Fast Flows.</a> Biotechnol Bioeng. 105, 600-610 (2010).</li><li>Vrouwenvelder, J. S., 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=Impact+of+flow+regime+on+pressure+drop+increase+and+biomass+accumulation+and+morphology+in+membrane+systems.">Impact of flow regime on pressure drop increase and biomass accumulation and morphology in membrane systems.</a> Water Res. 44, 689-702 (2010).</li><li>Mitchell, A. C., 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=Biofilm+enhanced+geologic+sequestration+of+supercritical+CO2.">Biofilm enhanced geologic sequestration of supercritical CO2.</a> International Journal of Greenhouse Gas Control. 3, 90-99 (2009).</li><li>Soleimani, S., Van Geel, P. J., Isgor, O. B., Mostafa, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+biological+clogging+in+unsaturated+porous+media.">Modeling of biological clogging in unsaturated porous media.</a> J Contam Hydrol. 106, 39-50 (2009).</li><li>Kumar, A., 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=Microscale+confinement+features+can+affect+biofilm+formation.">Microscale confinement features can affect biofilm formation.</a> Microfluid Nanofluid. 14, 895-902 (2013).</li><li>Neethirajan, S., et al., Bhushan, B., 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=.">.</a> Encylopedia of Nanotechnology. , (2012).</li><li>Barathi, S., Vasudevan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+petroleum+hydrocarbons+by+Pseudomonas+fluorescens+isolated+from+a+petroleum-contaminated+soil.">Utilization of petroleum hydrocarbons by Pseudomonas fluorescens isolated from a petroleum-contaminated soil.</a> Environ Int. 26, 413-416 (2001).</li><li>Das, S., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+post-formation+dynamics+of+bacterial+biofilm+streamers+as+highly+viscous+liquid+jets.">Formation and post-formation dynamics of bacterial biofilm streamers as highly viscous liquid jets.</a> arXiv preprint arXiv:1312.6056. , (2013).</li><li>Shaw, T., Winston, M., Rupp, C. J., Klapper, I., Stoodley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commonality+of+elastic+relaxation+times+in+biofilms.">Commonality of elastic relaxation times in biofilms.</a> Phys Rev Lett. 93, (2004).</li><li>Berejnov, V., Djilali, N., Sinton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab-on-chip+methodologies+for+the+study+of+transport+in+porous+media:+energy+applications.">Lab-on-chip methodologies for the study of transport in porous media: energy applications.</a> Lab Chip. 8, 689-693 (2008).</li></ol>

The authors have nothing to disclose.

Nós demonstramos um dispositivo micro simples que imita meios porosos para estudar o desenvolvimento de biofilme em habitats complexos. Existem vários passos críticos que ditam o resultado das experiências. Eles incluem a geometria do dispositivo. Embora a geometria pós podem variar, de poros adequado para o espaço de modo a formar serpentinas é necessário. Além disso, Valiei et al. 1 demonstraram que a formação da flâmula ocorre apenas em um determinado intervalo das taxas de fluxo. Em t...

Recentemente, demonstramos dinâmica de formação do biofilme bacteriano em um dispositivo micro que imita meios porosos 1. Biofilme bacteriano são essencialmente colônias de bactérias da superfície agregados que são revestidas por substâncias poliméricas extracelulares (EPS) 2-4. Estas películas finas de bactérias podem formar em quase todos os nichos concebível que varia de superfícies lisas ao habitat muito mais complexa de meios porosos. Valiei et al. Uma usado um dispositivo de microfluidos com uma matriz de micro-pilares para simular uma estrutura de meio poroso estudado a formação de biofilme e neste dispositivo, em função da taxa de fluxo do fluido. Eles descobriram que em um determinado regime de fluxo, os biofilmes filamentosos, conhecidos como flâmulas começaram a surgir entre os diferentes pilares. Flâmulas pode ser amarrado a uma ou ambas as extremidades a superfícies sólidas, mas o resto da estrutura é suspenso num líquido. Formação Streamer normalmente começa depois de uma primeira camada de biofilme foi formado e seu formatoion pode ditar a evolução a longo prazo de biofilme em tais habitats complexos. Recentemente, vários pesquisadores têm investigado a dinâmica de formação de serpentina. Yazdi et al. 5 mostrou que as serpentinas podem formar-se os fluxos de turbilhão originários a partir de uma bolha de oscilação. Numa outra experiência, Rusconi et al. 6 investigaram o efeito de curvatura do canal e da geometria do canal sobre a formação de fitas. Eles descobriram que as fitas podem se formar em trechos curvos de microcanais e morfologia de galhardetes está relacionada à mobilidade. Pesquisas recentes têm demonstrado que os galhardetes pode ter amplas repercussões em vários cenários naturais e artificiais como eles podem atuar como precursores da formação de estruturas maduras nas interfaces porosas, levar à proliferação de biofilme rápida e catastrófica em um sistemas biomédicos, e também causar fluxo-substancial interações estrutura, etc 1,7-9. Flâmulas biofilme muitas vezes formam ihabitats n complexos, tais como meios porosos. Crescimento do biofilme Entendimento em ambiente de meios porosos é relevante a vários processos ambientais e industriais, tais como o tratamento biológico de águas residuais 10, mantendo a integridade de poços em situações como a captura de CO 2 11 e entupimento dos poros no solo 12. Observando a formação de biofilme em tais habitats complexos, muitas vezes pode ser um desafio, devido à opacidade de meios porosos. Em tais situações, as plataformas de mídia porosos microfluídicos com base pode ser extremamente vantajoso, pois permitem em tempo real e monitoramento in situ. Outra vantagem da microfluídica é a capacidade de construir vários reatores em uma única plataforma bio-microfluídicos e, simultaneamente, permitir o monitoramento e / ou incorporação de sensores online. A flexibilidade para implementar várias experiências de laboratório em um dispositivo e a capacidade de recolher dados pertinentes significativas para a análise estatística rigorosa é um adv importanteantage de sistemas microfluídicos 13,14. No contexto da discussão acima, dinâmica de formação flâmula compreensão em um ambiente de meio poroso seria benéfico para várias aplicações. No presente estudo, nós desenvolvemos o protocolo para investigar a formação de um dispositivo de serpentina de em que mimetiza meios porosos. Confecção da plataforma microfluídica, são descritos os passos necessários para a cultura de células e experimentação. Nas nossas experiências, a estirpe bacteriana tipo selvagem de Pseudomonas fluorescens foi empregue. P fluorescens, que se encontra naturalmente no solo, tem um papel fundamental na manutenção da ecologia do solo 15. A estirpe bacteriana utilizada foi geneticamente manipulada para expressar a proteína fluorescente verde (GFP) constitutivamente.

<table><tbody><tr><td>Flourescent Microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>LB agar</td><td>Fisher</td><td>BP1425-500</td><td>suspend 40 g in 1 L of purified water</td></tr><tr><td>LB broth</td><td>Fisher</td><td>BP1427-500</td><td>suspend 20 g in 1 L of purified water</td></tr><tr><td>Biosafety hood</td><td>Microzone corporation</td><td></td><td></td></tr><tr><td>Petri dish</td><td>Fisher</td><td>875712</td><td>sterile 100 mm x 15 mm polystyrene Petri dish</td></tr><tr><td>Incubator shaker</td><td>New Brunswick Scientific</td><td></td><td>Excella E24 incubator shaker series</td></tr><tr><td>50 ml sterilized centrifuge tube</td><td>Corning</td><td>430828</td><td>Polypropylene RNase-/DNase-free</td></tr><tr><td>Tetracycline free base</td><td>MP Biomedicals</td><td>103012</td><td>50 &amp;#956;g/ml</td></tr><tr><td>SYLGARD 184 silicone</td><td>Dow Corning Corporation</td><td>68037-59-2</td><td>Elastomer Base and curing agent</td></tr><tr><td>Positive photoresist (AZ4620)</td><td></td><td></td><td></td></tr><tr><td>Plastic tube</td><td>Cole-Parmer</td><td></td><td></td></tr></tbody></table>

protocol for biofilm streamer formation in a microfluidic device with micro-pillars

Várias espécies bacterianas possuem a capacidade para ligar a superfícies e colonizar-los sob a forma de películas finas chamadas biofilmes. Os biofilmes que crescem em meios porosos são relevantes para vários processos industriais e ambientais, como o tratamento de águas residuais e sequestro de CO 2. Usamos Pseudomonas fluorescens, uma bactéria Gram-negativa aeróbica, para investigar a formação de biofilmes em um dispositivo de microfluidos, que imita meios porosos. O dispositivo de microfluidos consiste de uma matriz de micro-mensagens, que foram fabricados utilizando-litografia macia. Subsequentemente, a formação de biofilmes em estes dispositivos com o fluxo foi investigado e que demonstram a formação de biofilmes filamentosos conhecidos como serpentinas em nosso dispositivo. Os protocolos detalhados para fabricação e montagem de dispositivos microfluídicos são fornecidos aqui, juntamente com os protocolos de cultura de bactérias. Os procedimentos detalhados para a experimentação com o dispositivo micro também são apresentados juntamente com o representanteresultados.

Usando o protocolo de microfabricação acima mencionado, um dispositivo de microfluidos com base de PDMS foi construído. Figura 1 mostra o microscópio electrónico de varrimento (SEM) de imagens do dispositivo de PDMS. Figura 1a mostra a secção de entrada do dispositivo. Uma entrada de tipo forquilha é criado para equalizar a pressão do outro lado da cabeça do dispositivo. Outras imagens de SEM mostraram igualmente que as paredes da coluna são quase verticais (Figura 1b...

Watch this Scientific Journal Video about Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars at JoVE.com

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protocolo para a formação de biofilme Streamer em um dispositivo micro com micro-pilares

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

protocol-for-biofilm-streamer-formation-microfluidic-device-with

Research

JoVE Journal

Bioengineering

12.1K visualizações.  University of Alberta. Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated. 

Vídeo: Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa e Saccharomyces cerevisiae Biofilme em Células de Fluxo

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Imunologia e Infecção

Fabrication and Visualization of Capillary Bridges in Slit Pore Geometry

A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. High aspect ratio hydrophobic pillars are fabricated and functionalized to render their top surfaces hydrophilic. The combination of a physical feature (the pillar) with a chemical boundary (the hydrophilic film on the top of the pillar) provides both a physical and chemical heterogeneity that pins the triple contact line, a necessary feature to create stable long but narrow capillary bridges. The substrates with the pillars are attached to glass slides and secured into custom holders. The holders are then mounted onto four axis microstages and positioned such that the pillars are parallel and facing each other. The capillary bridges are formed by introducing a fluid in the gap between the two substrates once the separation between the facing pillars has been reduced to a few hundred micrometers. The custom microstage is then employed to vary the height of the capillary bridge. A CCD camera is positioned to image either the length or the width of the capillary bridge to characterize the morphology of the fluid interface. Pillars with widths down to 250 &#181;m and lengths up to 70 mm were fabricated with this method, leading to capillary bridges with aspect ratios (length/width) of over 1001.

A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. The creation of capillary bridges relies on the formation of pillars to provide a directional physical and chemical heterogeneity to pin the fluid. Capillary bridges are formed and manipulated using microstages and visualized using a CCD camera.

Fabricação e Visualização de Pontes Capilares em Geometria de Poros Cortados

A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. High aspect ratio hydrophobic pillars are fabricated and ...

Engenharia

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Bioengenharia

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

Combinando dispositivos fluidos com microscopia e citometria de fluxo para estudar transporte microbiano em mídia porosa em escalas espaciais

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

Ambiente

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

A utilização de uma alta taxa de transferência<em&gt; In Vitro</em&gt; Microfluidic Sistema de Desenvolvimento Oral Biofilms Multi-espécies

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Métodos de caracterização da Co-desenvolvimento de biofilme e Habitat Heterogeneidade

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

Uma plataforma microfluídica para o estudo do bioentupimento em meios porosos

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Em Situ Mapeamento das propriedades mecânicas de biofilmes por de rastreamento de partículas Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluídicos Co-cultura de células epiteliais e bactérias para a Investigação Solúvel sinal mediada por interações

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Biologia

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Padronização de Microrganismos e Micropartículas através da Montagem Sequencial assistida pela Capilaridade

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...