Name: culture of small colony variant of pseudomonas aeruginosa and quantitation of its alginate
Uploaded: 2020-02-22T12:00:00
Description: Watch this Scientific Journal Video about Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate at JoVE.com

这项工作得到了国家卫生研究院（NIH）授予R44GM113545和P20GM103434的支持。

1. SCV 生长条件及打捞路径的生理激活
<ol><li>SCV 检测。<ol>
<li>在预热的伪单一酶（PIA）板上，在37°C生长48小时，在预加热的伪单子分离琼脂（PIA）板上，将P.aeruginosa菌株PAO1、PAO1+pyrD、PAO581和PAO581_pyrD分发。在生长板上识别具有 SCV 表型的单一菌落隔离物（菌群大小为 1⁄3 mm，而不是正常的 3⁄5 mm 菌落大小）。</li>
		<li>重复步骤 1.1.1 以获得 SCV 的纯隔离。 注：由于SCV?...

<ol>
	<li>Govan, J. R., Deretic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+pathogenesis+in+cystic+fibrosis:+mucoid+Pseudomonas+aeruginosa+and+Burkholderia+cepacia.">Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.</a> Microbiological Reviews. 60 (3), 539-574 (1996).</li><li>Hogardt, M., Heesemann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+Pseudomonas+aeruginosa+during+persistence+in+the+cystic+fibrosis+lung.">Adaptation of Pseudomonas aeruginosa during persistence in the cystic fibrosis lung.</a> International Journal of Medical Microbiology. 300 (8), 557-562 (2010).</li><li>Evans, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+colony+variants+of+Pseudomonas+aeruginosa+in+chronic+bacterial+infection+of+the+lung+in+cystic+fibrosis.">Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis.</a> Future Microbiology. 10 (2), 231-239 (2015).</li><li>Johns, B. E., Purdy, K. J., Tucker, N. P., Maddocks, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+Genotypic+Characteristics+of+Small+Colony+Variants+and+Their+Role+in+Chronic+Infection.">Phenotypic and Genotypic Characteristics of Small Colony Variants and Their Role in Chronic Infection.</a> Microbiology Insights. 8, 15-23 (2015).</li><li>Pestrak, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+rugose+small-colony+variants+evade+host+clearance,+are+hyper-inflammatory,+and+persist+in+multiple+host+environments.">Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments.</a> PLoS Pathogones. 14 (2), e1006842 (2018).</li><li>Al Ahmar, R., Kirby, B. D., Yu, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrimidine+Biosynthesis+Regulates+Small+Colony+Variant+and+Mucoidy+in+Pseudomonas+aeruginosa+Through+Sigma+Factor+Competition.">Pyrimidine Biosynthesis Regulates Small Colony Variant and Mucoidy in Pseudomonas aeruginosa Through Sigma Factor Competition.</a> Journal of Bacteriology. 201 (1), e00575-e00618 (2019).</li><li>Ramsey, D. M., Wozniak, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+control+of+Pseudomonas+aeruginosa+alginate+synthesis+and+the+prospects+for+management+of+chronic+infections+in+cystic+fibrosis.">Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis.</a> Molecular Microbiology. 56 (2), 309-322 (2005).</li><li>Mathee, K., McPherson, C. J., Ohman, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranslational+control+of+the+algT+(algU)-encoded+sigma22+for+expression+of+the+alginate+regulon+in+Pseudomonas+aeruginosa+and+localization+of+its+antagonist+proteins+MucA+and+MucB+(AlgN).">Posttranslational control of the algT (algU)-encoded sigma22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN).</a> Journal of Bacteriology. 179 (11), 3711-3720 (1997).</li><li>Schurr, M. J., Yu, H., Martinez-Salazar, J. M., Boucher, J. C., Deretic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+AlgU,+a+member+of+the+sigma+E-like+family+of+stress+sigma+factors,+by+the+negative+regulators+MucA+and+MucB+and+Pseudomonas+aeruginosa+conversion+to+mucoidy+in+cystic+fibrosis.">Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.</a> Journal of Bacteriology. 178 (16), 4997-5004 (1996).</li><li>Rehm, B. H. A., Rehm, B. H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+Production:+Precursor+Biosynthesis,+Polymerization+and+Secretion.">Alginate Production: Precursor Biosynthesis, Polymerization and Secretion.</a> Alginates: Biology and Applications. , 55-71 (2009).</li><li>Remminghorst, U., Rehm, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+alginates:+from+biosynthesis+to+applications.">Bacterial alginates: from biosynthesis to applications.</a> Biotechnology Letters. 28 (21), 1701-1712 (2006).</li><li>Potvin, E., Sanschagrin, F., Levesque, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sigma+factors+in+Pseudomonas+aeruginosa.">Sigma factors in Pseudomonas aeruginosa.</a> FEMS Microbiology Reviews. 32 (1), 38-55 (2008).</li><li>Damron, F. H., Goldberg, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+regulation+of+alginate+overproduction+in+Pseudomonas+aeruginosa.">Proteolytic regulation of alginate overproduction in Pseudomonas aeruginosa.</a> Molecular Microbiology. 84 (4), 595-607 (2012).</li><li>Bowness, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+carbazole+reaction+to+the+estimation+of+glucuronic+acid+and+flucose+in+some+acidic+polysaccharides+and+in+urine.">Application of the carbazole reaction to the estimation of glucuronic acid and flucose in some acidic polysaccharides and in urine.</a> The Biochemical Journal. 67 (2), 295-300 (1957).</li><li>Fazio, S. A., Uhlinger, D. J., Parker, J. H., White, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimations+of+uronic+acids+as+quantitative+measures+of+extracellular+and+cell+wall+polysaccharide+polymers+from+environmental+samples.">Estimations of uronic acids as quantitative measures of extracellular and cell wall polysaccharide polymers from environmental samples.</a> Applied Environmental Microbiology. 43 (5), 1151-1159 (1982).</li><li>Knutson, C. A., Jeanes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+modification+of+the+carbazole+analysis:+application+to+heteropolysaccharides.">A new modification of the carbazole analysis: application to heteropolysaccharides.</a> Analytical Biochemistry. 24 (3), 470-481 (1968).</li></ol>

SCV 和藻酸盐都是与多种慢性感染相关的重要疾病标志物。因此，能够生长SCV以及研究由P.aeruginosa调节和生产的藻酸盐是发现这些慢性疾病的新疗法不可或缺的。
SCV菌株是出了名的难以生长，由于其缓慢的生长速度4相比，其他P.aeruginosa菌株，这有助于他们的抗微生物药物耐药性3。我们的工作确定了一种特定形式的P.aeruginosa<...

慢性肺部感染与伪单多尼亚龙是囊性纤维化 （CF） 患者发病率和死亡率的主要原因。在幼儿期，患者被多种细菌病原体殖民，包括P.aeruginosa1，2的非粘胶分离物。小菌群变异（SCV）分离物的出现以及粘水井分离物是慢性感染发病的标志。SCV分离物具有很强的抗药性3，因为它们的生长速度缓慢4，这使得他们在治疗团和其他慢性感染5由P.aeruginosa严重威慑。Al Ahmar等人6号的工作表明，SCV与通过新丙胺生物合成所联系的粘体之间存在联系。由于与苯丙胺生产有关的基因突变，苯丙胺饥饿导致非粘凝参考菌株PAO1和粘尿衍生物PAO581（PAO1mucA25）中的SCV表型。
尽管藻酸盐过度生产是CF慢性肺部感染的重要疾病标志物，但尚不清楚藻酸盐量与肺病理学之间是否存在直接相关性，也不清楚藻酸盐能否用作治疗7的预后标志物。藻酸盐生产主要由两个操作器调节，一个调节操作体（algUmucABCD）8，9和生物合成操作子（阿尔格Doperon）10，11。藻酸盐生产受到西格玛因子AlgU9、12（也称为AlgT）和反西格玛因子MucA13的降解的严格调节。从患者的痰样原位监测藻酸盐的产生的能力有助于开发新的治疗方案。
在这里，我们描述了一个生长条件，它检测由不能合成新热胺的突变体引起的SCV的存在。补充尿素和/或细胞氨酸，苯丙胺核苷核苷酸的氮碱，到介质激活抢救途径，从而恢复突变体的正常生长。这种针对这些特定SCV突变体的生长方法可用作筛选方法，以识别患者样本中的丙酰胺突变。此外，我们还讨论了两种检测和测量由P.aeruginosa产生和分泌的藻酸盐的方法。第一种是使用高浓度酸降解多糖的传统方法14、15、16，然后添加一个色度指标来定量样品中的浓度。第二种方法，在我们的实验室开发，利用酶链接免疫吸附测定（ELISA）使用QED生物科学开发的抗藻酸盐单克隆抗体（mAb）。ELISA 方法证明比尿酸测定更具体、更敏感，由于避免了高浓度硫酸，因此可以更安全地使用。ELISA 能够直接用于患者痰样测量藻酸盐，因此可以开发为监测诊断工具，以跟踪感染不同时期肺部存在的藻酸盐量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Step Ultra TMB-ELISA</td>
			<td>Thermo Scientific</td>
			<td>34028</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (200 Proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>Molecular Bio-grade</td>
		</tr>
		<tr>
			<td>Accu Block Digital Dry Bath</td>
			<td>Labnet</td>
			<td>NC0205808</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Assay Plates 96-well</td>
			<td>CoStar</td>
			<td>2021-12-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench Top Vortex-Genie 2</td>
			<td>Scientific Industries</td>
			<td>G560</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Research Products International Corp.</td>
			<td>10043-35-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cabinet Incubator</td>
			<td>VWR</td>
			<td>1540</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbazole</td>
			<td>Sigma</td>
			<td>C-5132</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate-Bicarbonate Buffer</td>
			<td>Sigma</td>
			<td>C3041</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Culture Test Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-956-6D</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Cuvette Polystyrene (1.5 ml)</td>
			<td>Fisher Scientific</td>
			<td>14955127</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Cytosine</td>
			<td>Acros Organics</td>
			<td>71-30-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Diposable Inoculation Loops</td>
			<td>Fisher Scientific</td>
			<td>22-363-597</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannuronic Acid Sodium</td>
			<td>Sigma Aldrich</td>
			<td>SMB00280</td>
			<td></td>
		</tr>
		<tr>
			<td>FMC Alginate</td>
			<td>FMC</td>
			<td>2133</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>For Molecular Biology</td>
		</tr>
		<tr>
			<td>Mouse Anti-Alginate Monoclonal Antibody</td>
			<td>QED Biosciences</td>
			<td>N/A</td>
			<td>Lot # :15725/15726</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline Powder (PBS)</td>
			<td>Sigma</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Goat Anti-Mouse Poly-HRP Antibody</td>
			<td>Thermo Scientific</td>
			<td>32230</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>1310-58-3</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas Isolation Agar (PIA)</td>
			<td>Difco</td>
			<td>292710</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Pseudomonas Isolation Broth (PIB)</td>
			<td>Alpha Biosciences</td>
			<td>P16-115</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Round Toothpicks</td>
			<td>Diamond</td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Seaweed alginate (Protanal CR 8133)</td>
			<td>FMC Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Skim Milk</td>
			<td>Difco</td>
			<td>232100</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>SmartSpec Plus Spectrophotometer</td>
			<td>BioRad</td>
			<td>170-2525</td>
			<td>or preferred vendor</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S-5886</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax i3x Multi-mode MicroPlate Reader</td>
			<td>Molecular Devices</td>
			<td>i3x</td>
			<td>or preferred vendor</td>
		</tr>
		<tr>
			<td>Sterile Petri Dish 100mm x 15mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>Fisher Scientific</td>
			<td>A298-212</td>
			<td>Technical Grade</td>
		</tr>
		<tr>
			<td>Sulfuric Acid (2 Normal -Stop Solution)</td>
			<td>R&#38;D Systems</td>
			<td>DY994</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Acros Organics</td>
			<td>66-22-8</td>
			<td></td>
		</tr>
	</tbody>
</table>

culture of small colony variant of pseudomonas aeruginosa and quantitation of its alginate

假单组动物，一种机会性革兰氏阴性细菌病原体，可以过度产生外多糖藻酸盐，从而产生一种称为粘凝的独特表型。藻酸盐与慢性肺部感染有关，导致囊性纤维化 （CF） 患者预后不佳。了解调节藻酸盐生产的途径有助于制定针对藻酸盐形成的新型治疗策略。另一种与疾病相关的表型是小菌群变异（SCV）。SCV 是由于细菌生长缓慢，并且经常与抗菌素耐药性增加有关。在本文中，我们首先展示了一种由于苯丙胺生物合成突变而具有遗传定义的P.aeruginosa SCV的培养方法。补充氮碱，尿素或细胞氨酸，返回这些突变体的正常生长，表明存在一个拯救途径，从环境中清除自由碱。接下来，我们将讨论两种测量细菌藻酸盐的方法。第一种方法依靠多糖的水解到其尿酸单体，然后衍生与色原试剂，卡巴佐尔，而第二种方法使用ELISA基于市售的，藻酸盐特异性mAb。两种方法都需要一个标准曲线进行定量。研究还表明，该免疫法是藻酸盐定量的特异性，可用于临床标本的藻酸盐测量。

图1显示了PAO1和PAO581的板，在pyrD基因（pyrimidine生物合成通路的一个基因）中，无论是否在帧内删除，导致SCV6。PAO1 SCV突变体在响应尿液补充时恢复正常生长（图1A，B）。此外，PAO581®pyrD SCV突变体通过相同的尿素处理返回到粘液，因为母菌株PAO581具有额外的MucA25突变（...

Watch this Scientific Journal Video about Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate at JoVE.com

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

在这里，我们描述了一个生长条件，以培养伪多蒙多尼亚小殖民地变种。我们还描述了使用传统的尿酸卡巴佐尔测定法和基于藻酸盐的单克隆抗体（mAb）的ELISA检测和定量由P.aeruginosa产生的外糖藻酸盐的两种独立方法。

伪多蒙多尼亚小殖民地文化及其藻酸盐的定量

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

小殖民地变种或SCV技术允许选择和生长小殖民地变种的伪多多尼亚亚鲁吉诺萨。该方法非常明确，允许比正常方法更容易选择。与标准尿酸卡巴佐尔法相比，该协议还详细说明了一种更安全、更敏感的 ELISA 扩增方法，使样品中的直接藻酸盐定量无需烧灼或样品操作。展示这个程序的将是实验室技术员布兰登·柯比和来自我实验室的研究生罗伊·阿尔·阿赫马尔。要检测小菌落变种或SCV，首先在预热PIA板上条纹P.aeruginosa应变PAO1三角洲PYD。在37摄氏度下种植菌株48小时。在生长板上，识别具有 SCV 表型的单一菌落分离物，其特征为一到三毫米的菌落大小，而不是正常的三到五毫米菌群大小。重新条纹所选菌落，以获得 SCV 的纯隔离。要对抢救途径进行生理激活，请使用无菌接种回路从PIA板中选取SCV菌落。在预热的 PIA 板上条纹选定的菌落，并辅以 0.1 毫摩尔化。在37摄氏度下将板生长24至48小时。要开始尿酸卡巴佐尔测定，从所需的菌株的纯培养中识别单个菌落，并使用无菌牙签挑选菌落。将牙签放在含有五毫升 PIB 的培养试管中。在37摄氏度的摇床孵化器中生长24小时。孵育后，在预热的PIA板上加入一个培养的PIB汤。使用无菌细胞扩散器，将汤在盘子上扩散，并在37摄氏度下生长24小时。使用移液器控制器和无菌 50 毫升移液器，在成人草坪上加入 0.85% 氯化钠，并使用细胞扩散器刮板收集样品。使用新鲜 50 毫升移液器吸气样品，并转移到 50 毫升收集管中。将样品高旋转，将样品混合并放在冰上。要测量样品的OD600，首先使用一毫升0.85%氯化钠将分光光度计空白。然后将样品的一毫升添加到新的一次性蛋黄酱中，然后读取 OD。重复此步骤两次，以获得每个样本的读取量三倍。现在，将三毫升硫酸玻酸玻酸溶液加入培养管，让坐冰上。将收集的样品中的350微升缓慢地加入试管中的酸性混合物中。在低度上短暂涡旋后，在酸样品混合物中加入100微升0.1%的卡巴佐和乙醇溶液。将管子和涡流盖住介质设置五秒钟。然后在55摄氏度的干浴中放置30分钟。孵育后，将管子短暂地旋转到高位，冷却五分钟。使用含有 0.85% 氯化钠的管作为分光光度计的空白。然后将一毫升混合物加入一个干净的库子中，并在分光光度计上读取530纳米样品的OD。通过测量已知浓度 d-曼努尼酸的连续稀释的 OD530 来准备标准曲线。重复两次，并从这些读数中提取线性方程。使用标准曲线计算每个样品中藻酸盐的浓度，将藻酸盐浓度从线性方程中除以OD600，以获得每OD600的藻酸盐总量。使用微管，将收集的样品的 50 微升添加到未经处理的 96 井板中。然后向油井中加入 50 微升 ELISA 涂层缓冲液。在37摄氏度下孵育板两小时。使用喷瓶，用 PBS-T 将板井用 PBS-T 洗涤两次，然后将板翻过来排干。在井中加入200微升的阻尼缓冲液，用微管在4摄氏度下孵育。第二天，用PBS-T和以前一样洗盘子两次。然后向井中加入100微升稀释原抗体，在37摄氏度下孵育一至两个小时。孵育后，与以前一样用PBS-T清洗板井三次。接下来，在井中加入100微升稀释的二次抗体，在37摄氏度下孵育一至两个小时。再次清洗板三次后，使用微管添加 100 微升 TMB ELISA 溶液。将板放入抽屉或用铝箔包裹，在室温下孵育板30分钟。然后添加 100 微升停止解决方案。使用板读卡器在 450 纳米时测量 OD。通过测量已知浓度 d-曼努尼酸的连续稀释的 OD450 来生成标准曲线。重复测量两次并提取线性方程。使用标准曲线计算每个样品中的藻酸盐浓度，将藻酸盐浓度从线性方程中除以 OD600，以获得每 OD600 的藻酸盐总量。此处显示的有PAO581和PAO581的样本，这些样本具有在PIA和PIA上生长的调节苯丙胺新生物合成基因突变，并辅以0.1毫摩尔的尿素。数据显示，介质中存在尿酸会导致突变菌株转换回粘膜，如恢复的藻酸盐生产所见。在这里，显示了抗藻酸盐单克隆抗体为基础的ELISA的结果。菌落生长在PIA板上，用阿拉伯糖诱导PBAD启动子和PHERD-20T质粒。图中所示为PAO1、PAO1携带表达质粒pHERD-20T，主要藻酸盐特异性西格玛因子algU，PAO1带algD基因在帧内删除，编码关键藻酸盐生物合成酶GDP-曼诺异质酶。这些数据比较了PAO1和PAO1三角洲algD测量的非粘液水平与PAO1用pHERD-20T algU测量的藻酸盐的粘液水平。抗藻酸盐ELISA也在患者痰样上进行了测试，没有在板上生长。三个具有粘液P.aeruginosa生长的CF痰样显示可检测的藻酸盐相比，两个患者痰样本，要么含有非粘液P.aeruginosa或没有P.aeruginosa生长。为藻酸盐定量准备标准曲线非常重要，因为这样可以精确准确地测量样品中的藻酸盐。卡巴佐尔方法中使用的酸溶液非常危险，因此必须使用适当的个人防护设备和预防措施来确保安全。隔离SCV后，可以进行进一步的基因分析，以更好地了解发生突变的发生和与此类突变相关的致病性。ELISA 方法可用于监测囊性纤维化患者的慢性肺部感染。此外，我们的生长方法可以帮助诊断由特定SCV引起的感染。

Research

JoVE Journal

Immunology and Infection

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

铜绿假单胞菌和酿酒酵母流细胞生物膜

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

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

在伴有海藻酸钠生产新基因的鉴定<em&gt;铜绿假单胞菌</em&gt;使用小型<em&gt; himar1</em&gt;水手转座子介导的诱变

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Pseudomonas aeruginosa Induced Lung Injury Model

Pseudomonas aeruginosa Induced Lung Injury Model

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...