Name: microtiter dish biofilm formation assay
Uploaded: 2011-01-30T18:00:00
Description: Watch this Scientific Journal Video about Microtiter Dish Biofilm Formation Assay at JoVE.com

Спасибо Шерри Кучма, Пит Ньюэлл и Роберт Шанкс для обеспечения изображения на рисунке 1. Эта работа была поддержана NIH грант R01AI083256 в ГАО

1. Рост биопленки <ol><li> Рост культуры дикого типа синегнойной палочки или мутантный штамм в течение ночи в богатой среде (т.е. LB) </li><li> Развести более 1:100 культуры ночь в свежую среду для биопленки анализов. Стандартной среде анализа биопленки для P. палочки М63 является минимальн...

<ol>
	<li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple,+convergent+signalling+pathways:+a+genetic+analysis.">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis.</a> Mol. Microbiol. 28, 449-461 (1998).</li><li>O'Toole, G. A., Doyle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. , 91-109 (1999).</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=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426, 306-310 (2003).</li><li>Kuchma, S. L., Connolly, J. P., 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+three-component+regulatory+system+regulates+biofilm+maturation+and+type+III+secretion+in+Pseudomonas+aeruginosa.">A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa.</a> J Bacteriol. 187, 1441-1454 (2005).</li><li>Caiazza, N. C., 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=SadB+is+required+for+the+transition+from+reversible+to+irreversible+attachment+during+biofilm+formation+by+Pseudomonas+aeruginosa+PA14.">SadB is required for the transition from reversible to irreversible attachment during biofilm formation by Pseudomonas aeruginosa PA14.</a> J Bacteriol. 186, 4476-4485 (2004).</li><li>Shanks, R. M., Sargent, J. L., Martinez, R. M., Graber, M. L., 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=Catheter+lock+solutions+influence+staphylococcal+biofilm+formation+on+abiotic+surfaces.">Catheter lock solutions influence staphylococcal biofilm formation on abiotic surfaces.</a> Nephrol Dial Transplant. 21, 2247-2255 (2006).</li><li>Caiazza, N. C., Merritt, J. H., Brothers, K. M., 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=Inverse+regulation+of+biofilm+formation+and+swarming+motility+by+Pseudomonas+aeruginosa+PA14.">Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14.</a> J. Bacteriol. 189, 3603-3612 (2007).</li><li>Hinsa, S. M., Espinosa-Urgel, M., Ramos, J. L., 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=Transition+from+reversible+to+irreversible+attachment+during+biofilm+formation+by+Pseudomonas+fluorescens+WCS365+requires+an+ABC+transporter+and+a+large+secreted+protein.">Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein.</a> Mol Microbiol. 49, 905-918 (2003).</li><li>Mack, D. <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+transposon+mutants+of+biofilm-producing+Staphylococcus+epidermidis+impaired+in+the+accumulative+phase+of+biofilm+production:+genetic+identification+of+a+hexosamine-containing+polysaccharide+intracellular+adhesin.">Characterization of transposon mutants of biofilm-producing Staphylococcus epidermidis impaired in the accumulative phase of biofilm production: genetic identification of a hexosamine-containing polysaccharide intracellular adhesin.</a> Infect. Immun. 62, 3244-3253 (1994).</li><li>Vidal, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+an+Escherichia+coli+K-12+mutant+strain+able+to+form+biofilms+on+inert+surfaces:+involvement+of+a+new+ompR+allele+that+increases+curli+expression.">Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression.</a> J. Bacteriol. 180, 2442-2449 (1998).</li><li>Junker, L. M., Clardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screens+for+small-molecule+inhibitors+of+Pseudomonas+aeruginosa+biofilm+development.">High-throughput screens for small-molecule inhibitors of Pseudomonas aeruginosa biofilm development.</a> Antimicrob Agents Chemother. 51, 3582-3590 (2007).</li></ol>

Этот метод может быть модифицирована для использования в самых разнообразных видов микроорганизмов. Подвижные микробы обычно придерживаются стен и / или днища колодцев, в то время как неподвижные микроорганизмы обычно придерживаются на дно скважины. Оптимальные условия для образова?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1 X M63</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Prepare as a 5X M63 stock by dissolving 15g KH2PO4, 35g K2HPO4 and 10g (NH4)2SO4 in 1 L of water. This stock does not need to be autoclaved and can be stored at room temperature. Dilute 5X stock 1:5, autoclave, cool, then add the desired components.</td>
</tr>
<tr>
<td>KH2PO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P285-500</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P288-500</td>
<td>&nbsp;</td>
<tr>
<td>(NH4)2SO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5132</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium sulfate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>M63-500</td>
<td>Add to 1 mM final concentration. Prepare as a 1 M stock in water and autoclave.</td>
</tr>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>D16-3</td>
<td>Add to 0.2% final concentration. Prepare as a 20% stock in water and autoclave.</td>
</tr>
<tr>
<td>Casamino acids</td>
<td>&nbsp;</td>
<td>Beckton-Dickinson</td>
<td>223050</td>
<td>Add to 0.5% final concentration. Prepare as a 20% stock in water and autoclave.</td>
</tr>
<tr>
<td>Arginine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5131</td>
<td>Add to 0.4% final concentration. Prepare as a 20% stock in water and filter sterilize. This alternative carbon/energy source can replace glucose and casamino acids</td>
</tr>
<tr>
<td>Microtiter plates</td>
<td>&nbsp;</td>
<td>Beckton-Dickinson</td>
<td>353911</td>
<td>Falcon 3911, Microtest III, Flexible assay plates, 96 well, U-bottom, non-sterile, non-tissue-culture treated.</td>
</tr>
<tr>
<td>Microtiter plate lids</td>
<td>&nbsp;</td>
<td>Beckton-Dickinson</td>
<td>353913</td>
<td>The lids can be reused by cleaning with 95% ethanol in water.</td>
</tr>
<tr>
<td>Crystal violet</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>229641000</td>
<td>Prepare as a 0.1% solution in water.</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Microtiter Dish Biofilm Formation Assay at JoVE.com

Microtiter Dish Biofilm Formation Assay

Анализ описывает быстрый способ для измерения раннего образования биопленки у бактерий и грибков. Этот метод использует планшет в качестве субстрата для микробной образование биопленки и биопленки визуализируется использованием штамма кристаллический фиолетовый. Препарат обеспечивает либо качественных или количественных тест для раннего образования биопленки.

Микротитровальные Блюдо образование биопленки Пробирной

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

Research

JoVE Journal

Immunology and Infection

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

96 Ну планшет метода, основанного на формировании мониторинга и тестирования Противогрибковые Восприимчивость Candida Albicans Биопленки

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Синегнойной палочки и Saccharomyces CEREVISIAE биопленки в Проточные ячейки

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

In vitro Biofilm Formation in an 8-well Chamber Slide

In vitro Biofilm Formation in an 8-well Chamber Slide

В пробирке образование биопленки в 8-и палаты слайдов

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

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Расшифровка и визуализации патогенез и запи-<em&gt; Микобактерии abscessus</em&gt; У эмбрионов данио рерио

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Ориентация биопленки Associated<em&gt; Стафилококк</em&gt; Использование ресазурин На основе лекарственной чувствительности анализа

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

&quot;Фагосоме Закрытие Анализ&quot; для визуализации фагосоме Формирование в трех измерениях с помощью полного внутреннего отражения флуоресцентной микроскопии (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

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

Визуализация Эффекты мокроте на биопленки развития с использованием модели Chambered покровного стекла

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 ...

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

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

Визуализация биопленки в Candida albicans с помощью автоматизированных Microfluidic устройства

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

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Желчь соли индуцированной биопленки в кишечных патогенов: методы для идентификации и количественной оценки

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Высок объём Assay оценить и количественно нейтрофилов внеклеточного ловушку формирования

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...