Ringraziamo Matt Parsek presso l&#39;Università di Washington (Seattle, WA) per la fornitura di P. aeruginosa e E. ceppi coli e Roger Nokes presso l&#39;Università di Canterbury (Nuova Zelanda) per fornire l&#39;accesso a software Streams. Questo lavoro è stato sostenuto dalla concessione R01AI081983 dal National Institutes of Health, National Institute of Allergy e Malattie infettive. Confocale è stata eseguita presso l&#39;impianto di Northwestern Imaging biologica (BIF).

Setup Cella 1. Flusso e inoculazione NOTA:. Utilizzare una cella di flusso microfluidica doppio ingresso descritto nel Canto et al, 2014 14 a crescere biofilm. Questa cella di flusso è in grado di creare gradienti chimici lisce ben definiti. Il disegno cella di flusso è mostrata in Figura 1 e flusso fabbricazione cella precedentemente descritto canzone et al. 2014 14. Qui dettaglio i nostri metodi utilizzando P. aeruginosa e E. coli per formare biofilm, ma altre specie può essere adatto anche. Abbiamo usato P. aeruginosa PAO1- GFP, che esprime costitutivamente una proteina verde fluorescente (GFP), come organismo modello per lo sviluppo di biofilm. Inoltre, abbiamo utilizzato E. coli DH5α per formare biofilm mixed-specie con P. aeruginosa. P. aeruginosa e E. ceppi coli sono state coltivate su piastre di agar LB. <ol><li> Preparare la cella di flusso utilizzando polidimetilsilossano (PDMS) legato ad unacoprioggetto di vetro con trattamento al plasma di ossigeno, come descritto in 14. Le dimensioni della camera di cella di flusso sono di 23 mm x 13 millimetri × 0,24 millimetri (lunghezza x larghezza × profondità). </li><li> Preparare modificato FAB mezzo di crescita 15. Per osservare la crescita di biofilm sotto un gradiente nutrizionale all&#39;interno della cella di flusso, introducono modifiche FAB media 15 con 0,6 micron di glucosio in un ingresso e di introdurre FAB media senza alcuna fonte di carbonio attraverso l&#39;altro ingresso. Filtro-sterilizzare (dimensione dei pori = 0,2 micron) la soluzione di glucosio magazzino (60 mm) prima di aggiungerlo al mezzo FAB. Autoclave Anche il mezzo FAB con ciclo 1 (liquido, 15 min; 121 ° C, 17 psi) prima dell&#39;uso. </li><li> Sterilizzare il sistema di flusso. Prima dell&#39;inoculo, autoclave l&#39;intero percorso di flusso (bottiglie medie, tubi, trappole bolla, celle di flusso) utilizzando ciclo 1, eccetto per le valvole a tre vie (plastica monouso e pre-sterilizzati) a monte della cella di flusso. (Valvole a tre vie sono utilizzati per l&#39;iniezione delle cellule culture, tracciante fluorescente e microsfere.) Per evitare la contaminazione in fase di montaggio, coprire tutte le aperture e tubi di collegamento con un foglio di alluminio o sacchetti di autoclave prima autoclave. </li><li> Collegare il sistema di flusso. Assemblare i componenti del sistema di cella di flusso accuratamente (vedi video per il montaggio del sistema di flusso) e fornire terreno di coltura alla cella di flusso tramite una pompa peristaltica che controlla con precisione la portata. </li><li> Preparare coltura cellulare per l&#39;inoculazione trasferendo una colonia di P. aeruginosa o E. coli dalle piastre LB a 3 ml di brodo LB e scuotere la cultura O / N a 225 giri al minuto e 37 ° C. Diluire gli O / colture cellulari N in 1 ml di acqua sterile ad una OD finale 600 = 0,01 come inoculo. (Cultura e diluire i batteri in una cappa a flusso laminare per evitare la contaminazione.) </li><li> Per gli esperimenti con biofilm mista specie, diluire le due colture batteriche ad un rapporto di 1: 1 in 1 ml con un equivalente OD 600 = 0,01 per ogni batterio. </li><li> Seminare la cella di flusso. Iniettare 1 ml di inoculo all&#39;ingresso cella di flusso dalla valvola a tre vie. Dopo l&#39;iniezione, mettere in pausa il flusso per 1 ora per consentire cellule batteriche di allegare al vetro di copertura. (Assicurarsi che la cella di flusso è posizionato con il lato coprioggetto basso per consentire alle cellule in sospensione di depositarsi sul vetrino.) </li><li> Dopo 1 ora, riprendere il flusso e pompare il mezzo di crescita alla cella di flusso ad una velocità costante di 0,03 ml / min per ciascun ingresso per 3 giorni. </li></ol> 2. Characterizing biofilm sviluppo in risposta a nutrienti sfumature Utilizzo Microscopia confocale NOTA: P. aeruginosa può essere ripreso con costitutivamente-espressa GFP, ma E. coli in biofilm misti specie deve essere ripreso dalla controcolorazione. <ol><li> Osservare le biofilm 3 giorni usando la microscopia confocale. Per i risultati rappresentativi, osservare biofilm utilizzando un microscopio confocale con un obiettivo 63X. Prima di imaging, markla cella doppio ingresso a flusso con una griglia sul lato vetro di copertura. (Lo scopo di questa griglia è di consentire lo sperimentatore per individuare le regioni di imaging nella camera cella di flusso.) </li><li> Per colorazione di contrasto, diluire 10 ml di rosso fluorescente macchia cellule permeante acidi nucleici, come l&#39;acido macchia verde fluorescente nucleico come STYO 62, soluzione madre (1 mm) in 990 ml di acqua sterilizzata e poi lentamente iniettare la macchia diluito con una siringa in la camera di cella di flusso dalla valvola a tre vie monte. Mantenere la cella di flusso stagnante e al buio per 30 minuti, quindi riprendere il flusso di lavare la macchia non legato per 15 min. </li><li> Eseguire confocale. Attivare la 488 nm Argon laser per l&#39;eccitazione e l&#39;emissione di raccolta canali per GFP (500-535 nm) e macchia acidi nucleici (per lampade fluorescenti colorazione verde acido nucleico, 650-750 nm). Regolare guadagni e offset per entrambi i canali per avere immagini brillanti e pulite. Selezionare xyz e modalità di imaging simultaneo. Utilizzare la manopola di z-controllo per idenduare parte inferiore e superiore del biofilm nel campo. Impostare la z-passo a 0,5 micron per l&#39;acquisizione di una serie di immagini 3-D. (Per garantire immagini di alta qualità, utilizzare una media linea di quattro per l&#39;acquisizione di immagini.) </li><li> Per mappare i modelli spaziali di sviluppo biofilm all&#39;interno della cella di flusso, biofilm immagine a tre o più longitudinale (x) distanze lungo l&#39;ingresso della cella di flusso, e alle tre trasversale (y) posizioni relative al gradiente nutrizionale imposto, come mostrato in Figura 1 . </li></ol> 3. Characterizing Soluto interno Trasporto Iniezioni di conservatore fluorescente Tracer NOTA: Un tracciante fluorescente conservatore, come Cy5, può essere utilizzato per caratterizzare modelli di trasporto dei soluti all&#39;interno del biofilm. <ol><li> Sciogliere Cy5 (Mono-Reactive NHS Ester) in acqua ad una concentrazione finale di 10 mg / ml come soluzione madre. Conservare la soluzione di riserva Cy5 in un -20 ° C freezer. NOTA: Come Cy5 è chiaro Sensitive, magazzini, diluite e iniettare soluzioni Cy5 nel buio. </li><li> Prima delle iniezioni del tracciante fluorescente, prendere una serie di immagini 3-D del biofilm di 3 giorni con la microscopia confocale (con un obiettivo 63X). (Regioni larghe delle colonie biofilm sono ideali per serie temporali osservazioni di trasporto colorante.) Anche scegliere un piano z con colonie ben separati per l&#39;imaging (come mostrato in Figura 5A). </li><li> Per ogni esperimento iniezione Cy5, diluire 2 ml di soluzione madre Cy5 in 998 microlitri di acqua sterilizzata per fornire una soluzione di iniezione avente una concentrazione Cy5 di 20 ug / ml. </li><li> Arrestare la pompa per interrompere il flusso e iniettare la soluzione Cy5 nel monte della cella di flusso utilizzando una siringa attraverso la valvola a 3 vie. (E &#39;importante per evitare bolle d&#39;aria di entrare nel percorso di flusso durante l&#39;iniezione. Bubble trappole devono essere tenuti aperti durante l&#39;iniezione posteriore.) </li><li> Dopo aver iniettato il soluzione Cy5, chiudere le trappole bolla, regolare la valvola a 3 vie, e riavviare ilpompa per fornire la soluzione Cy5 iniettato nella cella di flusso. </li><li> Passa alla modalità di imaging confocale a XYT. Attivare Cy5 e GFP canali sotto la modalità di imaging simultaneo. Regolare le impostazioni confocale su intensità Cy5 (intensità del laser, di guadagno e offset) per evitare segnali, che è fondamentale per la quantificazione saturazione. (Alta risoluzione temporale è preferito per la visualizzazione penetrazione del colorante. Tuttavia, la scansione veloce diminuisce la qualità dell&#39;immagine.) Scegliere la corretta velocità di scansione e media linea per bilanciare la risoluzione temporale dell&#39;imaging e la qualità dell&#39;immagine. Per questo protocollo utilizzare una media linea di quattro e un frame rate di 0,15 Hz. </li><li> Riprendere il flusso di consegnare Cy5 nella cella di flusso (ancora ad una portata di 0,03 ml / min). Contemporaneamente avviare serie temporali confocale. </li><li> Quantificare penetrazione Cy5 da radialmente media intensità Cy5 all&#39;interno di un 2-D biofilm colonia circolare. Per media, prima identificare il bordo di un cluster biofilm, quindi generare una mappa di distanza per una sezione 2-D del cluster, E infine modelli medi radiali di intensità Cy5 per generare una curva di penetrazione (vedere Figura 6). <ol><li> Per computazionalmente realizzare passo 3.8, utilizzare BioSPA (biofilm Spatial Pattern Analysis) pacchetto software (inedito, software e manuale disponibile su richiesta) o di altri programmi di analisi delle immagini. </li><li> Per utilizzare BioSPA, prima importare un&#39;immagine impostata in BioSPA. Quindi selezionare soglia adeguata per i canali GFP e Cy5 per rendere le immagini binarie. Nel menu di analisi / calcolo, selezionare Advanced Analysis e quindi Analysis diffusione. Utilizzare il canale GFP per definire il bordo di un cluster e utilizzare lo strumento poligono per selezionare un cluster come la regione di interesse (ROI). </li><li> Fare doppio clic sul gruppo biofilm selezionato per eseguire l&#39;analisi di diffusione. Salvare i risultati delle analisi di diffusione e calibrare l&#39;intensità Cy5 alla concentrazione Cy5 utilizzando una curva di calibrazione. <ol><li> Per generare Cy5 curva di calibrazione concentrazione, misurare intensità Cy5 su una conce Cy5gradiente ntration al microscopio confocale. (Intensità Cy5 e concentrazione hanno una relazione lineare.) </li></ol></li></ol></li></ol> 4. Caratterizzare il campo di moto circostante tracciando particelle fluorescenti NOTA: particelle fluorescenti, quali microsfere fluorescenti, può essere usato per caratterizzare il campo di flusso intorno biofilm. Campo di moto intorno biofilm può essere calcolata dalle osservazioni di serie temporali di posizioni delle particelle che utilizzano software di monitoraggio di particelle velocimetria. I risultati qui riportati sono stati ottenuti con il software Streams 2.02 pacchetto 16 (download del software e la guida a disposizione dell&#39;utente in <a href="http://www.civil.canterbury.ac.nz/streams.shtml">http://www.civil.canterbury.ac.nz/streams.shtml</a> ). <ol><li> Diluire le microsfere fluorescenti (diametro ~ 1 micron) in acqua sterile ad una concentrazione solida finale di 0,2% e un volume finale di 0,5 ml. Vortex per assicurarsi che le microsferesono ben dispersi. </li><li> Iniettare e fornire i microsfere fluorescenti diluito nella cella di flusso in base a procedure da passaggi 3,3-3,5. Per consentire un monitoraggio accurato del percorso particella, utilizzare un tasso relativamente basso flusso (0,01 ml / h per i risultati di monitoraggio di particelle in questo documento). </li><li> Passare impostazioni confocale modalità di XYT per tracciare il movimento delle particelle in un z-slice e prendere le immagini serie temporali. (Una frequenza di 1 Hz è stato utilizzato per i risultati rappresentativi riportati in questo articolo.) Iniezione di ripetizione di particelle e di immagini in diverse posizioni z di generare flusso di campo 3-D. </li><li> Pre-processo time-series immagini confocale sottraendo il segnale di fluorescenza di fondo (il segnale nella prima immagine acquisita del set di immagini) utilizzando ImageJ (download del software e manuale disponibile su <a href="http://imagej.nih.gov/ij/">http://imagej.nih.gov/ij/</a> ) o altri programmi. </li><li> In ImageJ, aprire la prima immagine di un set di immagini e quindi importare il set di immagini. Sotto processo / Immagine Calculator, sottrarre la prima immagine dal set di immagini. Salvare il set di immagini pre-trattati. </li><li> Per calcolare i vettori di flusso, import time-series immagini confocale in Streams 2.02. In &quot;vista del processo Apri&quot;, selezionare ed eseguire &quot;Identificare le particelle&quot;. Utilizzare soglia adeguata di identificare particelle. Utilizzare 0,5 e 5 micron come soglie di diametro minimo e massimo per 1 micron particelle. </li><li> Poi sotto &quot;view processo Open&quot;, selezionare ed eseguire &quot;PTV analisi condotta&quot; per generare percorsi di particelle. Controllare i percorsi di particelle per vedere se essi rappresentano il movimento delle particelle. Infine, selezionare ed eseguire &quot;Crea campo di velocità&quot; per generare una mappa di flusso vettoriale. </li></ol>

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The authors have nothing to disclose.

Abbiamo dimostrato una serie di metodi per la caratterizzazione di tre importanti interazioni biofilm-ambiente: risposta biofilm a gradienti chimici, gli effetti della crescita biofilm sul flusso microambiente circostante, e eterogeneità biofilm derivanti da limitazioni di trasporto interno. Per prima mostrato l&#39;uso di una cella di flusso microfluidica romanzo imporre un gradiente chimica ben definita per lo sviluppo biofilm. Per generare un gradiente chimica ben definita all&#39;intern...

Microrganismi attribuiscono alle superfici e formare biofilm - aggregati di cellule racchiuse in una matrice extracellulare-polimero 1. I biofilm si comportano in modo molto diverso dalle cellule microbiche individuali, perché biofilm hanno drammatico eterogeneità spaziale risultante da una combinazione di limitazioni del trasporto dei soluti interni e variazioni spaziali nel metabolismo cellulare 2,3. Le concentrazioni di ossigeno e nutrienti drasticamente diminuire all&#39;interfaccia tra biofilm e dintorni fluido e ottenere ulteriori impoverito all&#39;interno del biofilm 2. Variazioni spaziali in biofilm la respirazione e la sintesi proteica può verificarsi anche come una risposta all&#39;ossigeno localizzato e la disponibilità di nutrienti 2. In ambienti acquatici e del terreno, la maggior parte dei batteri abitano in biofilm. Biofilm naturali svolgono importanti processi biogeochimici compresa ciclo del carbonio e azoto e ridurre i metalli 4,5. Clinicamente, la formazione di biofilm è responsbile per polmonare prolungato e infezioni urinarie 6. Infezioni biofilm associate sono molto problematico perché le cellule in biofilm sono estremamente elevata resistenza agli antimicrobici rispetto ai loro omologhi planctonici 6. Perché biofilm sono importanti in contesti diversi, una notevole quantità di ricerca si è focalizzata sulla comprensione dei fattori ambientali che controllano le attività biofilm e l&#39;eterogeneità spaziale biofilm e microambiente circostante. Studi precedenti hanno dimostrato che lo sviluppo del biofilm è fortemente regolato da una serie di fattori ambientali: biofilm sviluppano differenti morfologie sotto varie condizioni di flusso; ossigeno e nutrienti disponibilità influenza biofilm morfologia; e sforzo di taglio idrodinamico colpisce l&#39;attaccamento di cellule planctoniche alle superfici e il distacco delle cellule dal biofilm 7-9. Inoltre, condizione di flusso esterno influenza la consegna dei substrati into ed entro 10 biofilm. La crescita di biofilm altera anche circostante condizioni fisiche e chimiche. Ad esempio, la crescita di biofilm porta alla deplezione locale di ossigeno e nutrienti 2; biofilm accumulano composti inorganici e organici dall&#39;ambiente circostante 11; e cluster biofilm deviano il flusso e l&#39;aumento della superficie di attrito 12,13. Perché biofilm interagiscono con il loro ambiente circostante in modi molto complessi, è fondamentale per ottenere contemporaneamente informazioni sulle proprietà biofilm e delle condizioni ambientali, e gli approcci multi-disciplinari devono essere utilizzati per caratterizzare completo interazioni biofilm-ambiente. Qui vi presentiamo una serie di metodologie integrate per caratterizzare i modelli spaziali in crescita microbica all&#39;interno mono-specie e biofilm dual-specie sotto una pendenza nutrizionale imposto, e di osservare la modifica conseguente della sostanza chimica locale e microambiente fluido. Noi FIRv descrivono l&#39;uso di una cella di flusso microfluidica doppia aspirazione recentemente sviluppato per osservare la crescita di biofilm sotto gradienti chimici ben definiti. Abbiamo poi dimostrare l&#39;uso di questa cella di flusso microfluidica per osservare la crescita di due specie di batteri, Pseudomonas aeruginosa ed Escherichia coli, in biofilm sotto una varietà di condizioni nutrizionali. Mostriamo come in visualizzazione situ fluorescente di propagazione tracciante in colonie biofilm può essere utilizzato per valutare quantitativamente i modelli di trasporto dei soluti in biofilm. Infine, si mostra come microscala velocimetria tracciamento di particelle, eseguita al microscopio confocale, può essere utilizzato per ottenere campo di moto locale intorno alle biofilm crescita.

<table border="0" cellpadding="0" cellspacing="0" width="586">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:89px;">Company</td>
			<td style="width:104px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;">Peristaltic Pump</td>
			<td style="width:89px;">Gilson</td>
			<td style="width:104px;">Miniplus 3</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">PUMP TUBING 0.50MM OVC, Orange/Yellow</td>
			<td style="width:89px;">Gilson</td>
			<td style="width:104px;">F117934</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Three-way Stopcock w/ Swivel male Luer lock</td>
			<td style="width:89px;">Smiths Medical&#160;</td>
			<td style="width:104px;">MX9311L</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Sylgard 184 Solar Cell Encapsulation for Making Solar Panels</td>
			<td style="width:89px;">ML Solar LLC</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Pyrex Medium Bottle, 1L, GL45</td>
			<td style="width:89px;">VWR</td>
			<td>16157-191</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">C-FLEX Tubing</td>
			<td style="width:89px;">Cole-Parmer</td>
			<td style="width:104px;">06422-02</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">1 mL TB Syringe</td>
			<td style="width:89px;">BD</td>
			<td style="width:104px;">309659</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Polymer Tubing</td>
			<td style="width:89px;">IDEX</td>
			<td style="width:104px;">1520G</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Sterile Intramedic Luer Stub Adapter</td>
			<td style="width:89px;">Clay Adams</td>
			<td style="width:104px;">427564</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">PrecisionGlide Needle</td>
			<td style="width:89px;">BD</td>
			<td style="width:104px;">305195</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Spectrophotometer</td>
			<td style="width:89px;">HACH</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">Syringe filters- sterile (0.2 &#956;m)</td>
			<td style="width:89px;">Fisherbrand</td>
			<td style="width:104px;">09-719A</td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:227px;">MAXQ Shaker</td>
			<td style="width:89px;">Thermo Scientific</td>
			<td style="width:104px;"></td>
			<td style="width:167px;">Flow cell setup and inoculation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Ammonium sulfate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">A4418</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium phosphate dibasic anhydrous</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">RES20908-A7</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Monobasic potassium phosphate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">P5655</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">S7653</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Magnisium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">M8266</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Calcium chloride</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C5670</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Calcium sulfate dihydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C3771</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Iron(II) sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">215422</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Manganese(II) sulfate monohydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">M7634</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Copper(II) sulfate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">451657</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Zinc sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">Z0251</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Cobalt(II) sulfate heptahydrate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">C6768</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sodium molybdate</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">243655</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Boric acid</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">B6768</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Dextrose</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">D9434</td>
			<td>Growth media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Luria Bertani Broth</td>
			<td style="width:89px;">Sigma Aldrich</td>
			<td style="width:104px;">L3022</td>
			<td>Growth media</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">TCS SP2 Confocal Microscopy</td>
			<td style="width:89px;">Leica</td>
			<td style="width:104px;"></td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:227px;">SYTO 62</td>
			<td style="width:89px;">Life Technology</td>
			<td style="width:104px;">S11344</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:227px;">Cy5</td>
			<td style="width:89px;">GE Healthcare Life Sciences</td>
			<td style="width:104px;">PA15100</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Red Fluorescent (580/605) FluoSphere</td>
			<td style="width:89px;">Life Technology</td>
			<td style="width:104px;">F-8801</td>
			<td>Fluorescent imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">BioSPA</td>
			<td style="width:89px;">Packman Lab</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">ImageJ</td>
			<td style="width:89px;">NIH</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:227px;">Volocity</td>
			<td style="width:89px;">PerkinElmer</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Streams 2.02</td>
			<td style="width:89px;">University of Cantebury</td>
			<td style="width:104px;"></td>
			<td>Image Processing</td>
		</tr>
	</tbody>
</table>

methods for characterizing the co-development of biofilm and habitat heterogeneity

I biofilm sono comunità microbiche-superficie collegata che hanno strutture complesse e producono significativi eterogeneità spaziali. Sviluppo di biofilm è fortemente regolata dal flusso circostante e l&#39;ambiente nutrizionale. Crescita biofilm aumenta anche l&#39;eterogeneità del microambiente locale generando campi di flusso complessi e modelli di trasporto dei soluti. Per studiare lo sviluppo di eterogeneità in biofilm e le interazioni tra biofilm e il loro locale micro-habitat, siamo cresciuti biofilm mono-specie di Pseudomonas aeruginosa e biofilm dual-specie di P. aeruginosa e Escherichia coli sotto gradienti nutrizionali in una cella di flusso microfluidica. Forniamo protocolli dettagliati per creare gradienti nutrienti all&#39;interno della cella di flusso e per la crescita e la visualizzazione sviluppo biofilm in queste condizioni. Abbiamo anche protocolli presenti per una serie di metodi ottici di quantificare modelli spaziali nella struttura biofilm, flusso Distributi su biofilm, e trasporto di massa intorno e all&#39;interno di colonie biofilm. Questi metodi supportano le indagini a tutto campo del co-sviluppo di biofilm e di habitat eterogeneità.

La cella doppia presa flusso microfluidica consente l&#39;osservazione della crescita biofilm sotto un gradiente chimica ben definita formata miscelando due soluzioni all&#39;interno della camera di flusso. Il gradiente chimica risultante è stato precedentemente osservato per iniezione colorante e caratterizzato in dettaglio da Song et al. 14. Gradienti di concentrazione lisce sono formate in direzione trasversale, come illustrato in Figura 1. Il profilo di concentrazione era ripida...

Watch this Scientific Journal Video about Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity at JoVE.com

Methods for Characterizing 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 ...

methods-for-characterizing-co-development-biofilm-habitat

Research

JoVE Journal

Bioengineering

9.8K Views.  Northwestern University. 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. 

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

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To this end, our laboratory has applied the principles of nanomedicine to simplify ART regimens and as such reduce toxicities while improving compliance and drug pharmacokinetics. Simple and reliable methods for manufacturing nanoformulated ART (nanoART) are shown. Particles of pure drug are encapsulated by a thin layer of surfactant lipid coating and produced by fractionating larger drug crystals into smaller ones by either wet milling or high-pressure homogenization. In an alternative method free drug is suspended in a droplet of a polymer. Herein, drug is dissolved within a polymer then agitated by ultrasonication until individual nanosized droplets are formed. Dynamic light scattering and microscopic examination characterize the physical properties of the particles (particle size, charge and shape). Their biologic properties (cell uptake and retention, cytotoxicity and antiretroviral efficacy) are determined with human monocyte-derived macrophages (MDM). MDM are derived from human peripheral blood monocytes isolated from leukopacks using centrifugal elutriation for purification. Such blood-borne macrophages may be used as cellular transporters for nanoART distribution to human immunodeficiency virus (HIV) infected organs. We posit that the repackaging of clinically available antiretroviral medications into nanoparticles for HIV-1 treatments may improve compliance and positively affect disease outcomes.

Nanoparticles of indinavir, ritonavir, efavirenz and atazanavir were manufactured using wet milling, homogenization and ultrasonication. These nanoformulations, collectively termed nanoformulated antiretroviral therapy (nanoART), assessed macrophage-based drug delivery. Monocyte-derived macrophage nanoART uptake, retention and sustained release were determined. These preliminary studies suggest the potential of nanoART for clinical use.

Sviluppo metodi per il trasporto dei macrofagi del sangue Borne Nanoformulated farmaci antiretrovirali

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

Ricerca

Bioingegneria

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Metodi di rilevamento a fluorescenza per piattaforme goccia microfluidica

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans Biofilm Chip (Ca BChip) per High-throughput screening farmaco antifungino

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

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

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 porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

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 ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Metodi per la Self-integrazione di Megamolecular biopolimeri sul asciugatura ad aria-LC Interfaccia

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Che caratterizzano la migrazione delle cellule all'interno del tridimensionale In Vitro ferita ambienti

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Due metodi per decellularizzazione di tessuti vegetali per applicazioni di ingegneria del tessuto

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

Valutazione globale dell'efficacia e della sicurezza di Placenta-Targeted Drug Delivery utilizzando tre metodi complementari

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Dispositivi elettrici e a campo magnetico per la stimolazione dei tessuti biologici

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Formulazione e caratterizzazione di nanoparticelle lipidiche per la somministrazione genica utilizzando una piattaforma di miscelazione microfluidica

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...