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
Summary
我们描述了一个简单，快速和可靠的方法形成<em>白色念珠菌</em>生物膜在细胞内生物膜的抗真菌药敏试验使用96孔板和它的效用。
Abstract
白色念珠菌仍然是在一个不断增长的人口对受损的病人和念珠菌的真菌感染最常见的原因，现在是第三大最常见的感染在美国的医院。与生物膜的形成，对宿主组织和/或医疗设备（如导尿管），不同的表现形式的念珠菌。生物膜形成负面的临床意义，生物膜内的细胞是从宿主的免疫反应，保护和抗真菌药物的行动。我们已经开发为C 的形成简单，快速和可靠的体外模型白色念珠菌生物膜使用96孔酶标板，也可用于生物膜抗真菌药物药敏试验。的基础上减少代谢活性真菌生物膜细胞的XTT法（四唑盐）比色法，这个实验的读数。一个典型的实验中生物膜的形成大约需要24小时，与其他抗真菌药物药敏试验的24 h。由于其简单和常用的实验材料和设备的使用，这种技术民主化生物膜研究和代表对真菌生物膜的抗真菌药敏试验标准化的重要一步。
Protocol
1。 C语言的制备白色念珠菌 白色念珠菌是一种风险集团1/BSL1微生物。一定要记住使用工作良好的无菌/灭菌技术，微生物和生物危害材料的妥善处置的后续机构的程序。 准备C.培养过夜C.单菌落接种的YPD（酵母蛋白胨葡萄糖）液体培养基中的白色念珠菌到25 mL YPD 中的白色念珠菌。 在轨道摇床（约180转）在30 ° C过?…
Discussion
在这里，我们描述了一个简单，快速，经济和高度重现96再加上一个比色法措施内使用XTT法的生物膜细胞的代谢活动的念珠菌生物膜的形成以及酶标板模型。这96孔酶标板模型生物膜的形成最初的 C 白色念珠菌 ，但可用于其他念珠菌 。和轻松地适用于其他真菌生物。该方法可用于检查多个参数和生物膜形成的影响因素，并估计多个真菌分离和/或突变株生物膜形成能力。…
Divulgations
The authors have nothing to disclose.
Acknowledgements
生物膜相关的实验室工作是由赠款资助，从美国国立牙科和颅面研究所和国立过敏和传染病（JLL – R）。研究所编号R21DE017294和R21AI080930。内容完全是作者的责任，并不一定代表NIDCR，NIAID的美国国立卫生研究院的官方意见。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Sabouraud-dextrose agar | Becton Dickinson | 211584 | To prepare plates for fresh subcultures of fungal isolates | |
| YPD: Yeast peptone dextrose | US Biological | Y2076 | Medium for propagation of overnight liquid cultures | |
| RPMI-1640 without sodium bicarbonate supplemented with L-glutamine | Cellgro | 50-020-PB | Liquid medium for biofilm formation | |
| Morpholinepropanesulfonic acid (MOPS) | Fisher | BP308 | To buffer RPMI 1640 | |
| Phosphate buffered saline, PBS | Sigma | P4417 | Buffer for washes | |
| XTT sodium salt | Sigma | X4251 | See above for preparation instructions | |
| Ringer’s lactate | Hospira | NDC0409-7953-09 | For preparation of XTT solution | |
| Menadione | Sigma | M5625 | Caution: hazardous by skin contact, inhalation or ingestion | |
| Petri dishes | Fisher | 08-757-12 | ||
| 15 ml conical centrifuge tubes | Corning | 430790 | ||
| 50 ml conical centrifuge tubes | Corning | 430828 | ||
| 96 well microtiter plates: Polystyrene, flat-bottomed, tissue culture treated | Corning | 3595 | ||
| Multichannel pipette and tips | Eppendorf | |||
| Incubator | Any | |||
| Microtiter plate reader | Any |
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Tags
96 Well Microtiter PlateMonitoringFormationAntifungal Susceptibility TestingCandida AlbicansBiofilmsCandidiasisFungal InfectionsMedical DevicesCathetersIn Vitro ModelColorimetric AssayXTTTetrazolium SaltMetabolically Active Fungal Biofilm CellsExperiment DurationAntifungal Susceptibility Testing Standardization
Citer Cet Article
Pierce, C. G., Uppuluri, P., Tummala, S., Lopez-Ribot, J. L. A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms. J. Vis. Exp. (44), e2287, doi:10.3791/2287 (2010).