Name: transurethral induction of mouse urinary tract infection
Uploaded: 2010-08-05T17:30:00
Description: Watch this Scientific Journal Video about Transurethral Induction of Mouse Urinary Tract Infection at JoVE.com

Os autores gostariam de agradecer Shelly Xie e Jennifer Payne por sua assistência técnica especializada. Este trabalho foi possível através de doações da Sociedade de Urologia Pediátrica e do Fundo de Investigação Pediátrica Stanford.

I. Elaboração de cateteres uretrais <ol><li> Para cada bexiga e rim do rato a amostrar, coloque 5-6 grânulos de aço inoxidável (bexiga, 1,6 mm ou tamanho 0,9-2,0 mistura mm) ou grânulos de óxido de zircônio (rim, tamanho 0,5 mm) em um tubo de cryovial, respectivamente. Autoclave os tubos contendo talão. </li><li> Após os tubos esfriarem até a temperatura ambiente, adicionar 200 microlitros de PBS estéril a cada tubo de aço inoxidável contendo talão e 400 microlitros de PBS estéril a cada tubo de óxido...

<ol>
	<li>Schaeffer, A. J., Schwan, W. R., Hultgren, S. J., Duncan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+type+1+pilus+expression+in+Escherichia+coli+to+ascending+urinary+tract+infections+in+mice.">Relationship of type 1 pilus expression in Escherichia coli to ascending urinary tract infections in mice.</a> Infect Immun. 55, 373-380 (1987).</li><li>Rouschop, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urothelial+CD44+facilitates+Escherichia+coli+infection+of+the+murine+urinary+tract.">Urothelial CD44 facilitates Escherichia coli infection of the murine urinary tract.</a> J Immunol. 177, 7225-7232 (2006).</li><li>Malaviya, R., Ikeda, T., Abraham, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+mast+cells+to+bacterial+clearance+and+their+proliferation+during+experimental+cystitis+induced+by+type+1+fimbriated+E.+coli.">Contribution of mast cells to bacterial clearance and their proliferation during experimental cystitis induced by type 1 fimbriated E. coli.</a> Immunol LettM. 91, 103-111 (2004).</li><li>Lane, M. C., Alteri, C. J., Smith, S. N., Mobley, H. 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Indução de rato infecção do trato urinário por inoculação de bactérias transuretral é um procedimento estabelecido há muito tempo, mas tecnicamente exigente 11. Hung e Hultgren publicou recentemente uma excelente revisão das técnicas de ressecção transuretral de mouse infecção do trato urinário 13. Neste trabalho, tentamos ilustrar ainda mais os pontos mais delicados desta abordagem experimental. Novatos podem praticar a sua técnica através da realiza...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cryovial tubes, 2.0 ml polypropylene (no caps)</td>
<td>&nbsp;</td>
<td>E&amp;K Scientific</td>
<td>640201</td>
<td>&nbsp;</td>
<tr>
<td>Caps for cryovial tubes, with 'O' ring</td>
<td>&nbsp;</td>
<td>E&amp;K Scientific</td>
<td>440011-N</td>
<td>&nbsp;</td>
<tr>
<td>Bullet Blender</td>
<td>&nbsp;</td>
<td> Next Advance</td>
<td>BBX24</td>
<td>&nbsp;</td>
<tr>
<td>Zirconium Oxide Beads (0.5mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>ZROB05</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Beads (1.6mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>SSB16</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel Bead Blend (0.9-2.0mm)</td>
<td>&nbsp;</td>
<td>Next Advance</td>
<td>SSB14B</td>
<td>&nbsp;</td>
<tr>
<td>CBA/J Mice, 8-12 week old female</td>
<td>&nbsp;</td>
<td>Jackson Laboratories</td>
<td>000656</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline (NaCl 137mM, KCl 2.7mM, Na2HPO4 4.3mM, KH2PO4 1.4mM)</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>EM-SX0420-13 (NaCl) EM-PX1405-1 (KCl) EM1.06585.0 (Na2HPO4) EM-5108-1 (KH2PO4)</td>
<td>&nbsp;</td>
<tr>
<td>LB agar</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LB broth</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>1.10285.0500</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane (Aerrane)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>NDC 10019-773-60</td>
<td>&nbsp;</td>
<tr>
<td>Inoculating loops</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>22-363-601</td>
<td>&nbsp;</td>
<tr>
<td>Eosin-methylene blue (EMB) agar powder</td>
<td>&nbsp;</td>
<td>Himedia Laboratories</td>
<td>MM022-500G</td>
<td>&nbsp;</td>
<tr>
<td>MacConkey agar powder</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>212123</td>
<td>&nbsp;</td>
<tr>
<td>LB agar powder</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>244520</td>
<td>&nbsp;</td>
<tr>
<td>Intramedic non-radiopaque polyethylene tubing, PE10 [inner dimension 0.28 mm (0.011 inch); outer dimension 0.61 mm (0.024 inch)]</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>427401</td>
<td>&nbsp;</td>
<tr>
<td>30 gauge needles, 1/2 inch</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>305106</td>
<td>&nbsp;</td>
<tr>
<td>1 ml tuberculin Slip-Tip syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309602</td>
<td>&nbsp;</td>
<tr>
<td>3 ml Luer-Lock syringe</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>309585</td>
<td>&nbsp;</td>		</tbody>
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transurethral induction of mouse urinary tract infection

Cepas bacterianas uropathogenic de interesse são cultivadas em ágar. Geralmente uropathogenic,<em&gt; E. coli</em&gt; (UPEC) e de outras variedades podem ser cultivadas durante a noite em Luria-Bertani (LB) ágar a 37 ° C no ar ambiente. Cepas UPEC crescer como branco-amarelada translúcida colônias em ágar LB. Após a confirmação da morfologia da colônia apropriado, colônias individuais são, então, escolhido para ser cultivado em caldo. LB pode ser usado para cepas mais uropathogenic bacteriana. Dois serial, culturas caldo LB noite pode ser empregada para aumentar a expressão do pili tipo I, um fator de virulência bem definidos para as bactérias uropathogenic. Culturas caldo são diluídos para a concentração desejada em tampão fosfato salino (PBS). De oito a 12 semanas de idade camundongos fêmeas são colocadas sob anestesia com isoflurano e transurethrally inoculadas com bactérias usando tubos de polietileno coberta de 30 seringas de calibre. Inóculos típico, que deve ser determinado empiricamente para cada combinação estirpe bacteriana / mouse, são 10<sup&gt; 6</sup&gt; A 10<sup&gt; 8</sup&gt; Ufc por rato em 10 a 50 microlitros de PBS. Após o período de infecção desejado (um dia a várias semanas), amostras de urina e da bexiga e os rins são colhidos. Cada órgão é picada, colocados em PBS e homogeneizadas em um homogeneizador de bala azul. Homogeneizados de urina e tecido são serialmente diluída em PBS e cultivadas em ágar apropriado. No dia seguinte, as unidades formadoras de colónias são contadas.

Watch this Scientific Journal Video about Transurethral Induction of Mouse Urinary Tract Infection at JoVE.com

Transurethral Induction of Mouse Urinary Tract Infection

Este vídeo vai demonstrar os métodos para induzir transurethrally infecções do trato urinário do mouse e quantificar a extensão das infecções resultantes.

Indução transuretral de rato infecção urinária

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

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