Name: visualizing the effects of sputum on biofilm development using a chambered coverglass model
Uploaded: 2016-12-14T14:00:00.000+00:00
Duration: 5 min 3 s
Description: Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

TB reconhece uma bolsa de pesquisa da Fibrose Cística no Canadá.

Note-se que Conselho de Ética em Pesquisa (REB) é necessário para coletar e armazenar amostras de escarro de seres humanos. Esses estudos foram aprovados pelo Hospital para Crianças Doentes REB # 1000019444. 1. preparação de amostras de expectoração CF <ol><li> Recolher amostra de escarro de pacientes durante as visitas de rotina à clínica fibrose cística e manter em gelo. </li><li> Transporte de amostra de escarro no gelo dentro da primeira hora da coleta, para o laboratório de pesquisa, submeter-se a tratamento. </li></ol> 2. escarro Processing <ol><li> Registrar o volume da amostra de expectoração obtidos. Adicionar salina tamponada com fosfato (PBS) para 2x o volume da amostra (isto é, 2 partes de PBS, uma parte de amostra). </li><li> Misturar a amostra bem com uma pipeta de transferência. Vortex da amostra na potência máxima por 1 min para misturar completamente. </li><li> Aliquota de 1 ml da mistura acima referida sobre o número 1,5 ml tubos de microcentrífuga adequadas e girar a 5000 xg durante20 min a 4 ° C. </li><li> A seguir à centrifugação, o sobrenadante remover e descartar o sedimento. </li><li> Filtrar esterilizar o sobrenadante através de um filtro de 0,22? M e recolher num tubo de microcentrífuga limpo. NOTA: A esterilidade do filtrado é testado por plaqueamento em LB agar e inocular meio líquido. </li><li> Armazenar o sobrenadante expectoração a -80 ° C para uso futuro. NOTA: O escarro de vários pacientes também podem ser agrupados seguindo filtração. </li><li> Antes de utilizar, dilui-se expectoração filtrado 10/1 v / v (100 ul de expectoração, a 900 ul de meio), em meio desejado. NOTA: Aqui, foi utilizado caldo lysogeny padrão (LB) de mídia. </li></ol> 3. Chambered Método lamela para formação de biofilme <ol><li> Cresça isolado bacteriano de interesse durante a noite em meio desejado a 37 ° C com agitação (200 rpm). Nota: Um número de bactérias diferentes foram usados, incluindo isolados clínicos Pseudomonas aeruginosa, Staphylococcus aureus,Complexo Burkholderia cepacia e Achromobacter xylosoxidans. Escolha dos meios depende tensões e condições de interesse, no entanto meio LB podem ser utilizados em experiências iniciais. </li><li> A partir da cultura durante a noite, colocar 40 mL de cultura em 4 ml de meio fresco e crescer durante 3-4 horas a 37 ° C com agitação (200 rpm) para se obter uma cultura com uma densidade óptica a 600 nm (OD600) de cerca de 0,5 -0.6. </li><li> Dilui-se a cultura a partir do passo 3.2 a 1/5 na mídia desejada com 10% filtrado escarro ou sem filtrado escarro (como controle). Outros concentração do filtrado expectoração pode ser testado (isto é, 50% ou 100%). </li><li> Utilizar 200 ul da diluição para semear poços das câmaras de deslizamento. </li><li> Permitir que as bactérias aderir durante 4 h a 37 ° C sem agitação. </li><li> Após 4 horas, remova a mídia e lavar cuidadosamente o biofilme por meio fresco 1x. Substitua por 200 mL de mídia fresco. NOTA: Para estudar os efeitos do escarro no biofilme, os fresh mídia deve conter escarro sobrenadante. </li><li> Permitir biofilmes a crescer para a quantidade desejada de tempo a 37 ° C sem agitação, substituindo todos os meios de comunicação 12 horas, sem a lavagem até que o tempo para microscopia. NOTA: Para estudar o efeito de sobrenadantes de escarro na susceptibilidade aos antibióticos biofilme, os antibióticos são adicionados à mídia após 24 horas de crescimento do biofilme e são mantidos nos meios de comunicação até a coloração e imagens de biofilmes. </li></ol> 4. Os biofilmes de coloração e microscopia confocal <ol><li> Seguindo tempo de crescimento desejado (24-48 h, funciona melhor), remova a mídia de poços de câmara e lavar cuidadosamente cada câmara duas vezes com 300 ul de PBS estéril. </li><li> Prepare a mistura de coloração de biofilme por mistura de 1 mL de cada corante (fornecido no kit de viabilidade) por cada ml de solução necessário. Fazer a tintura em água ou meios solução. NOTA: A água é recomendado pelo fabricante. </li><li> Adicionar 200 ul de mistura de corante a cada cavidade de covergla septadasSS e incubar à temperatura ambiente, no escuro, durante 45 min. </li><li> Remover mistura de coloração das câmaras e lavagem de cada poço com 300 ul de PBS estéril. Remover PBS e substituir com água doce ou de mídia. </li><li> Proceda com visualização de biofilme através de microscopia confocal. </li></ol> 5. Visualizando biofilmes com microscopia confocal <ol><li> Leia biofilmes manchado em câmaras imediatamente após a coloração (dentro de 1 hora). Minimizar atraso na visualização das lâminas por coloração de 1 a 2 câmaras de 8 poços de cada vez. </li><li> Realizar imagem usando microscópio confocal com lasers para excitação e filtros de conjuntos para a aquisição. NOTA: Aqui, o sistema confocal disco giratório com lasers borealis espectrais (verde: 491nm, Vermelho: 561 nm) foram utilizados para a excitação. conjuntos de filtros de emissão de 515/40 e 624/40 foram usadas para visualizar as manchas do kit de viabilidade do biofilme. </li><li> Captar imagens utilizando uma objectiva de água 25X no microscópio confocal com câmera. </li><li&gt; Use Z-Pilhas para modelar o biofilme. Captar imagens a cada 0,5-1 mm a partir do primeiro plano em foco para o último quadro em foco do biofilme (tipicamente abrangendo 30-80 mm para 48 biofilmes hr) </li><li> Tome 3-5 imagens de cada poço. NOTA: Assim, para um 8 lamela bem câmaras, serão gerados 24-40 imagens. </li><li> Salvar imagens para análise. NOTA: As imagens devem ser salvas como arquivos OME-TIFF a ser analisado usando COMSTAT 18,19. Instruções para a análise de imagem do biofilme pode ser encontrada em http://www.comstat.dk/. Uma vez que as imagens são importadas, parâmetros como a espessura média, biomassa e cobertura de superfície para cada canal (vermelho e verde) podem ser analisados. </li></ol>

<ol>
	<li>Beaudoin, T., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+with+biofilm+formation:+selection+of+antimicrobials+and+role+of+prolonged+antibiotic+therapy.">Infections with biofilm formation: selection of antimicrobials and role of prolonged antibiotic therapy.</a> Pediatr.Infect.Dis.J. , (2016).</li><li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+microbial+life+on+surfaces.">Biofilms: microbial life on surfaces.</a> Emerg.Infect.Dis. 8 (9), 881-890 (2002).</li><li>Hobley, L., Harkins, C., MacPhee, C. E., Stanley-Wall, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giving+structure+to+the+biofilm+matrix:+an+overview+of+individual+strategies+and+emerging+common+themes.">Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes.</a> FEMS Microbiol.Rev. 39 (5), 649-669 (2015).</li><li>Katharios-Lanwermeyer, S., Xi, C., Jakubovics, N. S., Rickard, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-review:+Microbial+coaggregation:+ubiquity+and+implications+for+biofilm+development.">Mini-review: Microbial coaggregation: ubiquity and implications for biofilm development.</a> Biofouling. 30 (10), 1235-1251 (2014).</li><li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation:+a+clinically+relevant+microbiological+process.">Biofilm formation: a clinically relevant microbiological process.</a> Clin.Infect.Dis. 33 (8), 1387-1392 (2001).</li><li>Bjarnsholt, T., 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=The+in+vivo+biofilm.">The in vivo biofilm.</a> Trends Microbiol. 21 (9), 466-474 (2013).</li><li>Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S., O'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=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+resistance.">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li><li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <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-specific+antibiotic+resistance+gene+ndvB+is+important+for+expression+of+ethanol+oxidation+genes+in+Pseudomonas+aeruginosa+biofilms.">The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li><li>Beaudoin, T., Aaron, S. D., Giesbrecht-Lewis, T., Vandemheen, K., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+clonal+strains+of+Pseudomonas+aeruginosa+isolated+from+cystic+fibrosis+patients+in+Ontario,+Canada.">Characterization of clonal strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Ontario, Canada.</a> Can. J. Microbiol. 56 (7), 548-557 (2010).</li><li>Vidya, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+infection+phenotypes+of+Pseudomonas+aeruginosa+are+associated+with+failure+of+eradication+in+children+with+cystic+fibrosis.">Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis.</a> Eur.J.Clin.Microbiol.Infect.Dis. , (2015).</li><li>Beaudoin, T., Lafayette, S., Nguyen, D., Rousseau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucoid+Pseudomonas+aeruginosa+caused+by+mucA+mutations+result+in+activation+of+TLR2+in+addition+to+TLR5+in+airway+epithelial+cells.">Mucoid Pseudomonas aeruginosa caused by mucA mutations result in activation of TLR2 in addition to TLR5 in airway epithelial cells.</a> Biochem.Biophys.Res.Commun. 428 (1), 150-154 (2012).</li><li>Beaudoin, T., 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=The+level+of+p38alpha+mitogen-activated+protein+kinase+activation+in+airway+epithelial+cells+determines+the+onset+of+innate+immune+responses+to+planktonic+and+biofilm+Pseudomonas+aeruginosa.">The level of p38alpha mitogen-activated protein kinase activation in airway epithelial cells determines the onset of innate immune responses to planktonic and biofilm Pseudomonas aeruginosa.</a> J.Infect.Dis. 207 (10), 1544-1555 (2013).</li><li>LaFayette, S. L., 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=Cystic+fibrosis-adapted+quorum+sensing+mutants+cause+hyperinflammatory+responses.">Cystic fibrosis-adapted quorum sensing mutants cause hyperinflammatory responses.</a> Sci.Adv. 1 (6), e1500199 (2015).</li><li>Staudinger, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+associated+with+the+cystic+fibrosis+defect+promote+chronic+Pseudomonas+aeruginosa+infection.">Conditions associated with the cystic fibrosis defect promote chronic Pseudomonas aeruginosa infection.</a> Am.J.Respir.Crit.Care Med. 189 (7), 812-824 (2014).</li><li>Kennedy, 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=Activity+of+Tobramycin+against+Cystic+Fibrosis+Isolates+of+Burkholderia+cepacia+Complex+Grown+as+Biofilms.">Activity of Tobramycin against Cystic Fibrosis Isolates of Burkholderia cepacia Complex Grown as Biofilms.</a> Antimicrob.Agents Chemother. 60 (1), 348-355 (2015).</li><li>Tom, S. K., Yau, Y. C., Beaudoin, T., LiPuma, J. J., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+High-Dose+Antimicrobials+on+Biofilm+Growth+of+Achromobacter+Species+Isolated+from+Cystic+Fibrosis+Patients.">Effect of High-Dose Antimicrobials on Biofilm Growth of Achromobacter Species Isolated from Cystic Fibrosis Patients.</a> Antimicrob.Agents Chemother. 60 (1), 650-652 (2015).</li><li>Heydorn, 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=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (Pt 10), 2395-2407 (2000).</li><li>Vorregaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Informatics and Mathematical Modelling. , (2008).</li><li>Jurcisek, J. A., Dickson, A. C., Bruggeman, M. E., Bakaletz, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Biofilm+Formation+in+an+8-well+Chamber+Slide.">In vitro Biofilm Formation in an 8-well Chamber Slide.</a> J. Vis. Exp. (47), e2481 (2011).</li></ol>

Os métodos aqui descritos permitem a visualização de biofilmes bacterianas cultivadas na presença de produtos exógenos. Não surpreendentemente, a produção dos exoproducts é de importância quando se utiliza este tipo de sistema. Por exemplo, ditiotreitol (DTT), é frequentemente utilizado em amostras de saliva humana para ajudar a liquefazer as amostras. No entanto, o efeito do DTT sozinho pode diminuir o desenvolvimento do biofilme e a viabilidade (dados não mostrados). Assim, controles adequados para todas a...

biofilmes bacterianas são grupos de microrganismos que estão ligados uns aos outros e incorporados numa matriz auto-secretado. 1,2 Classicamente, eles representam bactérias fisicamente ligados a uma superfície abiótica ou biótico formado sob condições de fluxo. Os biofilmes também têm sido mostrados para crescer em condições estáticas (ausência de fluxo) e distal de superfícies, tais como na interface ar-líquido de piscinas termais ou películas formadas nos tubos de ensaio. Estes biofilmes têm sido reconhecidos no meio ambiente e são um grande prejuízo para processos industriais, como eles podem se formar em reservatórios de água ou em tubos, resultando em biofouling, corrosão e entupimentos. 3,4 Os biofilmes também são críticas em ambientes de cuidados de saúde, como têm sido mostrado estar envolvidas em infecções relacionadas com cateteres, infecções pulmonares em doentes com fibrose cística, bem como em numerosas outras infecções. 5,6 Uma das marcas de infecções biofilme é a devincado susceptibilidade das bactérias aos antibióticos e auditivos depuração pelo sistema imune inato. 7-9 O mais bem estudadas, cenários clinicamente relevantes envolvendo infecção baseada biofilme ocorre em pacientes com fibrose cística (FC), que estão cronicamente infectados com biofilmes de Pseudomonas aeruginosa. P. aeruginosa podem ser submetidos a uma série de alterações durante o estabelecimento da infecção crónica que tornam muito difíceis de tratar. 10,11 biofilmes pode diferencialmente ativar a imunidade inata e conduzir a inflamação. 12-14 Como estas infecções levar ao aumento da morbidade e mortalidade em pacientes com FC, é crucial para entender fatores que podem afetar o desenvolvimento do biofilme neste contexto. Um estudo recente sugere que hospedeiras-factores são importantes na formação de agregados de biofilme de P. aeruginosa. 15 Estes biofilmes contribuir para a redução da susceptibilidade aos antibióticos e os mecanismos de defesa do hospedeiro. o preseNCE de factores derivados do hospedeiro, tais como a elastase de neutrófilos, assim como produtos secretados a partir de microrganismos presentes no pulmão CF, tem o potencial de modular significativamente a formação de biofilme e de desenvolvimento. 16 Além disso, os biofilmes interagir com o hospedeiro para modular a expressão de numerosos caminhos e iniciar a inflamação. Enquanto os métodos de alto rendimento, tal como o ensaio de violeta de cristal padrão, podem fornecer alguma informação no que diz respeito ao processo de biofilme, a visualização do biofilme em resposta a estes factores proporcionam mais informação em profundidade. Nesse artigo, nós descrevemos um método para a utilização de factores de expectoração de pacientes com FC para estudar o desenvolvimento de biofilmes in vitro. Este método permite a rápida visualização de biofilme expostos a expectoração contendo factores do hospedeiro, utilizando um kit comercial viabilidade do biofilme. Esta técnica pode ser usada para identificar visualmente as alterações que ocorrem durante o crescimento de biofilme, na presença de exógenonos produtos, e representa um método melhorado para analisar as alterações no desenvolvimento de biofilme sob várias condições.

<table><tbody><tr><td>Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td><td>Thermo Sicher Scientific</td><td>155409</td><td></td></tr><tr><td>Filmtracer Live/Dead Biofilm Viabilty Kit</td><td>Thermo Fisher Scientific</td><td>L10316</td><td></td></tr><tr><td>Blood agar plates</td><td>Thermo Fisher Scientific</td><td>R10215</td><td>Confirming viability via CFU counts or selecting colonies for innoculation</td></tr><tr><td>COMSTAT</td><td>Availble software online</td><td></td><td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&amp;nbsp;</td></tr><tr><td>Millers LB Broth</td><td>Thermo Fisher Scientific</td><td>12780-052</td><td>Standard media for overnight gowth/biofilm growth</td></tr><tr><td>Millex-GV Syringe Filters</td><td>Millipore</td><td>SLGV013SL</td><td>Filtering of sputum supernants</td></tr><tr><td>Phosphate Buffered Saline (Dulbecco A)</td><td>Oxoid</td><td>BR0014G</td><td>Washing of biofilm chambers after media removal</td></tr><tr><td>Zeiss AxioVert 200M</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Hamamatsu C9100-13 EM-CCD</td><td>QS Technologies Inc.</td><td></td><td></td></tr><tr><td>Spectral Borealis</td><td>Qs Technologies Inc.</td><td></td><td></td></tr><tr><td>Perkin Elmer Volocity</td><td>QS Technologies Inc.</td><td></td><td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td></tr></tbody></table>

visualizing the effects of sputum on biofilm development using a chambered coverglass model

Os biofilmes consistem de grupos de bactérias embebidas em uma matriz auto-secretado. Eles desempenham um papel importante na contaminação industrial, bem como no desenvolvimento e persistência de muitas infecções relacionadas com a saúde. Um dos biofilmes mais bem descrita e estudada em doenças humanas ocorre na infecção pulmonar crónica de pacientes com fibrose cística. Ao estudar biofilmes no contexto do hospedeiro, muitos factores que podem influenciar a formação e desenvolvimento de biofilme. A fim de identificar como factores do hospedeiro pode afetar a formação de biofilme e desenvolvimento, foi utilizado um método lamela chambered estática para crescer biofilmes na presença de fatores derivados do hospedeiro na forma de sobrenadantes de escarro. As bactérias são semeadas em câmaras e expostos a filtrados de expectoração. Na sequência de 48 horas de crescimento, os biofilmes estão manchadas com um kit de viabilidade de biofilme comercial antes da microscopia confocal e análise. Após a aquisição das imagens, as propriedades do biofilme pode ser avaliada através de diferentes plataformas de software.Este método nos permite visualizar as propriedades fundamentais do crescimento do biofilme na presença de diferentes substâncias, incluindo antibióticos.

A concepção geral da experiência está representada na Figura 1. A utilização deste protocolo fornece um método conveniente para visualizar as alterações em biofilmes cultivadas por diferentes períodos de tempo (por exemplo, 24, 48 ou 72 horas). Importante, sinais exógenos, tais como expectoração filtrados, pode ser adicionado para visualizar as alterações no desenvolvimento de biofilme. Como pode ser visto na Figura 2, a presença...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizando os Efeitos de escarro no desenvolvimento de biofilmes usando um modelo Chambered lamela

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

visualizing-effects-sputum-on-biofilm-development-using-chambered

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.3K visualizações.  Hospital for Sick Children. This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Vídeo: Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-cultura de Modelos Pseudomonas aeruginosa Biofilmes Grown no Live Células Airway Humanos

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Imunologia e Infecção

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 ...

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Dish microtitulação Assay formação de biofilme

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

In vitro a formação de biofilme em um slide Câmara de 8 poços

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

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 ...

Bioengenharia

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Metodologias para o estudo<em&gt; B. subtilis</em&gt; Os biofilmes como um modelo para a caracterização de pequena molécula biofilme Inibidores

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

O Modelo WinCF - Um Microcosmo barato e tratável de um muco Plugged Bronquíolo para estudar a Microbiologia do Pulmão Infecções

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Visualização da formação de biofilmes de Candida albicans , usando um dispositivo Microfluidic automatizada

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Visualização de Pseudomonas aeruginosa no Escarro de Pacientes com Fibrose Cística

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

Visualizando os Efeitos de escarro no desenvolvimento de biofilmes usando um modelo Chambered lamela

Resumo

Explore mais vídeos

Capítulos neste vídeo