Agradecemos Matt Parsek na Universidade de Washington (Seattle, WA) para a prestação de P. aeruginosa e E. coli e Roger Nokes da Universidade de Canterbury (Nova Zelândia) para fornecimento de acesso a software córregos. Este trabalho foi financiado pela concessão R01AI081983 dos Institutos Nacionais de Saúde, Instituto Nacional de Alergia e Doenças Infecciosas. Imagem confocal foi realizada no Facility Northwestern Imagem Biológica (BIF).

1. Fluxo Setup celular e Inoculação NOTA:. Use uma célula de fluxo microfluídicos dupla via de entrada descrito no Song et al, 2014 14 para crescer biofilmes. Esta célula de fluxo é capaz de criar gradientes químicos suaves bem definidos. O desenho da célula de fluxo é mostrado na Figura 1 e o fluxo de fabricação de células foi previamente descrito em Song et al., 2014 14. Aqui detalhes nossos métodos usando P. aeruginosa e E. coli para formar biofilmes, mas outras espécies podem também ser adequados. Usamos P. aeruginosa PAO1- GFP, que constitutivamente expressa uma proteína fluorescente verde (GFP), como um organismo modelo para o desenvolvimento do biofilme. Além disso, foi utilizado E. coli DH5ct de formar biofilmes mistos de espécies com P. aeruginosa. P. aeruginosa e E. estirpes de E. coli foram cultivadas em placas de LB-agar. <ol><li> Prepare a célula de fluxo usando polidimetilsiloxano (PDMS) vinculado a umlamela de vidro via tratamento de plasma de oxigênio, conforme descrito no 14. As dimensões da câmara de célula de fluxo são de 23 mm x 13 milímetros x 0,24 milímetros (comprimento x largura x profundidade). </li><li> Prepare modificado FAB meio de crescimento 15. Para observar o crescimento do biofilme sob um gradiente nutricional dentro da célula de fluxo, introduzem modificado FAB média 15 com glicose 0,6 M em uma entrada e introduzir médio FAB sem qualquer fonte de carbono através da outra entrada. Filtrar a esterilizar (tamanho dos poros = 0,2 m) da solução de estoque de glucose (60 mM) antes de ser adicionada ao meio de FAB. Também autoclave o meio FAB com ciclo 1 (líquido, 15 min; 121 ° C, 17 psi) antes do uso. </li><li> Esterilizar o sistema de fluxo. Antes da inoculação, autoclave o caminho inteiro fluxo (médias garrafas, tubos, retentores de bolhas, as células de fluxo), utilizando o ciclo 1, excepto para as válvulas de três vias de plástico descartáveis ​​(e pré-esterilizado) situados a montante da célula de fluxo. (Válvulas de três maneiras são usadas para injetar células cultura, marcador fluorescente e micropérolas.) Para evitar a contaminação durante a montagem, cubra todos os tubos e conectores aberturas com folha de alumínio ou sacos de autoclave antes da autoclavagem. </li><li> Ligue o sistema de fluxo. Montar os componentes do sistema de células de fluxo com cuidado (veja o vídeo para a montagem do sistema de fluxo) e entregar meio de crescimento para a célula de fluxo por meio de uma bomba peristáltica que controla com precisão a taxa de fluxo. </li><li> Prepare cultura de células para inoculação através da transferência de uma colônia de P. aeruginosa ou E. coli a partir de placas LB a 3 ml de caldo LB e agitar a cultura O / N a 225 rpm e 37 ° C. Diluir o / n de culturas de células em 1 ml de água estéril para uma DO final 600 = 0,01 como o inoculo. (Cultura e diluir as bactérias numa câmara de fluxo laminar para evitar contaminação). </li><li> Para as experiências com biofilmes de espécies mistas, diluir as duas culturas bacterianas a uma proporção de 1: 1 em 1 ml com uma DO 600 = 0,01 equivalente para cada bactéria. </li><li> Inocular a célula de fluxo. Injectar 1 mL de inoculo para a entrada da célula de fluxo da válvula de três vias. Após a injeção, pausar o fluxo de 1 hora para permitir que as células bacterianas para anexar a tampa de vidro. (Certifique-se de que a célula de fluxo é colocado com o lado da lamela para baixo para permitir que as células suspensas para resolver sobre a lamela.) </li><li> Após 1 h, retomar o fluxo e bombear o meio de crescimento para a célula de fluxo a uma taxa constante de 0,03 ml / min para cada entrada, durante 3 dias. </li></ol> 2. Caracterizar biofilme Desenvolvimento para a resposta aos nutrientes gradientes usando microscopia confocal NOTA: P. aeruginosa pode ser trabalhada com GFP-constitutivamente expresso, mas E. coli em biofilmes mistos de espécies deve ser trabalhada por contracoloração. <ol><li> Observe os biofilmes de 3 dias usando microscopia confocal. Para os resultados representativos, observar biofilmes utilizando um microscópio confocal com um objetivo 63X. Antes de imagem, marcaa célula de fluxo dupla via de entrada com uma grade do lado da tampa de vidro. (A finalidade desta grelha é para permitir que o experimentador para localizar as regiões de imagem no interior da câmara de célula de fluxo.) </li><li> Para contracoloração, dilui-se 10 ul de ácido nucleico mancha fluorescente vermelho célula-permeante, tal como mancha fluorescente verde ácido nucleico tal como STYO 62, solução-mãe (1 mM) em 990 ul de água esterilizada e depois injectar lentamente a mancha diluída utilizando uma seringa a câmara de célula de fluxo da válvula de três vias a montante. Manter a célula de fluxo estagnado e no escuro durante 30 min, em seguida, retomar o fluxo para lavar o corante libertado durante 15 min. </li><li> Realizar imagem confocal. Ative a 488 nm laser de argônio para a excitação ea emissão coleta canais para GFP (500-535 nm) e mancha de ácido nucleico (por fluorescente mancha verde ácido nucleico, 650-750 nm). Ajuste ganhos e compensações para ambos os canais para ter imagens mais brilhantes e limpas. Select xyz e modo de gravação simultânea. Use o botão de controle z para identificar a parte inferior e superior do biofilme no campo. Definir o z-passo a 0,5 mm para aquisição de uma imagem da pilha de 3-D. (Para garantir a alta qualidade de imagem, use uma linha média de quatro para aquisição de imagem.) </li><li> Para mapear os padrões espaciais de desenvolvimento de biofilme dentro da célula de fluxo, biofilmes imagem em três ou mais longitudinal (x) as distâncias ao longo da entrada da célula de fluxo, e em três transversal (y) posiciona em relação ao gradiente nutricional aplicada, como mostrado na Figura 1 . </li></ol> 3. Caracterizar Soluto Interno Transportes por injeções de Conservador Fluorescent Tracer NOTA: Um marcador fluorescente conservadora, tais como Cy5, pode ser utilizado para caracterizar o padrão de transporte de solutos dentro do biofilme. <ol><li> Dissolve-Cy5 (mono-reactiva NHS Éster) em água a uma concentração final de 10 mg / ml como uma solução estoque. Guardar a solução estoque Cy5 em um congelador a -20 ° C. Nota: Como Cy5 é light-SensITive, loja, diluir e injetar soluções Cy5 no escuro. </li><li> Antes das injecções do marcador fluorescente, tomar uma imagem da pilha de 3-D do biofilme de 3 dias com microscopia confocal (com uma objectiva 63X). (Regiões largas de colônias biofilme são ideais para observações de séries temporais de transporte corante.) Também escolher um z-plane com colônias bem separados para imagens (como mostrado na Figura 5A). </li><li> Para cada experiência injecção Cy5, diluir 2 uL da solução de stock de Cy5 em 998 ul de água estéril para se obter uma solução para injecção com uma concentração de Cy5 20 ug / ml. </li><li> Parar a bomba para interromper o fluxo e injectar a solução para o Cy5 a montante da célula de fluxo, utilizando uma seringa, através da válvula de 3 vias. (É importante evitar que as bolhas de ar que entra no percurso de escoamento durante a injecção. Retentores de bolhas deve ser mantida aberta durante a injecção de volta). </li><li> Depois de injetar a solução Cy5, feche as armadilhas bolha, ajustar a válvula de 3 vias, e reiniciar obomba para fornecer a solução de Cy5 injectado para dentro da célula de fluxo. </li><li> Mudar o modo de imagem confocal para XYT. Ative Cy5 e GFP canais sob o modo de gravação simultânea. Ajustar as definições confocal sobre Cy5 intensidade (intensidade do laser, ganhar e offset) para evitar a saturação sinais, o que é crítico para a quantificação. (Alta resolução temporal é o preferido para a visualização de penetração do corante. No entanto, digitalização rápida diminui a qualidade da imagem.) Escolha a velocidade de verificação adequada e média linha para equilibrar o tempo de resolução de imagem e qualidade de imagem. Para este protocolo usar uma média de linha de quatro e uma taxa de quadros de 0,15 Hz. </li><li> Retomar o fluxo para entregar Cy5 para dentro da célula de fluxo (ainda a um caudal de 0,03 ml / min). Simultaneamente a hora de início da série confocal de imagem. </li><li> Quantificar Cy5 penetração por radialmente média intensidade Cy5 dentro de uma colônia de biofilme circular 2-D. Para fazer a média, primeiro identificar a aresta de um cluster de biofilme, em seguida, gerar um mapa de distância para uma secção de 2-D do aglomeradoE, finalmente, os padrões de intensidade média radiais Cy5 para gerar uma curva de penetração (ver Figura 6). <ol><li> Para computacionalmente perceber o passo 3.8, utilize BioSPA (Análise de Padrões de biofilme Spatial) pacote de software (inédito pacote, software e manual disponível mediante pedido) ou outros programas de análise de imagem. </li><li> Para usar BioSPA, primeiro importar uma imagem colocada em BioSPA. Em seguida, selecione limite adequado para os canais de GFP e Cy5 para fazer imagens binárias. No menu de análise / cálculo, selecione Análise Avançada e, em seguida, Análise de Difusão. Usar o canal GFP para definir a aresta de um cluster e utilizar a ferramenta polígono para seleccionar um cluster como a região de interesse (ROI). </li><li> Clique duas vezes o cluster biofilme selecionado para realizar a análise de difusão. Salve os resultados da análise de difusão e calibrar a intensidade Cy5 a concentração Cy5 utilizando uma curva de calibração. <ol><li> Para gerar Cy5 curva de calibração de concentração, medir intensidades Cy5 durante um conce Cy5gradiente ntration sob microscopia confocal. (Intensidade e concentração Cy5 têm uma relação linear). </li></ol></li></ol></li></ol> 4. Caracterização torno campo do fluxo através do rastreamento Fluorescent Particles NOTA: partículas fluorescentes, tais como micro-esferas fluorescentes, podem ser utilizados para caracterizar o campo de fluxo de cerca de biofilmes. Campo de fluxo em torno de biofilmes pode ser calculado a partir das observações de séries temporais de posições partículas usando software de monitoramento de partículas velocimetria. Os resultados aqui apresentados foram obtidos com o Streams 2,02 software pacote de 16 (download de software e manual do utilizador disponível em <a href="http://www.civil.canterbury.ac.nz/streams.shtml">http://www.civil.canterbury.ac.nz/streams.shtml</a> ). <ol><li> Dilui-se as microesferas fluorescentes (diâmetro de ~ 1 um) em água esterilizada a uma concentração de sólidos final de 0,2% e um volume final de 0,5 ml. Vortex para se certificar de que as microesferassão bem dispersa. </li><li> Injectar e entregar as microesferas fluorescentes diluídas na célula de fluxo de acordo com procedimentos de passos 3.3 a 3.5. Para permitir um acompanhamento preciso do caminho partícula, use uma taxa relativamente baixa de fluxo (0,01 ml / h para os resultados de rastreamento de partículas neste artigo). </li><li> Mudar configurações confocal modo para XYT para acompanhar o movimento de partículas em um z-slice e tomar imagens de séries temporais. (A taxa de quadros de 1 Hz foi utilizado para os resultados representativos apresentados no presente relatório.) Repita a injeção de partículas e imagem em diferentes locais z para gerar fluxo de campo 3-D. </li><li> Pré-processo o tempo-series imagens confocal subtraindo-se o sinal de fluorescência de fundo (o sinal na primeira imagem obtida do conjunto imagem) usando ImageJ (download de software e manual disponível em <a href="http://imagej.nih.gov/ij/">http://imagej.nih.gov/ij/</a> ) ou outros programas. </li><li> Em ImageJ, abra a primeira imagem de um conjunto de imagens e depois importar o conjunto de imagens. Em Processo de Imagem / Calculator, subtrair a primeira imagem do conjunto imagem. Salve o pré-processado conjunto imagem. </li><li> Para calcular vetores de fluxo, de importação de séries temporais de imagens confocal em Streams 2,02. No âmbito da &quot;visão de processo Open&quot;, selecionar e executar &quot;Identificar partículas&quot;. Use limiar adequado para identificar partículas. Use 0,5 e 5 m como limites mínimos e diâmetro máximo de 1 mm partículas. </li><li> Em seguida, em &quot;view processo Open&quot;, selecionar e executar &quot;PTV análise pipeline&quot; para gerar caminhos de partículas. Confira os caminhos de partículas para ver se eles representam o movimento de partículas. Finalmente, selecionar e executar &quot;Criar campo de velocidade&quot; para gerar um mapa do vetor fluxo. </li></ol>

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The authors have nothing to disclose.

Nós demonstramos um conjunto de métodos para caracterizar três importantes interações biofilme-ambientais: resposta biofilme para gradientes químicos, efeitos do crescimento do biofilme sobre o microambiente fluxo envolvente, e heterogeneidade biofilme resultante de limitações de transporte interno. Nós mostramos pela primeira vez o uso de uma nova célula de fluxo de microfluidos para impor um gradiente de química bem definida para o desenvolvimento do biofilme. Para criar um grad...

Microorganismos se prendem às superfícies e biofilmes de formulário - agregados de células fechadas em uma matriz extracelular-polímero 1. Os biofilmes se comportam de forma muito diferente a partir de células microbianas individuais, pois biofilmes tem heterogeneidade espacial dramática resultante de uma combinação de limitações de transporte de soluto internos e variações espaciais no metabolismo celular 2,3. As concentrações de oxigênio e de nutrientes diminuem drasticamente na interface entre biofilme e em torno de fluido e se ainda mais empobrecido dentro no biofilme 2. As variações espaciais na respiração do biofilme e a síntese de proteínas também podem ocorrer como uma resposta ao oxigénio localizada a disponibilidade de nutrientes e 2. Em ambientes aquáticos e do solo, a maioria das bactérias habitam em biofilmes. Biofilmes naturais realizar processos biogeoquímicos importantes, incluindo o ciclo do carbono e nitrogênio e reduzindo metais 4,5. Clinicamente, a formação de biofilme é responsvel para pulmonar prolongada e infecções urinárias 6. Infecções associadas ao biofilme são altamente problemático porque as células em biofilmes tem altíssima resistência aos antimicrobianos em comparação com os seus homólogos planctônicas 6. Porque biofilmes são importantes em diversos contextos, uma quantidade substancial de investigação tem-se centrado na compreensão dos fatores ambientais que controlam as atividades de biofilme e da heterogeneidade espacial em biofilmes e do microambiente circundante. Estudos anteriores mostraram que o desenvolvimento do biofilme é fortemente regulado por uma série de factores ambientais: biofilmes desenvolver diferentes morfologias sob várias condições de fluxo; oxigênio e nutrientes disponibilidade influência morfologia biofilme; e tensões hidrodinâmicas afeta a fixação de células planctônicas a superfícies e o desprendimento de células de biofilmes 7-9. Além disso, a condição de escoamento externo influencia a entrega de substratos into e dentro de biofilmes 10. O crescimento de biofilmes também altera circundante condições físicas e químicas. Por exemplo, o crescimento de biofilme conduz à depleção local do oxigénio e nutrientes 2; biofilmes acumular compostos inorgânicos e orgânicos provenientes do ambiente circundante 11; e agrupamentos de biofilme desviar incremento da superfície de fricção do fluxo e 12,13. Porque biofilmes interagir com o seu ambiente circundante em formas muito complexas, é crítico para a obtenção simultânea das informações sobre as propriedades de biofilme e condições ambientais, e multidisciplinares precisam de ser usados ​​para caracterizar exaustivamente interacções-biofilme ambiente. Aqui apresentamos uma série de métodos integrados para caracterizar padrões espaciais em crescimento microbiano dentro mono-espécies e biofilmes dual-espécies sob um gradiente nutricional imposta, bem como observar a modificação resultante do produto químico local e microambiente fluido. Nós firr descrevem a utilização de uma célula de dupla via de entrada de fluxo de microfluidos recentemente desenvolvido para observar o crescimento do biofilme sob gradientes químicos bem definidos. Em seguida, demonstrar a utilização dessa célula de fluxo de microfluidos para observar o crescimento de duas espécies de bactérias Pseudomonas aeruginosa, Escherichia coli e, em biofilmes sob uma gama de condições nutricionais. Mostramos como na visualização situ de propagação marcador fluorescente em colônias biofilme pode ser usado para avaliar quantitativamente os padrões de transporte de soluto em biofilmes. Finalmente, mostra-se como microescala velocimetria de rastreamento de partículas, realizada sob microscopia confocal, pode ser usado para obter o fluxo de campo local em torno dos biofilmes em crescimento.

<table border="0" cellpadding="0" cellspacing="0" width="586">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:89px;">Company</td>
			<td style="width:104px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">Peristaltic Pump</td>
			<td style="width:89px;">Gilson</td>
			<td style="width:104px;">Miniplus 3</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">PUMP TUBING 0.50MM OVC, Orange/Yellow</td>
			<td style="width:89px;">Gilson</td>
			<td style="width:104px;">F117934</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Three-way Stopcock w/ Swivel male Luer lock</td>
			<td style="width:89px;">Smiths Medical&#160;</td>
			<td style="width:104px;">MX9311L</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Sylgard 184 Solar Cell Encapsulation for Making Solar Panels</td>
			<td style="width:89px;">ML Solar LLC</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Pyrex Medium Bottle, 1L, GL45</td>
			<td style="width:89px;">VWR</td>
			<td>16157-191</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">C-FLEX Tubing</td>
			<td style="width:89px;">Cole-Parmer</td>
			<td style="width:104px;">06422-02</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">1 mL TB Syringe</td>
			<td style="width:89px;">BD</td>
			<td style="width:104px;">309659</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Polymer Tubing</td>
			<td style="width:89px;">IDEX</td>
			<td style="width:104px;">1520G</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Sterile Intramedic Luer Stub Adapter</td>
			<td style="width:89px;">Clay Adams</td>
			<td style="width:104px;">427564</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">PrecisionGlide Needle</td>
			<td style="width:89px;">BD</td>
			<td style="width:104px;">305195</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Spectrophotometer</td>
			<td style="width:89px;">HACH</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Syringe filters- sterile (0.2 &#956;m)</td>
			<td style="width:89px;">Fisherbrand</td>
			<td style="width:104px;">09-719A</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">MAXQ Shaker</td>
			<td style="width:89px;">Thermo Scientific</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Ammonium sulfate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">A4418</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium phosphate dibasic anhydrous</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">RES20908-A7</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Monobasic potassium phosphate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">P5655</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">S7653</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Magnisium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">M8266</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Calcium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C5670</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Calcium sulfate dihydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C3771</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Iron(II) sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">215422</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Manganese(II) sulfate monohydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">M7634</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Copper(II) sulfate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">451657</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Zinc sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">Z0251</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Cobalt(II) sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C6768</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium molybdate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">243655</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Boric acid</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">B6768</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Dextrose</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">D9434</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Luria Bertani Broth</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">L3022</td>
			<td>Growth media</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">TCS SP2 Confocal Microscopy</td>
			<td style="width:89px;">Leica</td>
			<td style="width:104px;"></td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:227px;">SYTO 62</td>
			<td style="width:89px;">Life Technology</td>
			<td style="width:104px;">S11344</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:227px;">Cy5</td>
			<td style="width:89px;">GE Healthcare Life Sciences</td>
			<td style="width:104px;">PA15100</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Red Fluorescent (580/605) FluoSphere</td>
			<td style="width:89px;">Life Technology</td>
			<td style="width:104px;">F-8801</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">BioSPA</td>
			<td style="width:89px;">Packman Lab</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">ImageJ</td>
			<td style="width:89px;">NIH</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">Volocity</td>
			<td style="width:89px;">PerkinElmer</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Streams 2.02</td>
			<td style="width:89px;">University of Cantebury</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
	</tbody>
</table>

methods for characterizing the co-development of biofilm and habitat heterogeneity

Os biofilmes são comunidades microbianas em anexo de superfície que têm estruturas complexas e produzem heterogeneidades espaciais significativos. O desenvolvimento do biofilme é fortemente regulada pelo fluxo envolvente e ambiente nutricional. O crescimento do biofilme também aumenta a heterogeneidade do microambiente local, gerando campos de fluxo e os padrões complexos de transporte de solutos. Para investigar o desenvolvimento da heterogeneidade em biofilmes e interações entre biofilmes e sua micro-habitat local, nós crescemos biofilmes mono-espécies de Pseudomonas aeruginosa e biofilmes dual-espécies de P. aeruginosa e Escherichia Coli sob gradientes nutricionais em uma célula de fluxo de microfluidos. Nós proporcionam protocolos detalhados para a criação de gradientes de nutrientes dentro da célula de fluxo e para o crescimento e desenvolvimento de biofilme visualização sob estas condições. Nós também protocolos atuais para uma série de métodos ópticos para quantificar padrões espaciais na estrutura do biofilme, o fluxo distribuições sobre biofilmes, e transporte de massa ao redor e dentro de colônias de biofilme. Estes métodos apoiar investigações abrangentes do co-desenvolvimento de biofilme e habitat heterogeneidade.

A célula de fluxo microfluidico dupla via de entrada permite a observação do crescimento do biofilme sob um gradiente de química bem definida formada pela mistura de duas soluções no interior da câmara de fluxo. O gradiente químico resultante foi anteriormente observado por injeção de corante e caracterizados em detalhe por Song et al. 14. Gradientes de concentração lisas foram formados na direcção transversal, tal como mostrado na Figura 1. O perfil de concentração f...

Watch this Scientific Journal Video about Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity at JoVE.com

Methods for Characterizing 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 ...

methods-for-characterizing-co-development-biofilm-habitat

Research

JoVE Journal

Bioengineering

9.8K visualizações.  Northwestern University. 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. 

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

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.

Métodos de Desenvolvimento de Sangue Transporte Macrófago Borne de Nanoformulated medicamentos anti-retrovirais

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

Bioengenharia

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Métodos de detecção de fluorescência para as plataformas de gotículas microfluídicos

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans biofilme Chip (Ca BChip) para seleção de alto rendimento antifúngico

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

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

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 porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

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 ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Os métodos para a auto-integração da Megamolecular Biopolímeros na secagem ao ar-LC interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Caracterizando a migração celular no tridimensional em Vitro ferida ambientes

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Dois métodos para Decellularization de tecidos vegetais para aplicações de engenharia de tecidos

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

Avaliação global da eficácia e segurança de entrega de droga Placenta-alvo usando três métodos complementares

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Dispositivos de campo elétrico e magnético para estimulação de tecidos biológicos

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Formulando e caracterizando nanopartículas lipídicas para entrega de genes usando uma plataforma de mistura microfluida

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...