Name: growth of mycobacterium tuberculosis biofilms
Uploaded: 2012-02-15T13:00:00
Description: Watch this Scientific Journal Video about Growth of Mycobacterium tuberculosis Biofilms at JoVE.com

Arbeidet ble gjennomført med økonomisk støtte fra National Institute of Health og American Lung Association.

1. Voksende biofilm av M. tuberkulose i en 250ml skrue avkortet flaske <ol><li> Media forberedelse: Løs 0,5 g av KH 2 4 PO, 0,5 g av MgSO 4, 4g L-asparagin, 2G sitronsyre, 0.05g av Ferric Ammonium Citrate, 60ml av glyserol i 900mL vann. Juster pH til 7,0 med NaOH. Autoklav, kjølig og like før du starter eksperimentet, legge sterile ZnSO 4 til en endelig konsentrasjon på 0,1% w / v. Siden mc 2 7000 er en pantothenate auxotroph denne belastningen krever o...

<ol>
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Tuberkulose (TB), forårsaket av infeksjon av Mycobacterium tuberculosis, er fortsatt en stor trussel mot den globale folkehelsen. Nesten en tredjedel av verdens befolkning er anslått til å være asymptomatically smittet av patogen, ca 9 millioner nye tilfeller dukker opp i klinikken hvert år med symptomer på aktiv tuberkulose og om lag 1,7 millioner dør av infeksjonen hvert år 11. Den enorme byrden av sykdommen er først og fremst bidratt med mangel av en vaksine og en svært komplisert kjemot...

<table border="1">
	<tbody>
		<tr>
			<td>Equipment and supplies</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>VWR</td>
			<td>Model # 1923/25</td>
		</tr>
		<tr>
			<td>Polystyrene culture bottles</td>
			<td>Fisher Scientific</td>
			<td>03-374-300</td>
		</tr>
		<tr>
			<td>12-well tissue culture plate</td>
			<td>VWR</td>
			<td>62406-165</td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>VWR</td>
			<td>89039-660</td>
		</tr>
		<tr>
			<td>Rocker</td>
			<td>Thermo Scientific</td>
			<td>57019-662</td>
		</tr>
		<tr>
			<td>Chromatographic refrigerator</td>
			<td>VWR</td>
			<td>55702-520</td>
		</tr>
		<tr>
			<td>petri dish</td>
			<td>VWR</td>
			<td>25384-342</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>REAGENT</td>
			<td>SUPPLIER</td>
			<td>CATALOG NUMBER</td>
		</tr>
		<tr>
			<td>KH2PO4 (monobasic)</td>
			<td>EMD</td>
			<td>PX1565-1</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>M65-500</td>
		</tr>
		<tr>
			<td>L-asparagine</td>
			<td>Sigma</td>
			<td>A4284-100G</td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma</td>
			<td>C1857-100G</td>
		</tr>
		<tr>
			<td>ferric ammonium citrate</td>
			<td>Sigma</td>
			<td>F5879-100G</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>EMD</td>
			<td>GX0185-5</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>S8045-500G</td>
		</tr>
		<tr>
			<td>ZnSO4</td>
			<td>Sigma</td>
			<td>Z4750-500G</td>
		</tr>
		<tr>
			<td>D-pantothenic acid</td>
			<td>Sigma</td>
			<td>P2250-25G</td>
		</tr>
		<tr>
			<td>Difco Middlebrook 7H9 Broth</td>
			<td>Becton Dickinson</td>
			<td>271310</td>
		</tr>
		<tr>
			<td>Middlebrook OADC Enrichment</td>
			<td>BBL</td>
			<td>212351</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher</td>
			<td>T164-500</td>
		</tr>
		<tr>
			<td>250mL storage bottle</td>
			<td>Corning</td>
			<td>430281</td>
		</tr>
		<tr>
			<td>12 well plates</td>
			<td>Falcon (BD)</td>
			<td>353043</td>
		</tr>
		<tr>
			<td>rifampicin</td>
			<td>Sigma</td>
			<td>R3501-1G</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>J.T. Baker</td>
			<td>9070-05</td>
		</tr>
		<tr>
			<td>10mlLsyringe</td>
			<td>Becton Dickinson</td>
			<td>301604</td>
		</tr>
		<tr>
			<td>1-200&mu;L pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
		</tr>
		<tr>
			<td>parafilm M</td>
			<td>VWR</td>
			<td>PM-996</td>
		</tr>
		<tr>
			<td>15mL centrifuge tube</td>
			<td>Greiner Bio-One</td>
			<td>188-285</td>
		</tr>
		<tr>
			<td>Difco Mycobacteria 7H11 Agar</td>
			<td>Becton Dickinson</td>
			<td>283810</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-1</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333-500G</td>
		</tr>
		<tr>
			<td>Na2HPO4 (dibasic)</td>
			<td>Sigma</td>
			<td>S0876-500G</td>
		</tr>
	</tbody>
</table>

growth of mycobacterium tuberculosis biofilms

Mycobacterium tuberculosis, etiologic agent for human tuberkulose, har en forunderlig evne til å overleve mot miljømessige påkjenninger, inkludert antibiotika. Selv stresstoleranse av M. tuberkulose er en av de sannsynlige bidragsytere til den 6 måneder lange kjemoterapi av tuberkulose 1, de molekylære mekanismene bak dette karakteristiske fenotype av organismen fortsatt uklare. Mange mikrobielle arter har utviklet seg til å overleve i stressende miljøer ved selv-montering i svært organisert, overflate festet, og matrise innkapslet strukturer som kalles biofilm 2-4. Veksten i samfunn synes å være en foretrukket overlevelse strategi av mikrober, og oppnås gjennom genetiske komponenter som regulerer overflate vedlegg, intercellulær kommunikasjon, og syntese av ekstracellulære polymere stoffer (EPS) 5,6. Den toleranse mot miljøstress er sannsynligvis tilrettelagt av EPS, og kanskje ved physiolokirurgiske tilpasning av individuell basiller til heterogene microenvironments innenfor komplekset arkitektur biofilm 7. I en serie nye papirer etablerte vi at M. tuberkulose og Mycobacterium smegmatis har en sterk tilbøyelighet til å vokse i organiserte flercellede strukturer, kalt biofilm, som kan tåle mer enn 50 ganger de minste hemmende konsentrasjonen av legemidler mot tuberkulose isoniazid og rifampicin 8-10. M. tuberkulose krever imidlertid intrigere spesielle forhold for å danne modne biofilm, særlig 9:01 forholdet headspace: media samt begrenset utveksling av luft med atmosfæren 9. Krav til spesialiserte miljøforhold kan muligens knyttes til det faktum at M. tuberkulose er en obligat menneskelig patogen og dermed har tilpasset vev miljøer. I denne publikasjonen viser vi metoder for dyrking M. tuberkulosebiofilm i en flaske og en 12-brønns plate format, noe som er praktisk for bakteriologiske samt genetiske studier. Vi har beskrevet protokollen for en svekket stamme av M. tuberkulose, mc 2 7000, med sletting i de to loci, panCD og RD1, som er kritiske for in vivo vekst av patogen 9. Denne belastningen kan trygt brukes i en BSL-2 containment for å forstå grunnleggende biologi tuberkulose patogen og dermed unngår kravet om en kostbar BSL-3 anlegg. Metoden kan utvides, med passende endringer i media, for å vokse biofilm av andre culturable mykobakterielle arter. Samlet vil en uniform protokoll av dyrking mykobakterielle biofilmer hjelpe etterforskerne interessert i å studere de grunnleggende elastiske egenskapene til mykobakterier. I tillegg vil en klar og konsis metode for voksende mykobakterielle biofilm også hjelpe klinisk og farmasøytisk investigators å teste effekten av en potensiell narkotika.

Watch this Scientific Journal Video about Growth of Mycobacterium tuberculosis Biofilms at JoVE.com

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis danner narkotika tolerante biofilm når dyrket i visse vilkår. Her beskriver vi metoder for dyrking M. tuberkulose biofilmer og bestemme frekvensen av narkotika tolerante persisters. Disse protokollene vil være nyttig for videre studier i mekanismene for narkotika toleranse i M. tuberkulose.

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