Biofilm liés travailler dans le laboratoire est financé par des subventions et numérotées R21DE017294 R21AI080930 de l&#39;Institut national de recherche dentaire et craniofaciale et l&#39;Institut national des maladies allergiques et infectieuses (pour JLL-R.). Le contenu est uniquement la responsabilité de leurs auteurs et ne représentent pas nécessairement les vues officielles de l&#39;NIDCR, le NIAID ou du NIH.

1. Préparation de C. albicans C. albicans est un microorganisme 1/BSL1 des risques du Groupe. Rappelez-vous toujours à la bonne utilisation des techniques aseptiques / stériles pour travailler avec ce micro-organisme et de suivre les procédures institutionnelles pour l&#39;élimination appropriée des matériaux biologiques dangereux. <ol><li> Préparer une culture de nuit de C. albicans dans YPD (levure-peptone dextrose) milieu liquide en inoculant une seule colonie de C. albicans dans 25 ml de YPD. </li><li> Incuber la culture dans un agitateur orbital (environ 180 rpm) à 30 ° C pendant la nuit. C. La plupart des albicans souches augmentera à mesure que les cellules de levure dans ces conditions. </li><li> Centrifuger les cultures de la nuit (environ 3000 rpm, 5-10 minutes), se laver les cellules deux fois avec du PBS stérile, et remettre le culot final dans environ 20-25 ml de milieu RPMI 1640 tamponné avec 165 mM d&#39;acide morpholinepropanesulfonic à pH 7,0 et préchauffé à 37 ° C (à partir de maintenant sur ce milieu est appelé simplement &quot;RPMI 1640&quot;). </li><li> Compter les cellules en utilisant un hématimètre. Après le dépouillement, préparer une suspension de cellules à une densité finale de 1,0 x 10 6 cellules / ml dans du RPMI 1640. Remarque: Parce que les cellules ont tendance à s&#39;agréger, il est important de vortexer vigoureusement entre les lavages et avant le pipetage. </li></ol> 2. Configuration de la plaque de 96 puits de microtitration pour la formation du biofilm <ol><li> En utilisant une pipette multicanaux ajouter 100 uL de C. la suspension albicans dans les puits sélectionnés de la plaque de 96 puits de microtitration (-s). Ne pas ajouter des cellules à des puits dans la colonne 12, car ceux-ci servent de témoins négatifs. </li><li> Recouvrir la plaque de microtitration entière avec son couvercle d&#39;origine, joint avec du parafilm, place à l&#39;intérieur d&#39;un incubateur et incuber pendant 24 h à 37 ° C. La durée d&#39;incubation peut être adapté à la conception spécifique expérimentale. Par exemple, il est possible d&#39;examiner la cinétique de formation d&#39;un biofilm sur une période de 24 - 48 heures par ensemencement des plaques multiples et le traitement de chaque plaque à différents moments (c. 2, 4, 8, 12, 24 et 48 h) </li><li> Après la formation du biofilm, à l&#39;aide d&#39;une pipette multicanaux aspirer le support de soin que de ne pas toucher et perturber le biofilm qui se sont formés dans chacun des puits. </li><li> En utilisant une pipette multicanaux plaques laver trois fois dans du PBS stérile (200 ul par puits). Sinon, utilisez une rondelle de microtitration automatisé assiette. Entre les lavages, et notamment après le dernier lavage, égouttez les plaques en position inversée en épongeant avec du papier absorbant pour enlever tout résidu de PBS. A ce point biofilms formés sur le fond des puits doit être clairement visible, même à l&#39;œil nu et peut également être visualisés à l&#39;aide d&#39;un microscope inversé (figure 1). Les biofilms sont maintenant prêts à être traitées pour des dosages des tests de sensibilité antifongique. (Si le but principal de l&#39;expérience est d&#39;évaluer l&#39;étendue de la formation de biofilms, les plaques sont prêtes à être traitées en utilisant la méthode colorimétrique. Pour cela, le réactif XTT / ménadione (voir l&#39;étape 3 ci-dessous) peuvent être ajoutés et la couleur résultante lus en utilisant un lecteur de plaque de microtitration). </li></ol> 3. Test de sensibilité aux antifongiques des biofilms <ol><li> Des solutions stock ou en poudre, préparer une solution de travail en milieu RPMI 1640 de chaque antifongiques à tester. Typiques des concentrations élevées sont 1024 pg / ml pour le fluconazole, et 16 pg / ml à la fois pour l&#39;amphotéricine B et la caspofungine. D&#39;autres concentrations peuvent être utilisées pour différents agents. </li><li> En utilisant une pipette multicanaux, ajouter 200 ul de la forte concentration de travail des antifongiques dans les puits correspondants dans la colonne 1 de chaque plaque de microtitration contenant des biofilms fongiques. </li><li> Ajouter 100 ul de RPMI 1640 à chacun des puits dans les colonnes 2 à 10. </li><li> Ajouter 100 ul de RPMI 1640 aux puits dans la colonne 11. </li><li> Retirer 100 uL d&#39;agent antifongique dans les puits de la colonne 1 et l&#39;ajouter au puits adjacents dans la colonne 2 (contenant déjà 100 ul de milieu). </li><li> Mélangez le contenu bien par aspiration et refoulement d&#39;effectuer une double dilution en série, et de supprimer les pointes. </li><li> Répétez jusqu&#39;à ce que le déplacement vers la droite des puits de la colonne 10, après quoi la finale du 100 volumes uL dans les puits de la colonne 10 après le mélange est jeté. De cette façon, une série de dilutions de votre agent (-s) d&#39;intérêt ont été créés; de la plupart concentrées dans les puits de la colonne 1 de moins concentrées dans les puits de la colonne 10. Biofilms incontesté dans la colonne 11 servira de contrôles positifs, et les puits vides dans la colonne 12 servira de contrôles négatifs. </li><li> Couvrir les plaques avec leur couvercle, joint avec du parafilm et incuber pendant 24 h -48 à 37 ° C. </li><li> Après la période d&#39;incubation, laver les assiettes que dans l&#39;étape 2.4 avant (3 x PBS). </li><li> En utilisant une pipette multicanaux ajouter 100 ul de XTT / ménadione solution à chaque puits contenant un biofilm pré-lavés ainsi que les puits de contrôle négatif pour la mesure de X de fondTT-colorimétrique niveaux. <ol><li> Le XTT est préparée comme une solution saturée à 0,5 g / L dans le lactate de Ringer stérile, PBS ou de solution saline, qui doit être stérilisée par filtration en utilisant un filtre de 0,22 um de taille des pores. La solution est sensible à la lumière XTT, donc il devrait être couvert avec du papier aluminium pendant la préparation. Une fois préparé et stérilisé par filtration, partie aliquote dans 10 mL volumes de travail, et conserver à -70 ° C. Protéger les tubes de la lumière en utilisant du papier d&#39;aluminium. Décongelez uniquement comme de nombreux tubes comme nécessaire pour une expérience particulière, juste avant utilisation. </li><li> Ménadione est préparée comme une solution à 10 mM dans l&#39;acétone à 100%, répartie dans des petits volumes (environ 50 pi) et conservés à -70 ° C. Préparer la solution XTT / ménadione juste avant l&#39;utilisation, en ajoutant 1 pl de la solution stock de ménadione dans un tube contenant 10 ml de la solution XTT décongelé. </li></ol></li><li> Couvrir les plaques dans du papier aluminium et laisser incuber dans l&#39;obscurité pendant 2 h à 37 ° C. </li><li> Découvrez les assiettes. En utilisant une pipette multicanaux retirer 80 ul du surnageant résultant de couleur de chaque puits et le transfert dans les puits correspondants d&#39;une plaque de microtitration nouvelles. </li><li> Lire la plaque (-s) dans un lecteur de microplaque à 490 nm. </li><li> Calculer les concentrations minimales inhibitrices sessiles SMIC50 et SMIC80, qui sont les concentrations antifongiques au cours de laquelle une diminution de 50% ou 80% dans les lectures colorimétriques sont détectés en comparaison avec le contrôle des biofilms formés en l&#39;absence de médicament antifongique (dans ce cas les valeurs de la colonne 11 , rappelez-vous aussi à soustraire des valeurs de contrôle négatif à partir de puits dans la colonne 12 contenant XTT seulement). Les résultats du SMIC peut être présenté comme un tableau (ie multiples contre des isolats antifongiques multiples) ou, alternativement, les résultats individuels pour chaque champignon d&#39;isoler les uns contre les antifongiques peuvent être présentés sous forme de graphique en traçant la concentration d&#39;inhibition pour cent par rapport antifongique. </li></ol> Exemple: Après avoir soustrait les valeurs dans le contrôle négatif, la DO moyenne de biofilms formés de contrôle dans la colonne 11 est de 1,32. Le SMIC50 est la plus faible concentration antifongique conduisant à une réduction&gt; 50% dans les lectures colorimétriques, dans ce cas moins de 1,32 x 50 / 100 = 0,66. De même, le SMIC80 est la plus faible concentration antifongique conduisant à une réduction&gt; 80% dans les lectures colorimétriques, dans ce cas moins de 1,32 x 20/100 = 0.264. 4. Les résultats représentatifs La figure 1 montre une microphotographie d&#39;un C. albicans biofilm formé sur le fond d&#39;un puits dans une plaque de microtitrage à 96 ainsi prises à l&#39;aide d&#39;un microscope inversé. La figure 2 montre XTT-colorimétrique des lectures (DO 490 valeurs) pour chacun des 11 biofilms d&#39;un C. souche de type sauvage albicans formé dans chacune des huit lignes différentes de la même plaque de microtitration 96 puits. La figure 3 montre l&#39;activité de l&#39;amphotéricine B à des concentrations différentes contre C. albicans biofilms; flèches indiquent SMIC50 et SMIC80 valeurs. <img src="/files/ftp_upload/2287/2287fig1.jpg" alt="Figure 1" /> La figure 1 (A). Panneau A montre une microphotographie prise avec une caméra reliée à un microscope inversé d&#39;un C. albicans biofilm formé sur le fond du puits après aspiration du milieu RPMI et lavages successifs au PBS. (B) une micrographie d&#39;un typique C. albicans biofilm visualisé par microscopie électronique à balayage. Les bars sont 100 um et 10 um pour les panneaux A et B respectivement. <img src="/files/ftp_upload/2287/2287fig2.jpg" alt="Figure 2" /> Figure 2. Formation de multiples équivalents C. albicans biofilms dans les plaques 96 puits de microtitration. lectures colorimétriques (DO 490 valeurs) à partir de réduction des dosages XTT des biofilms formés par un C. albicans type sauvage dans des puits de microplaques. Les valeurs sont indépendantes de 11 biofilms formés dans chacune des huit lignes différentes de la même plaque de microtitration 96 puits. Résultats pour les différentes lignes ont été comparés par une analyse de variance et en utilisant le test de Bartlett pour l&#39;homogénéité des variances et des comparaisons multiples de Bonferroni du post-test. Aucune différence statistiquement significative n&#39;a été notée en comparant toutes les paires de lignes à l&#39;autre (p&gt; 0,05). <img src="/files/ftp_upload/2287/2287fig3.jpg" alt="Figure 3" /> Figure 3. Les résultats typiques des tests de sensibilité antifongique contre C. albicans biofilms. graphique illustrant les résultats typiques de l&#39;efficacité de différentes concentrations d&#39;amphotéricine B contre les biofilms d&#39;un C. souche de type sauvage albicans. Les valeurs sont exprimées en pour cent en moyenne des lectures colorimétriques pour XTT de réduction par rapport aux tests de contrôle de puits. SMIC50 et SMIC80 valeurs sont indiquées par des flèches.

<ol>
	<li>Bachmann, S. P. <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+activity+of+caspofungin+against+Candida+albicans+biofilms.">In vitro activity of caspofungin against Candida albicans biofilms.</a> Antimicrob Agents Chemother. 46, 3591-3596 (2002).</li><li>Hawser, S. P., Norris, H., Jessup, C. J., Ghannoum, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+a+2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-t+etrazolium+hydroxide+(XTT)+colorimetric+method+with+the+standardized+National+Committee+for+Clinical+Laboratory+Standards+method+of+testing+clinical+yeast+isolates+for+susceptibility+to+antifungal+agents.">Comparison of a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-t etrazolium hydroxide (XTT) colorimetric method with the standardized National Committee for Clinical Laboratory Standards method of testing clinical yeast isolates for susceptibility to antifungal agents.</a> J Clin Microbiol. 36, 1450-1452 (1998).</li><li>Kuhn, D. M., Balkis, M., Chandra, J., Mukherjee, P. K., Ghannoum, M. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+albicans+transcription+factor+Bcr1p:+a+regulator+of+cell-surface+genes+and+biofilm+formation.">C. albicans transcription factor Bcr1p: a regulator of cell-surface genes and biofilm formation.</a> Current Biol. 15, 1150-1155 (2005).</li><li>Ramage, G., Saville, S. P., Thomas, D. P., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+biofilms:+an+update.">Candida biofilms: an update.</a> Eukaryot Cell. 4, 633-638 (2005).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., p, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob Agents Chemother. 45, 2475-2479 (2001).</li><li>Bachmann, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+combinations+against+Candida+albicans+biofilms+in+vitro.">Antifungal combinations against Candida albicans biofilms in vitro.</a> Antimicrob Agents Chemother. 47, 3657-3659 (2003).</li><li>Ramage, G., VandeWalle, K., Bachmann, S. P., Wickes, B. L., Lopez-Ribot, J. L. <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+pharmacodynamic+properties+of+three+antifungal+agents+against+preformed+Candida+albicans+biofilms+determined+by+time-kill+studies.">In vitro pharmacodynamic properties of three antifungal agents against preformed Candida albicans biofilms determined by time-kill studies.</a> Antimicrob Agents Chemother. 46, 3634-3636 (2002).</li><li>Ramage, G., VandeWalle, K., Wickes, B. L., L&#243;pez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Rev. Iberoam. Micol. 18, 163-170 (2001).</li><li>Tellier, R., Krajden, M., Grigoriew, G. 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JLL-R possède une participation dans les technologies MicrobeHTS, Inc, qui est le développement d&#39;agents antifongiques. Technologies MicrobeHTS, Inc fourni aucune aide financière pour ces études.

Nous décrivons ici un moyen simple, rapide, économique et hautement reproductibles 96 modèle bien microplaque pour la formation de biofilms à Candida couplée à une méthode colorimétrique qui mesure l&#39;activité métabolique des cellules au sein du biofilm en utilisant XTT. Ce modèle bien 96 microplaque pour la formation de biofilms a été initialement développé pour C. albicans, mais peut être utilisé pour d&#39;autres espèces de Candida. et facilement adaptable à d&#39;autr...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>Sabouraud-dextrose agar</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>211584</td>
<td>To prepare plates for fresh subcultures of fungal isolates</td>
</tr>
<tr>
<td>YPD: Yeast peptone dextrose</td>
<td>&nbsp;</td>
<td>US Biological </td>
<td>Y2076</td>
<td>Medium for propagation of overnight liquid cultures</td>
</tr>
<tr>
<td>RPMI-1640 without sodium bicarbonate supplemented with L-glutamine</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>50-020-PB</td>
<td>Liquid medium for biofilm formation</td>
</tr>
<tr>
<td>Morpholinepropanesulfonic acid (MOPS)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP308</td>
<td>To buffer RPMI 1640</td>
</tr>
<tr>
<td>Phosphate buffered saline, PBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>Buffer for washes</td>
</tr>
<tr>
<td>XTT sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>X4251</td>
<td>See above for preparation instructions</td>
</tr>
<tr>
<td>Ringer's lactate</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>NDC0409-7953-09</td>
<td>For preparation of XTT solution</td>
</tr>
<tr>
<td>Menadione</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5625</td>
<td>Caution: hazardous by skin contact, inhalation or ingestion</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430790</td>
<td>&nbsp;</td>
<tr>
<td>50 ml conical centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>96 well microtiter plates: Polystyrene, flat-bottomed, tissue culture treated</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3595</td>
<td>&nbsp;</td>
<tr>
<td>Multichannel pipette and tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microtiter plate reader</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a 96 well microtiter plate-based method for monitoring formation and antifungal susceptibility testing of candida albicans biofilms

Candida albicans reste la cause la plus fréquente des infections fongiques chez une population en expansion des patients compromise et la candidose est maintenant le troisième plus fréquente d&#39;infection dans les hôpitaux américains. Différentes manifestations de la candidose sont associés à la formation de biofilm, à la fois sur tissus de l&#39;hôte et / ou des dispositifs médicaux (cathéters par exemple). La formation du biofilm porte négatives implications cliniques, comme les cellules dans le biofilm sont protégées par des réponses immunitaires de l&#39;hôte et de l&#39;action des antifongiques. Nous avons développé un modèle simple, rapide et robuste in vitro pour la formation de C. biofilms albicans aide de 96 puits de microtitration-plaques, qui peut également être utilisé pour tester la sensibilité aux antifongiques biofilm. La lecture de ce dosage est colorimétrique, basée sur la réduction du XTT (un sel de tétrazolium) par les cellules métaboliquement active biofilm fongique. Une expérience typique dure environ 24 h pour la formation de biofilms, avec un supplément de 24 h pour les tests de sensibilité aux antifongiques. En raison de sa simplicité et l&#39;utilisation de matériel de laboratoire couramment disponibles et des équipements, cette technique démocratise la recherche biofilm et représente une étape importante vers la normalisation des tests de sensibilité aux antifongiques des biofilms fongiques.

Watch this Scientific Journal Video about A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms at JoVE.com

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

Nous décrivons une méthode simple, rapide et robuste pour la formation de<em&gt; Candida albicans</em&gt; Biofilms en utilisant des plaques de microtitration 96 puits et son utilité dans les tests de sensibilité antifongique de cellules au sein des 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 ...

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Research

JoVE Journal

Immunology and Infection

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

44.5K vues.  University of Texas San Antonio - UTSA. Nous décrivons une méthode simple, rapide et robuste pour la formation de Candida albicans Biofilms en utilisant des plaques de microtitration 96 puits et son utilité dans les tests de sensibilité antifongique de cellules au sein des biofilms.

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

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 ...

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Immunologie et infection

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). ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Préparation d&#39;une hémoculture Pellet pour Rapid bactérienne identification et les tests de sensibilité aux antibiotiques

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Une méthode de référence de microdilution de bouillon pour dalbavancine<em&gt; In Vitro</em&gt; Tests de sensibilité des bactéries qui se développent Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

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. ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Profonde cutanée Injection comme un modèle d’Infection de la peau de Candida albicans pour les Analyses histologiques

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Photothérapie quotidienne avec lumière rouge pour réglementer Candida albicans biofilms croissance

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

Clarification et imagerie Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Thérapie photodynamique médiée par rose Bengal pour inhiber Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...