Le travail a été financé par NIDCD / NIH R01DC003915 à LO Bakaletz.

1. Dans la formation de biofilms in vitro <ol><li> Les colonies bactériennes (qui ont été cultivés sur gélose appropriée durant la nuit), ont été suspendues dans les médias et la DO 490 a été ajusté à 0,65 </li><li> La suspension résultante bactérienne est ensuite diluée 1:6 (1 ml de suspension bactérienne + 5 ml préchauffé moyenne) et incubées à 37 ° C avec 5% de CO 2 pendant environ 3 heures pour atteindre la mi-phase log. </li><li> Diluer le 1:2500 mi-log croissance en suspension avec des pré-chauffé les médias et le lieu 200 ul de dilution dans chaque puits d&#39;une lame de chambre 8-même. </li><li> Incuber à 37 ° C avec 5% de CO 2. </li><li> Après environ 16 heures, moyenne aspirer à partir du coin de chaque chambre et ajouter 200 ul frais, pré-chauffé moyen - long dispenser paroi de la chambre afin de ne pas créer des forces de cisaillement dans la chambre qui pourrait perturber le biofilm. </li><li> Si l&#39;incubation de plus de 24 heures, changement de milieu tous les 12 heures ou au besoin pour maintenir la viabilité bactérienne. </li></ol> 2. Visualisation des biofilms <ol><li> Aspirer moyennes de chaque chambre, comme décrit ci-dessus, et laver deux fois par biofilm résidents doucement avec une solution saline stérile. </li><li> Ajouter 200 ul de BacLight Live / Dead tache (3 uL composante A + 3 B composante ul par ml de solution saline) dans chaque puits et incuber à température ambiante pendant 15 minutes. Protéger la culture de la lumière à partir de ce point. </li><li> Aspirer la tache et laver deux fois délicatement avec une solution saline stérile comme avant. </li><li> Ajouter 200 ul du formol tamponné neutre à chaque puits et incuber pendant 30 minutes à température ambiante pour fixer spécimen. </li><li> Retirer fixateur et éliminer correctement conformément aux directives institutionnelles. </li><li> Laver deux fois avec une solution saline et d&#39;éliminer les fluides de lavage qui contiennent du formol comme ci-dessus. </li><li> Retirez le puits en plastique de la lame, ajouter suffisamment de solution saline pour chaque puits de sorte que quand une lamelle est placée sur le joint, pas de bulles d&#39;air sont présents. </li><li> Lamelle et sceller les bords de la lamelle avec un milieu de montage. Le vernis à ongles peut être utilisée pour sceller les diapositives cependant les diapositives ne seront pas aussi permanente. </li><li> Laisser le scellant sécher pendant environ 1 heure avant d&#39;examiner par microscopie. </li></ol> 3. Résultats <img src="/files/ftp_upload/2481/2481fig1.jpg" alt="Figure 1" /> Figure 1. Représentant 3D des images composites d&#39;un biofilm formé dans une chambre de diapositives 8-même. Les bactéries ont été marqués avec Live / Dead tache où des bactéries vivantes fluorescence verte et les bactéries mortes émettent une fluorescence rouge. (A) Image composite du biofilm ensemble montrant l&#39;architecture distincte avec des tours et des canaux d&#39;eau. (B) Même que dans le biofilm (A), maintenant vu en coupe transversale.

<ol>
	<li>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=Bacterial+biofilms+in+otitis+media:+evidence+and+relevance.">Bacterial biofilms in otitis media: evidence and relevance.</a> Pediatr Infect Dis J. 26, S17-S19 (2007).</li><li>Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K., Molin, S. <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, 2395-2407 (2000).</li></ol>

Comprendre le mécanisme par lequel former des biofilms, ainsi que la caractérisation de leurs composants de la matrice, est un domaine d&#39;intérêt intense. Ce protocole peut être utilisé pour aider à identifier les protéines ou d&#39;autres constituants qui contribuent à l&#39;intégrité structurale du biofilm, ainsi que d&#39;identifier et de cibler des protéines qui peuvent se prêter à une application thérapeutique. Le temps d&#39;incubation et des dilutions dans le protocole ci-dessus décrits ont été établies pour la formation de biofilm optimale par l&#39;Haemophilus influenzae non typables (NTHi). Certains travaux préliminaires pourraient être nécessaires afin d&#39;optimiser ces étapes pour d&#39;autres espèces bactériennes (ie dilution initiale et le temps d&#39;incubation nécessaire pour atteindre la mi-log phase de croissance). La dilution 1:2500 pour l&#39;ensemencement initial des chambres a été établi pour s&#39;assurer que le biofilm robuste se développe dans la chambre de diapositives comme induite par cet organisme et dans le temps d&#39;incubation a déclaré, sans le biofilm envahit la chambre et / ou épuiser les éléments nutritifs disponibles . En conséquence, la dilution initiale et ou de temps d&#39;incubation peut être nécessaire d&#39;ajuster pour les autres espèces bactériennes.

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<td>Chocolate II agar</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>B21267X</td>
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<td>8-well chamber slides</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-656-18</td>
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<td>BacLight Live/Dead bacterial viability kit</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>NC9439023</td>
<td>&nbsp;</td>
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<td>BHI</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>211059</td>
<td>&nbsp;</td>
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<td>Hemin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H5533</td>
<td>&nbsp;</td>
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<td>&beta;-NAD</td>
<td>&nbsp;</td>
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<td>N7004</td>
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in vitro biofilm formation in an 8-well chamber slide

La nature chronique de nombreuses maladies est attribué à la formation de biofilms bactériens qui sont récalcitrants à l&#39;antibiothérapie classique. Les biofilms sont communautaires associés bactéries attachées à une surface et enfermé dans une matrice. Le rôle de la matrice extracellulaire est multiforme, notamment en facilitant l&#39;acquisition des nutriments, et offre une protection significative contre les stress environnementaux (réponses de l&#39;hôte, par exemple immunitaire). Dans un effort pour acquérir une meilleure compréhension de comment les bactéries dans un biofilm de répondre aux contraintes environnementales, nous avons utilisé un protocole dans lequel nous visualisons les biofilms bactériens, qui se sont formées dans une chambre de diapositives 8-même. Les biofilms ont été colorés avec du BacLight Live / Dead taches et examinés à l&#39;aide d&#39;un microscope confocal pour caractériser la taille du biofilm relative, et la structure sous différentes conditions d&#39;incubation. Z-stack images ont été recueillies par microscopie confocale et analysées par COMSTAT. Ce protocole peut être utilisé pour aider à élucider le mécanisme et la cinétique par lequel former des biofilms, ainsi que d&#39;identifier les éléments qui sont importants pour la structure du biofilm et la stabilité.

Watch this Scientific Journal Video about In vitro Biofilm Formation in an 8-well Chamber Slide at JoVE.com

In vitro Biofilm Formation in an 8-well Chamber Slide

Cet article décrit la procédure pour la formation et la visualisation d&#39;un biofilm bactérien grandi au sein d&#39;une chambre de glisser de 8 puits

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. ...

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Research

JoVE Journal

Immunology and Infection

In vitro Biofilm Formation in an 8-well Chamber Slide

39.9K vues.  The Research Institute at Nationwide Children's Hospital. Cet article décrit la procédure pour la formation et la visualisation d'un biofilm bactérien grandi au sein d'une chambre de glisser de 8 puits

Vidéo: In vitro Biofilm Formation in an 8-well Chamber Slide

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 ...

Recherche

Immunologie et infection

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.

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 ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Quantitative<em&gt; In vitro</em&gt; Test pour mesurer l&#39;adhésion des neutrophiles à Activé cellules endothéliales microvasculaires humaines primaires dans des conditions statiques

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

Entérique invasion bactérienne des cellules épithéliales intestinales<em&gt; In Vitro</em&gt; Est considérablement améliorée en utilisant une chambre diffusion verticale du modèle

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

High Throughput Mesure de l&#39;ADN extracellulaire de sortie et quantitative Formation NET dans les neutrophiles humains<em&gt; In vitro</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

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

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 development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

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.

Visualisation des effets des expectorations sur le développement du biofilm Utilisation d&#39;un modèle Chambered lamelle couvre-objet

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 ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Méthodes in vitro pour la comparaison de liaison cible et CDC induction entre les anticorps thérapeutiques: Applications dans l&#39; analyse biosimilarité

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

Un<em&gt; In Vitro</em&gt; Ensemble de liaison de caséine qui prédit la pénétration des drogues dans les lésions tuberculeuses

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

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, ...