Name: visualization of biofilm formation in candida albicans using an automated microfluidic device
Uploaded: 2017-12-14T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device at JoVE.com

我们感谢金钗实验室的所有成员对生物膜测定进行有益的讨论。这项研究得到了国立卫生研究院 (NIH) 授予 R21 AI125801 (C.J.N.) 的支持。D.L.R. 得到了加利福尼亚大学墨西哥和美国研究所 (UC-MEXUS) 和理事会国家 Ciencia y Technologia (委员会) 的博士奖学金的支持。

1. 真菌细胞培养制剂
注意: 在生物安全柜内进行细胞培养工作 (即打开低温储存管、细胞培养管和烧瓶)。在工作前打开机箱的紫外线 (uv) 杀菌灯至少1小时, 并在机柜中积极工作时关闭紫外线灯。在实验开始前, 戴上手套、安全眼镜和适当的个人防护设备, 清除工作台表面的污管和70% 乙醇。建议使用无菌过滤器提示和熟悉基本无菌微生物技术。
<ol>
	<li>条纹白念珠菌菌株...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+biofilms:+development,+regulation,+and+molecular+mechanisms.">Candida albicans biofilms: development, regulation, and molecular mechanisms.</a> Microbes Infect. 18 (5), 310-321 (2016).</li><li>Uppuluri, P., 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=Dispersion+as+an+important+step+in+the+Candida+albicans+biofilm+developmental+cycle.">Dispersion as an important step in the Candida albicans biofilm developmental cycle.</a> PLoS Pathog. 6 (3), e1000828 (2010).</li><li>Andes, D., 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=Development+and+characterization+of+an+in+vivo+central+venous+catheter+Candida+albicans+biofilm+model.">Development and characterization of an in vivo central venous catheter Candida albicans biofilm model.</a> Infect Immun. 72 (10), 6023-6031 (2004).</li><li>Nett, J. E., Marchillo, K., Spiegel, C. A., Andes, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+in+vivo+Candida+albicans+biofilm+denture+model.">Development and validation of an in vivo Candida albicans biofilm denture model.</a> Infect Immun. 78 (9), 3650-3659 (2010).</li><li>Nett, J. E., 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=Rat+indwelling+urinary+catheter+model+of+Candida+albicans+biofilm+infection.">Rat indwelling urinary catheter model of Candida albicans biofilm infection.</a> Infect Immun. 82 (12), 4931-4940 (2014).</li><li>Krom, B. P., Willems, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Models+for+Candida+Biofilm+Development.">In Vitro Models for Candida Biofilm Development.</a> Methods Mol Biol. 1356, 95-105 (2016).</li><li>Hawser, S. P., Douglas, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+Candida+species+on+the+surface+of+catheter+materials+in+vitro.">Biofilm formation by Candida species on the surface of catheter materials in vitro.</a> Infect Immun. 62 (3), 915-921 (1994).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., Lopez-Ribot, J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+candidal+biofilm+microtiter+assay.">Optimized candidal biofilm microtiter assay.</a> J Microbiol Methods. 68 (2), 421-423 (2007).</li><li>Lohse, M. B., 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=Assessment+and+Optimizations+of+Candida+albicans+In+Vitro+Biofilm+Assays.">Assessment and Optimizations of Candida albicans In Vitro Biofilm Assays.</a> Antimicrob Agents Chemother. 61 (5), (2017).</li><li>Winter, M. B., 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=Global+Identification+of+Biofilm-Specific+Proteolysis+in+Candida+albicans.">Global Identification of Biofilm-Specific Proteolysis in Candida albicans.</a> mBio. 7 (5), (2016).</li><li>Nobile, C. 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=A+recently+evolved+transcriptional+network+controls+biofilm+development+in+Candida+albicans.">A recently evolved transcriptional network controls biofilm development in Candida albicans.</a> Cell. 148 (1-2), 126-138 (2012).</li><li>Fox, E. P., 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=An+expanded+regulatory+network+temporally+controls+Candida+albicans+biofilm+formation.">An expanded regulatory network temporally controls Candida albicans biofilm formation.</a> Mol Microbiol. 96 (6), 1226-1239 (2015).</li><li>Nobile, C. J., Mitchell, A. 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本文所描述的可定制微流控生物膜法可以在一个固定速率的层流和恒定温度下, 在单个细胞水平上 real-time 生物膜形成的可视化。它为研究野生型和突变菌株的生物膜的发展提供了有力的手段, 以及在模拟临床环境中观察到的生理条件下抗菌剂处理对生物膜的影响。不同于大多数的体外生物膜测定法, 这种方法允许检查在 real-time 形成的生物膜的发展。
这种方法可用于高?...

白色念珠菌是人类微生物的一个共生成员, 但它也是一种机会病原体, 能够引起表面和严重的真菌感染1,2。一个主要的毒力性状的白色白念珠菌是它的能力形成弹性和抗药性生物膜, 细胞群落附着在一个表面和封闭在一个细胞外基质材料1,3。白色念珠菌生物膜是高度结构化的, 包含多个细胞类型的多层 (圆芽酵母形态细胞, 椭圆形菌丝细胞, 和管状菌丝细胞)4。C. 白色念珠菌生物膜的发育始于圆的酵母形态细胞附着在表面 (播种生物膜), 其次是这些细胞在表面的增殖, 然后将未成熟的生物膜结构成熟为由胞外基质材料包围的完全形成的生物膜4。成熟的生物膜主要由形成致密和互连网络的细长菌丝细胞组成, 为生物膜4提供了结构稳定性。在整个生物膜的生命周期中, 圆芽酵母细胞从成熟的生物膜中分散开来, 并可能前往其他部位引起播散性感染或种子新的生物膜在其他站点4,5。白色念珠菌"可以在生物表面形成生物膜, 如粘膜表面和整个寄主组织, 以及在非生化表面, 如导管、心脏起搏器、假牙和假肢接头。由于生物膜的顽固性质, 它们非常难以根除, 而且在许多情况下, 唯一有效的治疗策略是去除感染的设备4。因此, 在类似于临床环境中观察到的条件下, 研究生物膜的形成至关重要。
有几个关键的体内动物模型用于研究C. 白色念珠菌生物膜形成6,7,8;然而, 这些研究可能是昂贵的, 耗时, 并受到限制的菌株和抗菌剂的数量, 可以在给定的时间进行测试。体外生物膜测定, 另一方面, 允许快速, 高通量的抗真菌化合物和突变株的评估, 并更 cost-effective 和伦理比在动物模型中进行的生物膜测定9, 10,11,12,13,14。在这里, 我们描述了一个体外分析, 我们开发和优化, 以观察在层流下的生物膜形成使用可定制的微流控设备14,15。该方法可以对生物膜形成的各个阶段进行可视化, 包括初始黏附步骤、细胞增殖、生物膜成熟和细胞分散。该分析也有助于可视化细胞形态学的变化, 在整个发展的生物膜。
孔板, 通常用于体外生物膜测定, 而高吞吐量, 不允许受控流条件。传统的层流细胞系统允许在受控流条件下对生物膜的形成进行连续的评估, 但通常这些都是建立起来的, 并且往往有有限的动态范围控制和吞吐量。这里使用的微流控装置克服了这些限制, 将高吞吐量板 (含48口井) 与内置层流室结合起来, 具有高度的重现性、通用性和可定制性。
在这里, 我们描述了一个使用商业上可用的自动微流控装置的协议来评估野生型的C. 白念珠菌菌株的生物膜形成, 已知的抗真菌剂对生物膜的发育和生物膜的影响在两个突变株的形成 (bcr1δ/δ和efg1 δ/δ), 以前报告有生物膜缺陷和在体内16,17, 18。所述的协议可用于测试抗菌剂在生物膜形成过程中抑制生物膜生成的功效, 并通过筛选突变文库来识别正常生物膜发育所需的基因。

<table>
	<tbody>
		<tr>
			<td>BioFlux 1000z</td>
			<td>Fluxion</td>
			<td></td>
			<td>Automated microfluidic device for live cell analysis</td>
		</tr>
		<tr>
			<td>48-well plate 0-20 dyne</td>
			<td>Fluxion</td>
			<td>910-0047</td>
			<td>Microfluidic plate</td>
		</tr>
		<tr>
			<td>Montage Software</td>
			<td>Fluxion</td>
			<td>Version 7.8.4.0</td>
			<td>Visualization analysis software</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>NIH</td>
			<td>https://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Criterion</td>
			<td>C7341</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose (D-Glucose)</td>
			<td>Fisher Scientific</td>
			<td>D163</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma-Aldrich</td>
			<td>R6504</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M3183</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Broth</td>
			<td>Criterion</td>
			<td>C6471</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco D-Mannitol</td>
			<td>BD Biosciences</td>
			<td>217020</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Criterion</td>
			<td>C5001</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Corning</td>
			<td>30-003-CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Inoculating Loops</td>
			<td>VWR</td>
			<td>30002-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes with Clear Lid</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Cuvettes</td>
			<td>Fisher Scientific</td>
			<td>14-955-127</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Paper</td>
			<td>VWR</td>
			<td>52846-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate and Cuvette Spectrophotometer</td>
			<td>BioTek</td>
			<td>EPOCH2TC</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking Incubator</td>
			<td>Eppendorf</td>
			<td>M12820004</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of biofilm formation in candida albicans using an automated microfluidic device

白色念珠菌是人类最常见的真菌病原体, 导致大约15% 的医院感染败血症病例。一个主要的毒力属性的白色白念珠菌是它的能力, 形成生物膜, 结构化群落的细胞附着在生物和非生物学表面。白色念珠菌生物膜可以在宿主组织 (如粘膜层) 和医疗设备 (如导管、心脏起搏器、假牙和关节修复体) 上形成。生物膜具有重大的临床挑战, 因为它们对物理和化学的扰动具有很强的抵抗力, 并且可以作为水库种子传播感染。各种体外检测方法已被用于研究白色念珠菌生物膜的形成, 如孔板测定、干重测量、细胞活力分析和共聚焦扫描激光显微镜。所有这些化验都是单一的 end-point 化验, 其中生物膜的形成是评估在特定的时间点。在这里, 我们描述了一个在层流条件下使用自动微流控装置研究 real-time 生物膜形成的协议。这种方法允许观察生物膜的形成随着时间的推移, 生物膜的发展, 使用可定制的条件, 模仿宿主, 如那些在血管导管中遇到的。该协议可用于评估遗传突变体的生物膜缺陷以及抗菌剂对 real-time 生物膜发育的抑制作用。

我们在两种介质条件 (RPMI-1640 和蜘蛛介质) 下使用野生型的C. 白念珠菌菌株进行微流控生物膜测定, 这种菌株存在于已知的抗真菌药物两性霉素 B (16 µg/毫升) 的 RPMI,和两个变种菌株以前报告的生物膜形成缺陷 (bcr1δ/δ和efg1 δ/δ) 在蜘蛛媒体。
视频1显示了野生型生物膜在 RPMI-1640 培养基中的发展, 以及抗真菌?...

Watch this Scientific Journal Video about Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device at JoVE.com

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

全自动微流控装置在白色念珠菌生物膜形成中的可视化

本协议描述了在宿主生理条件下, 使用可定制的自动微流控装置来可视化白色念珠菌中的生物膜形成。

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Research

JoVE Journal

Immunology and Infection

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

一个96孔微孔板为基础的方法监测的形成和抗真菌的药敏试验白色念珠菌生物膜

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

科研

免疫与感染

Microtiter Dish Biofilm Formation Assay

酶标菜生物膜形成实验

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

In vitro Biofilm Formation in an 8-well Chamber Slide

In vitro Biofilm Formation in an 8-well Chamber Slide

在体外生物膜的形成以及8商会幻灯片

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

深部真皮注射液作为白色念珠菌皮肤感染模型的组织学分析

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

红光光疗法调节白色念珠菌生物膜生长

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Clarifying and Imaging Candida albicans Biofilms

Clarifying and Imaging Candida albicans Biofilms

澄清和成像念珠菌生物膜

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

前体内分析研究胃 肠道的 念珠菌血脂磷

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

孟加拉玫瑰介导的光动力疗法抑制 白色念珠菌

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

使用 体内 成像筛选哺乳动物宿主活动性感染期间 白色念珠 菌突变菌株的形态发生表型

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic ...