Mes remerciements à Sherry Koutchma, Pete Newell et Robert Shanks pour fournir les images de la figure 1. Ce travail a été soutenu par NIH R01AI083256 au GAO

1. Growing un biofilm <ol><li> Cultiver une culture de l&#39;aeruginosa de type sauvage ou de souche mutante de Pseudomonas pendant la nuit dans un milieu riche (LB-dire) </li><li> Diluer le 1:100 nuit sur la culture dans un milieu frais pour des essais de biofilm. Un milieu de biofilm standards de dosage pour P. aeruginosa est M63 milieu minimal supplémenté avec du sulfate de magnésium, du glucose et des acides casamino (voir tableau). Comme un biofilm-promotion d&#39;autres moyens qui stimule la croissance moins planctoniques et un biofilm plus robuste, le glucose et des acides casamino peut être remplacé par l&#39;arginine comme le carbone unique et source d&#39;énergie. </li><li> Ajouter 100 ul de la dilution par puits dans un plat de 96 puits. Pour les tests quantitatifs, nous avons généralement recours à des puits 4-8 réplicats pour chaque traitement. </li><li> Incuber la microplaque pour 4-24 heures à 37 ° C. </li></ol> 2. La coloration des biofilms <ol><li> Après incubation, vider les cellules par retournement de la plaque et en secouant le liquide. </li><li> Doucement plonger la plaque dans une petite baignoire d&#39;eau (à savoir, l&#39;utilisation des fonds des boîtes de pointes de pipette pour P1000 pipetmen que la baignoire). Secouez l&#39;eau. Répétez ce processus une deuxième fois. Cette étape permet d&#39;éliminer les cellules seules et des composants média qui peut être teinté dans la prochaine étape, et réduit considérablement les taches de fond. </li><li> Ajouter 125 ul d&#39;une solution à 0,1% de cristal violet dans l&#39;eau à chaque puits de la microplaque. Porter des gants et une blouse de laboratoire tout en faisant la solution. Faites preuve de prudence lors de la pesée sur le CV que la poudre est hygroscopique et facilement tache les vêtements, la peau, etc </li><li> Incuber la microplaque à température ambiante pendant 10-15 min. </li><li> Rincer la plaque de 3-4 avec de l&#39;eau par immersion dans un bain d&#39;eau comme indiqué ci-dessus, secouer vigoureusement et éponger sur une pile de serviettes en papier pour débarrasser le plateau de toutes les cellules excédentaires et de teinture. </li><li> Tournez la plaque de microtitration à l&#39;envers et sec pendant quelques heures ou jusqu&#39;au lendemain. </li><li> Pour les essais qualitatifs, les puits peuvent être photographiés à l&#39;état sec. </li></ol> 3. Quantifier le biofilm <ol><li> Ajouter 125 ul d&#39;acide acétique à 30% dans de l&#39;eau dans chaque puits de la microplaque pour solubiliser le CV. </li><li> Incuber la microplaque à température ambiante pendant 10-15 min. </li><li> Transfert 125 ul du CV solubilisé à un nouveau plat de microtitration à fond plat. </li><li> Quantifier l&#39;absorbance dans un lecteur de plaque à 550 nm en utilisant 30% d&#39;acide acétique dans l&#39;eau comme le vide. </li></ol> 4. Les résultats représentatifs: <img src="/files/ftp_upload/2437/2437fig1.jpg" alt="Figure 1" /> Figure 1. Montre un résultat représentatif pour des essais de formation de biofilm effectué pour Pseudomonas aeruginosa, Pseudomonas fluorescens et Staphylococcus aureus. (A) Une vue de côté du puits avec un biofilm de P. aeruginosa (8 heures, 37 ° C). (B) Une vue de côté du puits avec un biofilm de P. fluorescens (6 h, 30 ° C). (C) Une vue de haut en bas du biofilm formé par S. aureus dans une plaque de microtitration à fond plat (deux puits, 24 h, 37 ° C). P. aeruginosa et P. fluorescens sont deux organismes motiles et forment un biofilm à l&#39;interface air-liquide. S. aureus est non mobile et des formes un biofilm sur le fond du puits.

<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>

Cette méthode peut être modifié pour être utilisé avec une grande variété d&#39;espèces microbiennes. Microbes mobiles généralement adhérer aux parois et / ou fond des puits, tandis que non mobiles microbes normalement adhérer au fond des puits. Les conditions optimales pour la formation du biofilm (ie, le milieu de croissance, température, temps d&#39;incubation) doit être déterminée empiriquement pour chaque microbe. Je vous recommandons d&#39;effectuer plusieurs répétitions pour chaque souche ou la ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

Les biofilms sont des communautés de microbes attachés à des surfaces, qui peuvent être trouvés dans les milieux médicaux, industriels et naturels. En fait, la vie dans un biofilm représente probablement le mode prédominant de la croissance des microbes dans la plupart des environnements. Biofilms matures ont quelques caractéristiques distinctes. Microbes biofilm sont généralement entourés d&#39;une matrice extracellulaire qui fournit la structure et la protection de la communauté. Les microbes de plus en plus dans un biofilm ont aussi une architecture caractéristique généralement composé de macrocolonies (contenant des milliers de cellules) entourés par des canaux remplis de fluide. Biofilm cultivés microbes sont également connus pour leur résistance à une gamme d&#39;agents antimicrobiens, y compris des antibiotiques cliniquement pertinents. Le dosage de plaque de microtitrage est un outil important pour l&#39;étude des premières étapes dans la formation de biofilms, et a été appliquée essentiellement pour l&#39;étude des biofilms bactériens, bien que ce test a également été utilisée pour étudier la formation de biofilms fongiques. Parce que ce test utilise statique, par lots des conditions de croissance, il ne permet pas à la formation des biofilms matures généralement associés aux systèmes de cellule d&#39;écoulement. Cependant, le dosage a été efficace pour identifier les nombreux facteurs requis pour l&#39;initiation de la formation du biofilm (ie, les flagelles, pili, adhésines, enzymes impliquées dans cyclique-di-GMP contraignant et métabolisme) et ainsi que des gènes impliqués dans la production de polysaccharide extracellulaire. Par ailleurs, des travaux publiés indiquent que les biofilms cultivés dans des plaques de microtitrage ne développent certaines propriétés des biofilms matures, une telle tolérance aux antibiotiques et la résistance aux effecteurs du système immunitaire. Ce test microtitration simples plat permet la formation d&#39;un biofilm sur les parois et / ou fond d&#39;un plat de microtitration. La nature à haut débit de l&#39;essai le rend utile pour les écrans de génétique, ainsi que la formation de biofilms par des souches multiples tests dans des conditions de croissance différentes. Des variantes de ce test ont été utilisés pour évaluer la formation de biofilm tôt pour une grande variété de microbes, y compris mais non limité à, les pseudomonades, Vibrio cholerae, Escherichia coli, staphylocoques, les entérocoques, les mycobactéries et les champignons. Dans le protocole décrit ici, nous allons nous concentrer sur l&#39;utilisation de ce test pour étudier la formation de biofilm par le aeruginosa Pseudomonas organisme modèle. Dans cet essai, l&#39;étendue de la formation du biofilm est mesuré en utilisant le colorant cristal violet (CV). Cependant, un certain nombre d&#39;autres taches colorimétriques et métaboliques ont été rapportées pour la quantification de la formation de biofilms en utilisant le dosage de plaque de microtitrage. La facilité, son faible coût et la flexibilité de l&#39;essai de plaque de microtitrage en a fait un outil essentiel pour l&#39;étude des biofilms.

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

Microtiter Dish Biofilm Formation Assay

Le test décrit un moyen rapide pour mesurer la formation de biofilm au début de bactéries et de champignons. Cette méthode utilise une plaque de microtitration comme substrat pour la formation de biofilms microbiens et le biofilm est visualisée en utilisant la souche de cristal violet. Le test fournit soit un test qualitatif ou quantitatif pour la formation de biofilm tôt.

Plaque de microtitrage biofilm essai de formation

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

106.7K vues.  Dartmouth Medical School. Le test décrit un moyen rapide pour mesurer la formation de biofilm au début de bactéries et de champignons. Cette méthode utilise une plaque de microtitration comme substrat pour la formation de biofilms microbiens et le biofilm est visualisée en utilisant la souche de cristal violet. Le test fournit soit un test qualitatif ou quantitatif pour la formation de biofilm tôt.

Vidéo: Microtiter Dish Biofilm Formation Assay

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

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

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

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Une plaque de microtitrage à 96 puits à base Méthode de formation suivi et les tests de sensibilité antifongique des Candida albicans Les biofilms

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

Recherche

Immunologie et infection

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 et le biofilm Saccharomyces cerevisiae en cellules à flux

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

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

Dans la formation de biofilms in vitro dans une diapositive Chambre de 8 puits

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

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Test de sensibilité aux antimicrobiens du complexe Mycobacterium tuberculosis des drogues de première et deuxième par dilution en bouillon dans un format microplaque

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

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

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

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

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Décrypter et de l&#39;imagerie et la pathogenèse de Cording<em&gt; Mycobacterium abscessus</em&gt; Dans embryons de poissons zèbres

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

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

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

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Ciblage biofilm Associated<em&gt; Staphylococcus aureus</em&gt; Utilisation de résazurine Basé Essai de sensibilité aux médicaments

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)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

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

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Phagosome Clôture Assay&quot; pour visualiser Formation Phagosome en trois dimensions en utilisant la réflexion interne totale Fluorescent Microscopy (TIRFM)

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

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.

Visualisation de la Formation de biofilms Candida albicans à l’aide d’un dispositif automatisé de microfluidique

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

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

La Formation de Biofilm induite sur les sels biliaires chez les agents pathogènes entériques : Techniques d’Identification et de Quantification

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

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Un test haut débit pour évaluer et quantifier les neutrophiles piège extracellulaire Formation

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