Biofilm attività relative al laboratorio è finanziata da sovvenzioni numerati R21DE017294 e R21AI080930 dal National Institute of Dental Research &amp; cranio-facciali e l&#39;Istituto nazionale per le allergie e malattie infettive (a JLL-R.). I contenuti sono di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale della NIDCR, il NIAID o il NIH.

1. Preparazione di C. albicans C. albicans è un microrganismo Group Risk 1/BSL1. Ricordate sempre di usare buone tecniche asettiche / sterile per lavorare con questo microrganismo e seguire le procedure istituzionali per il corretto smaltimento dei materiali a rischio biologico. <ol><li> Preparare una cultura durante la notte di C. albicans in YPD (Lievito peptone destrosio) mezzo liquido inoculando una singola colonia di C. albicans in 25 ml di YPD. </li><li> Incubare cultura in un agitatore orbitale (circa 180 giri al minuto) a 30 ° C durante la notte. La maggior parte C. albicans ceppi crescerà come cellule di lievito in queste condizioni. </li><li> Centrifugare le culture durante la notte (circa 3.000 rpm, 5-10 minuti), lavare le cellule due volte con PBS sterile e risospendere il pellet in finale di circa 20-25 ml di RPMI 1640 medium tamponato con l&#39;acido mm 165 morpholinepropanesulfonic a pH 7,0 e pre-riscaldata a 37 ° C (da ora in poi questo mezzo è definita semplicemente come &quot;RPMI 1640&quot;). </li><li> Contare le cellule utilizzando un emocitometro. Dopo il conteggio, preparare una sospensione di cellule ad una densità finale di 1,0 x 10 6 cellule / ml in RPMI 1640. Nota: Poiché le cellule hanno la tendenza ad aggregarsi, è importante vortice vigorosamente tra lavaggi e prima di pipettaggio. </li></ol> 2. Impostazione del 96-ben micropiastra per la formazione del biofilm <ol><li> Utilizzando una pipetta multicanale, aggiungere 100 ml di C. sospensione albicans nei pozzetti selezionate del-96 e micropiastra (-s). Non aggiungere alle cellule di pozzi in colonna 12, in quanto questi serviranno come controlli negativi. </li><li> Coprire l&#39;intera piastra microtiter con il suo coperchio originale, sigillare con parafilm, posto all&#39;interno di un incubatore e incubare per 24 ore a 37 ° C. La lunghezza di incubazione può essere adattata alla struttura specifica sperimentale. Per esempio, è possibile esaminare la cinetica della formazione di biofilm in un periodo di 24 - 48 ore dalla semina placche multiple e l&#39;elaborazione di un piatto in momenti diversi (es. 2, 4, 8, 12, 24 e 48 ore) </li><li> Dopo la formazione di biofilm, utilizzando una pipetta multicanale aspirare il terreno con attenzione per non toccare e disturbare il biofilm che si sono formati in ciascuno dei pozzi. </li><li> Usando una pipetta lavare piatti multicanale tre volte in PBS sterile (200 ul per pozzetto). In alternativa, utilizzare un sistema automatico lavaggio micropiastra. Tra un lavaggio, e in particolare dopo l&#39;ultimo lavaggio, le piastre di scarico in posizione invertita tamponando con carta assorbente per eliminare ogni residuo PBS. A questo punto biofilm formato sul fondo dei pozzetti deve essere chiaramente visibile anche ad occhio nudo e possono anche essere visualizzati utilizzando un microscopio invertito (Figura 1). Biofilm sono ora pronti per essere elaborati per le analisi antimicotico test di sensibilità. (Se lo scopo principale dell&#39;esperimento è quello di valutare l&#39;entità della formazione di biofilm, i piatti sono pronti per essere trattati con il metodo colorimetrico. Per questo, il XTT / menadione reagente (vedi punto 3 di seguito) possono essere aggiunti e il colore risultante letti utilizzando un lettore di micropiastra). </li></ol> 3. Test di suscettibilità antifungina di biofilm <ol><li> Dalle soluzioni stock o in polvere, preparare una soluzione di lavoro in RPMI 1640 medium di ogni antimicotico da testare. Tipici livelli di concentrazione elevati 1.024 mg / ml per fluconazolo, e 16 mg / ml per entrambe le amfotericina B e caspofungin. Altre concentrazioni possono essere utilizzati per diversi agenti. </li><li> Utilizzando una pipetta multicanale, aggiungere 200 microlitri della alta concentrazione di lavoro antimicotica nei pozzetti corrispondenti nella colonna 1 di ogni micropiastra contenente biofilm fungine. </li><li> Aggiungere 100 ml di RPMI 1640 in ciascun pozzetto in colonne da 2 a 10. </li><li> Aggiungere 100 ml di RPMI 1640 ai pozzi nella colonna 11. </li><li> Rimuovere 100 l di agente antimicotico dai pozzetti di colonna 1 e aggiungere ai pozzetti delle colonne 2 (già contenente 100 ml di media). </li><li> Mescolare il contenuto ben pipettando su e giù per eseguire una diluizione seriale raddoppio, e rimuovere le punte. </li><li> Ripetere il movimento a destra fino a quando i pozzi di colonna 10, dopo di che viene eliminato l&#39;ultimo volume di 100 microlitri dai pozzetti della colonna 10 dopo la miscelazione. In questo modo, una serie di doppie diluizioni del vostro agente (-s) di interesse sono stati creati, da più concentrata in pozzetti di colonna 1 al meno concentrato in pozzetti di colonna 10. Biofilm incontrastato nella colonna 11 serviranno come controlli positivi, e pozzi vuoti nella colonna 12 serviranno come controlli negativi. </li><li> Coprire le piastre con i loro coperchi, sigillare con parafilm e incubare per 24 -48 ore a 37 ° C. </li><li> Dopo il periodo di incubazione lavare i piatti, come nel passo 2,4 prima (3 x PBS). </li><li> Utilizzando una pipetta multicanale, aggiungere 100 ml di XTT / menadione soluzione in ogni pozzetto contenente una pre-lavati biofilm nonché di pozzetti di controllo negativo per la misura di X sfondoTT-colorimetrico livelli. <ol><li> Il XTT è pronta come una soluzione satura a 0,5 g / L in sterile di Ringer lattato, PBS o soluzione salina, che deve essere sterilizzata per filtrazione con un 0,22 micron, dimensioni dei pori del filtro. La soluzione XTT è sensibile alla luce, quindi dovrebbe essere coperto con un foglio di alluminio durante la preparazione. Una volta preparato e sterilizzata per filtrazione, aliquota in 10 ml di volume di lavoro, e conservare a -70 ° C. Proteggere i tubi di luce utilizzando un foglio di alluminio. Scongelare solo come tubi quante necessarie per un particolare esperimento appena prima dell&#39;uso. </li><li> Menadione è pronta come una soluzione al 10 mM magazzino in 100% acetone, frazionare in piccoli volumi (circa 50 mL) e conservati a -70 ° C. Preparare il XTT / menadione soluzione al momento dell&#39;uso, con l&#39;aggiunta di 1 ml di soluzione madre di menadione in una provetta contenente 10 mL della soluzione XTT scongelati. </li></ol></li><li> Coprire le piastre in alluminio ed incubare al buio per 2 ore a 37 ° C. </li><li> Scoprire i piatti. Utilizzando una pipetta multicanale eliminare l&#39;80 l di supernatante risultante colorate da ogni pozzetto e trasferire nei pozzetti corrispondenti della micropiastra nuovo. </li><li> Leggere la piastra (-s) in un lettore di micropiastre a 490 nm. </li><li> Calcolare il sessili concentrazioni minime inibenti SMIC50 e SMIC80, che sono le concentrazioni antimicotico in cui vengono rilevati il ​​50% o 80% di diminuzione nella lettura colorimetrica in confronto al controllo biofilm formati in assenza di farmaci antifungini (in questo caso i valori di colonna 11 , ricordate anche per sottrarre i valori da controlli negativi da pozzi nella colonna 12 contenente XTT solo). Risultati SMIC può essere presentato come una tabella (isola cioè multipla nei confronti delle antimicotici multipla) o, in alternativa, i risultati per ogni singolo fungine isolare l&#39;uno contro l&#39;antimicotico può essere presentato come un grafico disegnando inibizione per cento rispetto concentrazione antimicotico. </li></ol> Esempio: Dopo aver sottratto i valori del controllo negativo, il valore medio dei biofilm di controllo formata in colonna 11 è 1,32. Il SMIC50 è la più bassa concentrazione antimicotico che porta ad una riduzione&gt; 50% nelle letture colorimetrici, in questo caso meno di 1,32 x 50/100 = 0.66. Allo stesso modo, il SMIC80 è la più bassa concentrazione antimicotico che porta ad una riduzione&gt; 80% nelle letture colorimetrici, in questo caso meno di 1,32 x 20/100 = 0,264. 4. Rappresentante Risultati La figura 1 mostra una Microfotografia di una C. albicans biofilm formato sul fondo di un pozzo in una piastra da 96 microtitolazione preso bene utilizzando un microscopio invertito. La Figura 2 mostra XTT-colorimetrico letture (OD 490 valori) per ciascuna di 11 biofilm di C. ceppo di tipo selvatico albicans formate in ciascuna delle 8 righe diverse dello stesso 96 micropiastra bene. La figura 3 mostra l&#39;attività di amfotericina B in funzione della concentrazione contro C. albicans biofilm; frecce indicano SMIC50 e SMIC80 valori. <img src="/files/ftp_upload/2287/2287fig1.jpg" alt="Figura 1" /> Figura 1. (A) Un pannello mostra un Microfotografia scattate con una videocamera collegata ad un microscopio invertito di C. albicans biofilm formata sul fondo del pozzo dopo l&#39;aspirazione di medie RPMI e successivi lavaggi con PBS. (B) Una microfotografia di un tipico C. albicans biofilm visualizzati usando la microscopia elettronica a scansione. I bar sono 100 micron e 10 micron per i pannelli A e B rispettivamente. <img src="/files/ftp_upload/2287/2287fig2.jpg" alt="Figura 2" /> Figura 2. Formazione di molteplici equivalente C. albicans biofilm in piastre da 96 pozzetti microtiter. letture colorimetriche (OD 490 valori) da XTT riduzione analisi dei biofilm formato da una C. albicans tipo selvatico in pozzetti di piastre microtiter. I valori sono per 11 biofilm indipendente formata in ciascuna delle 8 righe diverse dello stesso 96 micropiastra bene. Risultati per le varie file sono stati confrontati con una analisi della varianza e utilizzando il test della Bartlett per l&#39;omogeneità delle varianze e confronto multiplo del Bonferroni post-test. Nessuna differenza statisticamente significativa è stata notata nel confronto tra tutte le coppie di righe tra loro (p&gt; 0,05). <img src="/files/ftp_upload/2287/2287fig3.jpg" alt="Figura 3" /> Figura 3. Risultati tipici di test di suscettibilità antifungina contro C. albicans biofilm. grafico raffigurante risultati tipici della efficacia di differenti concentrazioni di amfotericina B contro i biofilm di C. albicans ceppo selvatico. I valori sono espressi come valori colorimetrici per cento in media d&#39;XTT riduzione test rispetto al controllo pozzi. SMIC50 e SMIC80 valori sono indicati da frecce.

<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 possiede partecipazioni in MicrobeHTS Technologies, Inc., che sta sviluppando agenti antifungini. MicrobeHTS Technologies, Inc. ha fornito alcun supporto finanziario per questi studi.

Qui si descrive un semplice, rapido, economico e altamente riproducibili 96 pozzetti modello micropiastre per la formazione di biofilm Candida accoppiato con un metodo colorimetrico che misura l&#39;attività metabolica delle cellule all&#39;interno del biofilm utilizzando XTT. Questo modello ben 96 micropiastre per la formazione di biofilm è stato originariamente sviluppato per C. albicans ma può essere utilizzato per altre Candida spp. e facilmente adattato per altri organismi fungini. Il ...

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		<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>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 rimane la causa più frequente di infezioni fungine in una popolazione in espansione di pazienti compromessi e candidosi è ora la terza più comune infezione negli ospedali degli Stati Uniti. Diverse manifestazioni della candidiasi sono associati con la formazione di biofilm, sia su tessuti dell&#39;ospite e / o dispositivi medici (cateteri per esempio). Formazione di biofilm svolge negative implicazioni cliniche, come le cellule all&#39;interno del biofilm sono protetti dalla risposta immunitaria e dall&#39;azione di antimicotici. Abbiamo sviluppato un modello semplice, veloce e robusto in vitro per la formazione di C. biofilm albicans con 96 pozzetti per microtitolazione piastre, che può anche essere utilizzato per test di suscettibilità biofilm antimicotico. La lettura di questo saggio è colorimetrico, basato sulla riduzione delle XTT (un sale di tetrazolio) da cellule metabolicamente attive biofilm fungine. Un tipico esperimento dura circa 24 ore per la formazione di biofilm, con ulteriori 24 ore per i test di suscettibilità antifungina. A causa della sua semplicità e l&#39;utilizzo di materiali di laboratorio e attrezzature comunemente disponibili, questa tecnica democratizza ricerca biofilm e rappresenta un passo importante verso la standardizzazione dei test di suscettibilità antifungina di biofilm fungine.

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

Descriviamo un metodo semplice, rapido e robusto per la formazione di<em&gt; Candida albicans</em&gt; Biofilm su 96 micropiastre bene e la sua utilità nei test di suscettibilità antifungina di cellule all&#39;interno di biofilm.

A 96 Beh microtest piastra a base di Metodo per la Formazione di monitoraggio e test di suscettibilità antifungini Candida albicans Biofilm

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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Immunology and Infection

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

44.7K Views.  University of Texas San Antonio - UTSA. Descriviamo un metodo semplice, rapido e robusto per la formazione di Candida albicans Biofilm su 96 micropiastre bene e la sua utilità nei test di suscettibilità antifungina di cellule all'interno di biofilm.

Video: 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.

Microtitolazione Dish Biofilm saggio Formazione

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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Immunologia e infezioni

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

Nella formazione di biofilm in vitro in un 8-bene scorrere Camera

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 di suscettibilità antimicrobica del complesso Mycobacterium tuberculosis delle droghe prima e seconda linea per diluizione brodo in un formato micropiastra

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.

Preparazione di un Sangue cultura pellet per il Rapid batterica identificazione e test di sensibilità agli antibiotici

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.

Un riferimento brodo di microdiluizione per Dalbavancina<em&gt; In Vitro</em&gt; Test della Sensibilità di batteri che crescono aerobico

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.

Visualizzazione della formazione di Biofilm in Candida albicans utilizzando un dispositivo microfluidico automatizzato

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.

Profondo cutaneo iniezione come un modello di infezione della pelle Candida albicans per analisi istologiche

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.

Quotidiano fototerapia con luce rossa per sviluppo di Biofilm di regolare dei albicans del Candida

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 ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Visualizzazione degli albicani Candida nel tratto gastrointestinale Murine utilizzando l'ibridazione fluorescente in Situ

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

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.

Chiarire e 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 ...