Name: protocol for biofilm streamer formation in a microfluidic device with micro-pillars
Uploaded: 2014-08-20T14:00:00.000+00:00
Duration: 7 min 19 s
Description: Watch this Scientific Journal Video about Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars at JoVE.com

The authors would like to thank Professor Howard Ceri from the Biological Sciences Department of the University of Calgary for providing bacterial strains. A. Kumar acknowledges support from NSERC. T. Thundat acknowledges financial support from the Canada Excellence Research Chair (CERC) program. The authors would also like to acknowledge help from Ms. Zahra Nikakhtari for help with videography.

Eseguire i protocolli sperimentali qui nell&#39;ordine descritto di seguito. Protocolli di microfabbricazione per la creazione della piattaforma microfluidica sono discussi nel Passo 1 Passo 2 descrive il protocollo di coltura batterica (Figura 2), e punto 3 riguarda il montaggio del setup sperimentale (Figura 3). Infine, la fase sperimentale attuale è descritta al punto 4. 1 Chip Fabrication Procedura NOTA: La corretta procedure di sicurezza devono essere seguite per i processi descritti di seguito. Consultare il responsabile della sicurezza istituzionale per i dettagli. <ol><li> Progettare la maschera con un software appropriato (ad esempio, L-Edit). La progettazione canale è composto da un micro-canale di larghezza di 625 micron. La regione centrale del canale contiene una serie di micro-messaggi di 50 micron di diametro, distanziati di 25 micron a parte (vedi Supplemental Files). </li><li> Stampa questo disegno su vetro (5 &quot;x 5&quot; vetro soda lime), Che ha uno spessore di 0.09 &quot;ed è rivestito da circa 70 nm spesso strato di cromo (Cr), utilizzando mascheratura per preparare una maschera fotografico. Autore Utilizzo AZ400K per 1 min. Poi, etch strato Cr utilizzando Cr mordenzante per circa 1 min Usare acetone per togliere il resistere e pulirlo con soluzione piranha freddo. (H 2 SO 4 e H 2 O 2 in un rapporto di 3: 1). </li><li> Fotolitografia <ol><li> Pulire un wafer da 4 &quot;silicio standard chimicamente con una soluzione piranha per 20 min. </li><li> Sciacquare la cialda con acqua deionizzata e asciugare. </li><li> Riscaldare il wafer su una piastra calda (200 ° C per 15 min). </li><li> Rivestire il wafer di silicio con photoresist. Qui, il fotoresist AZ4620 positivo è stato spin-rivestito su un wafer di silicio a 2.000 rpm per 25 secondi per ottenere uno strato di spessore di 12,5 micron. </li><li> Rimuovere tutto il solvente dalla cottura morbida della fetta su una piastra calda per galleggiamento della fetta per 90 secondi in flusso di azoto a 100 ° C. Poi, tenerlo in vuoto a tegli stessa temperatura per 60 sec. </li><li> Posizionare la cialda in una scatola scura per 24 ore per disidratazione. </li><li> Esporre il wafer alla luce UV per trasferire il pattern progettato per il fotoresist. </li><li> Immergere il wafer in soluzione di sviluppo di fotoresist (AZ400K) per 240 sec. Poi, risciacquare il wafer con alcool isopropilico e asciugare mettendo in corrente di azoto. </li></ol></li><li> ICP-DRIE (plasma accoppiato induttivamente - Deep Reactive Ion Etching) Processo <ol><li> Applicare DRIE incisione. Scegliere la profondità etch appropriata in base alla profondità finale richiesta per il dispositivo (50 micron in questa indagine). Photoresist agisce come uno strato di mascheratura durante questo processo. </li><li> Rimuovere il photoresist rimanente con acetone e pulire la cialda. </li></ol></li><li> PDMS (polidimetilsilossano) Casting <ol><li> Utilizzare trichloromethylsilane (TCMS) per silanizzanti stampo maestro di silicio. Versare 2 o 3 gocce di trichloromethylsilane in un flaconcino e metterlo in un essiccatore accanto alstampo maestro silicio. Consenti 2-3 ore per il processo silanizzante per completare. </li><li> In un contenitore separato, mescolare la base di silicone Sylgard 184 con induritore dal rapporto in peso di 10: 1 per preparare PDMS. Degassarla PDMS sottoponendolo a vuoto condizioni (circa 2 ore). </li><li> Mettete lo stampo maestro di silicio in un supporto. Poi, versare il PDMS sullo stampo maestro di silicio per formare il timbro PDMS. Assicurarsi che le bolle non si formano nei PDMS durante questo processo. </li><li> Curare le PDMS per 2 ore a 80 ° C. </li><li> Staccare il timbro PDMS dallo stampo master. Quindi, tagliare le PDMS bollo in microchip separati. Infine, utilizzare un nucleo di taglio per perforare fori per l&#39;ingresso (s) e di uscita (s). </li></ol></li><li> Incollaggio di PDMS al vetro <ol><li> Esporre il vetrino e PDMS bollo di plasma di ossigeno per 30 sec. Legame PDMS bollo per la polizza di copertura. </li><li> Per ottenere una corretta tenuta tra il timbro PDMS e il vetrino, temprare il dispositivo mettendolo in forno a 70 ° C per 10 min. </li&gt; </ol></li></ol> 2 batterica Cultura NOTA: i protocolli di corretta biosicurezza deve essere seguita per i passaggi 2-4. Consultare il responsabile della sicurezza istituzionale per i dettagli. <ol><li> Preparare piastre LB Agar <ol><li> Aggiungere 20 g di Luria-Bertani (LB) agar (Miller) polveri e 500 ml di acqua ultrapura per un pallone da 1 L. Mescolare per sciogliere la polvere. </li><li> Sterilizzare in autoclave a 15 psi, 121 ° C per 15 min. </li><li> Lasciare raffreddare il pallone fino a 50-55 ° C su una panchina o in un bagno d&#39;acqua nella cappa di biosicurezza. </li><li> Aggiungere l&#39;antibiotico tetraciclina per ottenere una concentrazione finale di 50 mg / ml. Mescolare bene agitando. </li><li> Versare il composto in lastre. Riempire ogni piatto fino a 1/2 - 2/3 pieno. </li><li> Aria Fiamma bolle brevemente per farli scoppiare se fanno. Bolle d&#39;aria solidificati sono difficili da diffondere la cultura batterica sopra. </li><li> Lasciare le piastre a raffreddare a temperatura ambiente per una notte. </li><li> Quando sono freschi,mettere lastre di nuovo nella loro manica, sigillare il sacchetto, etichetta (antibiotico e data), e conservare a 4 ° C. NOTA: Coprire lo stock di piastre con carta stagnola, come la luce disattiva molti antibiotici. </li></ol></li><li> LB Broth Preparazione <ol><li> Aggiungere 20 g Luria-Bertani (LB) brodo (Miller) polveri e 1 L di acqua ultrapura in un pallone. Mescolare per sciogliere la polvere. </li><li> Sterilizzare in autoclave a 15 psi, 121 ° C per 15 min. </li><li> Lasciare pallone raffreddare a 50-55 ° C su una panchina o in un bagno d&#39;acqua nella cappa di biosicurezza. </li><li> Aggiungere l&#39;antibiotico tetraciclina per ottenere una concentrazione finale di 50 mg / ml. Mescolare bene agitando. </li><li> Lasciar raffreddare, posizionare la bottiglia etichettata a 4 ° C. NOTA: Coprire la bottiglia con carta stagnola, come la luce disattiva molti antibiotici. </li></ol></li><li> Coltura dei batteri su una piastra di LB Agar (Questo protocollo utilizza Pseudomonas fluorescens) <ol><li> Prendete il magazzino batterica dal congelatore (-80 °, C) e posizionarlo sul ghiaccio. </li><li> Posizionare l&#39;° C magazzino batterica -80 e un piatto di agar LB all&#39;interno di una cappa di biosicurezza. </li><li> Streak il ceppo batterico su una piastra di agar LB a zig-zag. Coprire la piastra di agar e incubare a 30 ° C durante la notte. Infine, riporre il piatto in frigorifero a 4 ° C. </li></ol></li><li> Preparare la soluzione batterica (S1) <ol><li> Versare 50 ml di brodo LB supporto a un pallone autoclavato. Eseguire questa operazione all&#39;interno di una cappa di biosicurezza. </li><li> Trasferire una singola colonia batterica dalla piastra di agar LB al pallone. Questa operazione deve essere eseguita all&#39;interno di una cappa di biosicurezza. </li><li> Mettere il pallone in un incubatore shaker a 30 ° C e 150 rpm per un tempo adeguato (4 ore). </li></ol></li><li> Preparare la soluzione batterica Diluire (S2) <ol><li> Versare 5 ml di LB supporti brodo in un tubo di plastica sterilizzata. </li><li> Diluire S1 mescolando con mezzi brodo LB. Quindi, agitare la soluzione. Diluire la soluzione raggiungere l&#39;ottica desiderataAl densità (OD misurata a 600 nm = 0,1). NOTA: esperimenti Biofilm tipicamente impiegano valori di OD in questa zona. </li></ol></li></ol> 3 Preparare il setup sperimentale <ol><li> Utilizzando le pinzette collegano tubi di plastica flessibili (0.20 &quot;ID) nella presa (s) e di uscita (s) del microchip. Gli ingressi e le uscite sono state precedentemente perforati nella porzione PDMS del microchip (fase 1.5.5). In questa indagine , il microchip è costituito da due ingressi ed una uscita. </li><li> Riempire la siringa (s) con la soluzione batterica (soluzione S2) e rimuovere tutte le bolle nella siringa (s). </li><li> Collegare punta della siringa (s) (30 G 0.5 &quot;ago smussato) in tubo di ingresso (s). Quindi, collegare il tubo di uscita (s) per i rifiuti del contenitore. </li></ol> 4 Esecuzione dell&#39;esperimento <ol><li> Collegare la siringa (s) per la punta della siringa (s). </li><li> Luogo e siringa fix (s) sulla pompa a siringa. Poi mettere il microchip sotto un microscopio ottico con lenti dell&#39;obiettivo di Desired ingrandimento (ad esempio, 40X). Coprire il microchip con un dispositivo di camera di cellule vive per mantenere un ambiente a temperatura costante per la crescita batterica (30 ° C per P. fluorescens). </li><li> Impostare la pompa al livello di portata desiderata (per esempio 10 ml / ora) ed avviare il pompaggio del fluido. </li><li> Una volta che i batteri vengono introdotti nella camera, è anche avviata la formazione di biofilm. Formazione di biofilm e maturazione si verificano in genere in un periodo di diverse ore o addirittura giorni. Osservare e prendere le immagini della crescita di biofilm attraverso il microscopio. </li></ol>

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The authors have nothing to disclose.

Abbiamo dimostrato un semplice dispositivo di microfluidica che imita mezzi porosi per lo studio dello sviluppo del biofilm in ambienti complessi. Ci sono diversi passaggi critici che determinano l&#39;esito degli esperimenti. Essi comprendono la geometria del dispositivo. Mentre la geometria del palo può variare, adeguato poro-spazio per bandierine per formare è necessario. Inoltre, Valiei et al. 1 hanno dimostrato che la formazione streamer si verifica solo in un determinato campo di portata. A p...

Recentemente, abbiamo dimostrato la dinamica di formazione di biofilm batterici in un dispositivo di microfluidica che imita mezzi porosi 1. Biofilm batterici sono essenzialmente colonie di batteri di superficie aggregati che sono racchiusi da sostanze polimeriche extracellulari (EPS) 2-4. Questi film sottili di batteri possono formare in quasi ogni nicchia immaginabile che vanno da superfici lisce all&#39;habitat molto più complesso di mezzi porosi. Valiei et al. 1 usato un dispositivo microfluidico con una matrice di micro-pilastro per simulare una struttura di supporto porosa e studiato la formazione di biofilm in questo dispositivo in funzione della portata del fluido. Essi hanno scoperto che in un certo regime di flusso, biofilm filamentosi noti come stelle filanti hanno cominciato ad emergere tra i diversi pilastri. Streamers possono essere legati ad una o entrambe le estremità per superfici solide, ma il resto della struttura è sospesa in un liquido. Formazione Streamer inizia in genere dopo un primo strato di biofilm ha formato e il suo formatoione può dettare l&#39;evoluzione a lungo termine di biofilm in tali habitat complessi. Recentemente, alcuni ricercatori hanno studiato le dinamiche di formazione streamer. Yazdi et al. 5 ha mostrato che le fiamme possono formare nei flussi vorticosi provenienti da una bolla oscillante. In un altro esperimento, Rusconi et al. 6 studiato l&#39;effetto di curvatura del canale e della geometria del canale sulla formazione di stelle filanti. Essi hanno scoperto che le fiamme possono formare nelle sezioni curve di microcanali, e streamer morfologia è correlata alla motilità. Recenti ricerche hanno dimostrato che le fiamme possono avere ampie ripercussioni in vari scenari naturali e artificiali in quanto possono fungere da precursori della formazione di strutture maturi in interfacce porosi, portare a una rapida e catastrofica proliferazione di biofilm in sistemi biomedici, e anche causare notevoli flow interazioni struttura, ecc 1,7-9. Filanti Biofilm spesso formano ihabitat n complesse come mezzi porosi. Crescita biofilm intesa in ambiente mezzi porosi è rilevante per diversi processi ambientali e industriali come il trattamento biologico delle acque reflue 10, mantenendo ben-bore integrità in situazioni come la cattura di CO 2 11 e collegare dei pori nel terreno 12. Osservando la formazione di biofilm in tali habitat complessi spesso può essere difficile a causa della opacità dei mezzi porosi. In tali situazioni, le piattaforme multimediali porosi microfluidica base possono rivelarsi estremamente vantaggiosa in quanto consentono in tempo reale e monitoraggio in situ. Un altro vantaggio della microfluidica è la possibilità di costruire più bioreattori su un&#39;unica piattaforma bio-microfluidica e contemporaneamente consentire il monitoraggio e / o integrazione di sensori on-line. La flessibilità per implementare più esperimenti di laboratorio in un unico dispositivo e la capacità di raccogliere importanti dati pertinenti per l&#39;analisi statistica accurata è un adv importanteantage di sistemi microfluidici 13,14. Nel contesto della discussione di cui sopra, le dinamiche di formazione streamer comprensione in un ambiente mezzi porosi sarebbe utile per diverse applicazioni. In questo studio, sviluppiamo il protocollo per indagare la formazione streamer in un dispositivo che imita mezzi porosi. Realizzazione della piattaforma microfluidica, sono descritti i passi necessari per la cultura e la sperimentazione di cellule. Nei nostri esperimenti, il ceppo batterico tipo selvaggio di Pseudomonas fluorescens è stato impiegato. P. fluorescens, che si trova naturalmente nel suolo, svolge un ruolo chiave nel mantenimento ecologia del suolo 15. Il ceppo batterico impiegato era stato geneticamente per esprimere la proteina fluorescente verde (GFP) costitutivamente.

<table><tbody><tr><td>Flourescent Microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>LB agar</td><td>Fisher</td><td>BP1425-500</td><td>suspend 40 g in 1 L of purified water</td></tr><tr><td>LB broth</td><td>Fisher</td><td>BP1427-500</td><td>suspend 20 g in 1 L of purified water</td></tr><tr><td>Biosafety hood</td><td>Microzone corporation</td><td></td><td></td></tr><tr><td>Petri dish</td><td>Fisher</td><td>875712</td><td>sterile 100 mm x 15 mm polystyrene Petri dish</td></tr><tr><td>Incubator shaker</td><td>New Brunswick Scientific</td><td></td><td>Excella E24 incubator shaker series</td></tr><tr><td>50 ml sterilized centrifuge tube</td><td>Corning</td><td>430828</td><td>Polypropylene RNase-/DNase-free</td></tr><tr><td>Tetracycline free base</td><td>MP Biomedicals</td><td>103012</td><td>50 &amp;#956;g/ml</td></tr><tr><td>SYLGARD 184 silicone</td><td>Dow Corning Corporation</td><td>68037-59-2</td><td>Elastomer Base and curing agent</td></tr><tr><td>Positive photoresist (AZ4620)</td><td></td><td></td><td></td></tr><tr><td>Plastic tube</td><td>Cole-Parmer</td><td></td><td></td></tr></tbody></table>

protocol for biofilm streamer formation in a microfluidic device with micro-pillars

Diverse specie batteriche possiedono la capacità di attaccare alle superfici e colonizzare in forma di film sottili chiamati biofilm. I biofilm che crescono in mezzi porosi sono rilevanti per diversi processi industriali e ambientali come il trattamento delle acque reflue e di CO 2 sequestro. Abbiamo usato Pseudomonas fluorescens, un batterio aerobico Gram-negativi, per indagare la formazione di biofilm in un dispositivo di microfluidica che imita mezzi porosi. Il dispositivo di microfluidica è costituito da una serie di micro-posti, che sono stati fabbricati utilizzando soft-litografia. In seguito, la formazione di biofilm in questi dispositivi a flusso è stato studiato e ci dimostrano la formazione di biofilm filamentosi note come stelle filanti nel nostro dispositivo. Qui vengono fornite insieme ai protocolli di coltura batterica I protocolli dettagliati per la fabbricazione e l&#39;assemblaggio del dispositivo di microfluidica. Le procedure dettagliate per la sperimentazione con il dispositivo di microfluidica sono anche presentati insieme con il rappresentanterisultati.

Utilizzando il protocollo microfabbricazione sopra menzionato, un dispositivo microfluidico basato PDMS è stato costruito. Figura 1 mostra il microscopio elettronico a scansione (SEM) immagini del PDMS dispositivo. Figura 1a mostra la sezione di ingresso del dispositivo. Un ingresso a forcella è stato creato per equalizzare la testa di pressione attraverso il dispositivo. Ulteriori immagini SEM ha anche mostrato che le pareti pilastro sono quasi verticale (Figura 1b)....

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Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protocollo per il Biofilm Streamer formazione in un dispositivo a microfluidi con Micro-pilastri

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

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12.1K Views.  University of Alberta. Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated. 

Video: Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

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 e biofilm Saccharomyces cerevisiae in celle di flusso

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

Ricerca

Immunologia e infezioni

Fabrication and Visualization of Capillary Bridges in Slit Pore Geometry

A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. High aspect ratio hydrophobic pillars are fabricated and functionalized to render their top surfaces hydrophilic. The combination of a physical feature (the pillar) with a chemical boundary (the hydrophilic film on the top of the pillar) provides both a physical and chemical heterogeneity that pins the triple contact line, a necessary feature to create stable long but narrow capillary bridges. The substrates with the pillars are attached to glass slides and secured into custom holders. The holders are then mounted onto four axis microstages and positioned such that the pillars are parallel and facing each other. The capillary bridges are formed by introducing a fluid in the gap between the two substrates once the separation between the facing pillars has been reduced to a few hundred micrometers. The custom microstage is then employed to vary the height of the capillary bridge. A CCD camera is positioned to image either the length or the width of the capillary bridge to characterize the morphology of the fluid interface. Pillars with widths down to 250 &#181;m and lengths up to 70 mm were fabricated with this method, leading to capillary bridges with aspect ratios (length/width) of over 1001.

A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. The creation of capillary bridges relies on the formation of pillars to provide a directional physical and chemical heterogeneity to pin the fluid. Capillary bridges are formed and manipulated using microstages and visualized using a CCD camera.

Fabbricazione e visualizzazione di ponti capillari in geometria dei pori a fessura

A procedure for creating and imaging capillary bridges in slit-pore geometry is presented. High aspect ratio hydrophobic pillars are fabricated and ...

Ingegneria

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Bioingegneria

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

Combinazione di dispositivi fluidici con microscopia e citometria di flusso per studiare il trasporto microbico nei mezzi porosi attraverso le scale spaziali

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

Ambiente

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

L&#39;uso di un high-throughput<em&gt; In Vitro</em&gt; Microfluidic sistema per sviluppare orale biofilm Multi-specie

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Metodi per caratterizzare il co-sviluppo di biofilm e Habitat Eterogeneità

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

Una piattaforma microfluidica per studiare il bioclogging in mezzi porosi

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

In Situ mappatura delle proprietà meccaniche del biofilm da-Particle tracciamento Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidica Co-coltura di cellule epiteliali e batteri per le indagini solubile del segnale mediato da Interazioni

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Biologia

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Modellazione di microrganismi e microparticelle attraverso l'assemblaggio sequenziale assistito da capillarità

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...