Name: methods for characterizing the co-development of biofilm and habitat heterogeneity
Uploaded: 2015-03-11T14:00:00
Description: Watch this Scientific Journal Video about Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity at JoVE.com

Vi takker Matt PARSEK ved University of Washington (Seattle, Washington) for å gi P. aeruginosa og E. coli-stammer og Roger Nokes ved Universitetet i Canterbury (New Zealand) for å gi tilgang til Streams programvare. Dette arbeidet ble støttet av tilskuddet R01AI081983 fra National Institutes of Health, National Institute of Allergy og smittsomme sykdommer. Konfokal bildebehandling ble utført ved Northwestern Biological Imaging Facility (BIF).

1. Flow Cell Setup og Inoculation MERK:. Bruk en dobbel-innløp microfluidic flyt celle beskrevet i Song et al, 2014 14 for å vokse biofilm. Denne flyten celle er i stand til å skape veldefinerte glatte kjemiske gradienter. Strømningscellekonstruksjon er vist i figur 1 og strømningscellen fabrikasjon ble tidligere beskrevet i Song et al., 2014 14. Her detalj våre fremgangsmåter ved hjelp av P. aeruginosa og E....

<ol>
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Vi viste en pakke med metoder for å karakterisere tre viktige biofilm-miljø interaksjoner: biofilm respons på kjemiske gradienter, effekter av biofilm vekst på rundt flyt mikromiljøet, og biofilm heterogenitet som følge av interne transport begrensninger. Vi først viste bruk av en roman mikrofluidstrømning celle å ilegge en veldefinert kjemisk gradient for biofilm utvikling. For å generere en veldefinert kjemisk gradient i strømningscellen, er det viktig å opprettholde den samme ...

Mikroorganismer feste til overflater og danner biofilm - cellleaggregater vedlagt i et ekstracellulært-polymer matrise 1. Biofilmer oppføre seg svært forskjellig fra individuelle mikrobielle celler, fordi biofilmer har dramatiske romlig heterogenitet som skyldes en kombinasjon av oppløst stoff interne transportbegrensninger og romlige variasjoner i cellulær metabolisme 2,3. Oksygen og næringsstoffer konsentrasjoner drastisk nedgang i grenseflaten mellom biofilm og omkringliggende væske og få ytterligere utarmet innenfor i biofilm to. Romlige variasjoner i biofilm respirasjon og proteinsyntese kan også oppstå som en respons på lokaliserte oksygen og næringsstoffer 2. I akvatiske og jord miljøer, de fleste bakterier bor i biofilm. Naturlige biofilmer utføre viktige biogeokjemiske prosesser, inkludert sykling karbon og nitrogen og redusere metaller 4,5. Klinisk er biofilmdannelse responsusynlige for langvarig lunge og urinveisinfeksjoner 6. Biofilmassosierte infeksjoner er svært problematisk fordi cellene i biofilmer har ekstremt høy motstand mot antimikrobielle midler i forhold til sine planktoniske kolleger 6. Fordi biofilm er viktige i ulike innstillinger, har en betydelig mengde forskning vært fokusert på å forstå de miljømessige faktorene som styrer biofilm aktiviteter og romlig heterogenitet i biofilmer og det omkringliggende mikromiljøet. Tidligere studier har funnet at biofilm utvikling er sterkt regulert av en rekke miljøfaktorer: biofilmer utvikle ulike morfologi under ulike strømningsforhold; oksygen og næringsstoffer innflytelse biofilm morfologi, og hydrodynamiske skjærspenning påvirker bindingen av planktoniske celler til overflater og løsgjøring av celler fra biofilmer 7-9. Videre påvirker flyten tilstand ekstern levering av underlag into og innen biofilm 10. Vekst av biofilm endrer også omkringliggende fysiske og kjemiske forhold. For eksempel fører biofilmveksten av lokal utarming av oksygen og næringsstoffer 2; biofilmer akkumulere uorganiske og organiske forbindelser fra omgivelsene 11; og biofilm klynger avlede strømmen og øker overflatefriksjonen 12,13. Fordi biofilmer samhandle med deres omkringliggende miljø i svært komplekse måter, er det viktig å samtidig få informasjon om biofilm egenskaper og miljømessige forhold, og flerfaglige tilnærminger må brukes til omfattende karakter biofilm-miljø interaksjoner. Her presenterer vi en serie av integrerte metoder for å karakterisere romlige mønstre i mikrobiell vekst innen mono-arter og dual-arter biofilm under en pålagt ernæringsmessig gradient, og å observere den resulterende endring av lokal kjemisk og væske mikromiljøet. Vi first beskriver anvendelse av et nylig utviklet doble innløpsmikrofluidstrømningscellen for å observere biofilm vekst under veldefinerte kjemiske gradienter. Vi demonstrerer bruken av denne mikrofluidstrømningscellen for å observere vekst av to arter av bakterier, Pseudomonas aeruginosa og Escherichia coli, i biofilmer under en rekke næringsforhold. Vi viser hvordan in situ visualisering av fluoriserende spor forplantning inn i biofilm kolonier kan brukes til å kvantitativt vurdere mønstre av oppløst stoff transport i biofilmer. Endelig viser vi hvordan mikropartikkelsporings velocimetry, utført under konfokal mikroskopi, kan anvendes for å oppnå lokal strømningsfeltet rundt de voksende biofilmer.

<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

Biofilmer er overflate festet mikrobielle samfunn som har komplekse strukturer og produserer betydelige romlige heterogeniteter. Biofilm utvikling er sterkt regulert av den omgivende strømmen og næringsmiljø. Biofilmvekst øker også heterogenitet i den lokale mikromiljøet ved å generere komplekse strømningsfelt og oppløst stoff transportmønstre. For å undersøke utviklingen av heterogenitet i biofilmer og interaksjoner mellom biofilmer og deres lokale mikro-habitat, vokste vi mono-arter biofilm av Pseudomonas aeruginosa og dual-arter biofilm av P. aeruginosa og Escherichia coli i henhold til ernæringsmessige gradienter i et mikrofluidstrømningscellen. Vi gir detaljerte protokoller for å skape nærings gradienter innen strømningscellen og for dyrking og visualisere biofilm utvikling under disse forholdene. Vi presenterer også protokoller for en serie av optiske metoder for å kvantifisere romlige mønstre i biofilm struktur, flyte distributions løpet biofilm, og massetransport rundt og innenfor biofilm kolonier. Disse metodene støtter omfattende undersøkelser av co-utvikling av biofilm og habitat heterogenitet.

Den doble innløpsmikrofluidstrømningscelle tillater observasjon av biofilmveksten under en veldefinert kjemisk gradient dannet ved blanding av to løsninger inne i strømningskammeret. Den resulterende kjemiske gradient ble tidligere observert av fargestoff-injeksjonen og karakterisert i detalj ved Song et al., 14. Glatte konsentrasjonsgradienter ble dannet i den tverrgående retning, som vist i figur 1. Konsentrasjonsprofilen var bratt nær innløpet og fikk avslappet nedstrøms p...

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.

Metoder for å karakterisere Co-utvikling av Biofilm og Habitat Heterogenitet

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

Research

JoVE Journal

Bioengineering

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 ...

Fluorescence detection methods for microfluidic droplet platforms

Fluorescens deteksjon metoder for microfluidic dråpe plattformer

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

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

Protokoll for Biofilm Streamer-formasjonen i en mikrofluid enhet med Micro-søyler

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

Metoder for Self-integrering av Megamolecular Biopolymere på Tørking Air-LC-grensesnitt

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

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Karakterisere celle migrasjon tredimensjonale In Vitro sår miljøer

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

To metoder for Decellularization av anlegget vev for Tissue Engineering programmer

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

Omfattende evaluering av effektiviteten og sikkerheten til morkaken målrettede narkotika-leveranser med tre komplementære metoder

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

Elektriske og magnetiske feltenheter for stimulering av biologisk vev

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

Formulere og karakterisere Lipid nanopartikler for genlevering ved hjelp av en mikrofluidisk blandeplattform

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

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Multi-tidsskala mikroskopi metoder for karakterisering av fluorescerende merket mikrobobler for ultralyd-utløst legemiddel frigjøring

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...