Pierce Christopher G, Uppuluri Priya, Tristan Amanda R, Wormley Floyd L, Mowat Eilidh, Ramage Gordon, Lopez-Ribot Jose L
Department of Biology and South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, Texas 78249, USA.
Nat Protoc. 2008;3(9):1494-500. doi: 10.1038/nport.2008.141.
The incidence of fungal infections has increased significantly over the past decades. Very often these infections are associated with biofilm formation on implanted biomaterials and/or host surfaces. This has important clinical implications, as fungal biofilms display properties that are dramatically different from planktonic (free-living) populations, including increased resistance to antifungal agents. Here we describe a rapid and highly reproducible 96-well microtiter-based method for the formation of fungal biofilms, which is easily adaptable for antifungal susceptibility testing. This model is based on the ability of metabolically active sessile cells to reduce a tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide) to water-soluble orange formazan compounds, the intensity of which can then be determined using a microtiter-plate reader. The entire procedure takes approximately 2 d to complete. This technique simplifies biofilm formation and quantification, making it more reliable and comparable among different laboratories, a necessary step toward the standardization of antifungal susceptibility testing of biofilms.
在过去几十年中，真菌感染的发生率显著增加。这些感染常常与植入生物材料和/或宿主表面上的生物膜形成有关。这具有重要的临床意义，因为真菌生物膜表现出与浮游（自由生活）群体截然不同的特性，包括对抗真菌剂的耐药性增加。在此，我们描述了一种基于96孔微量滴定板的快速且高度可重复的真菌生物膜形成方法，该方法易于适用于抗真菌药敏试验。该模型基于代谢活跃的固着细胞将四唑盐（2,3-双（2-甲氧基-4-硝基-5-磺基苯基）-2H-四唑-5-羧基苯胺）还原为水溶性橙色甲臜化合物的能力，并可使用微量滴定板读数器测定其强度。整个过程大约需要2天完成。该技术简化了生物膜的形成和定量，使其在不同实验室之间更可靠且具有可比性，这是生物膜抗真菌药敏试验标准化的必要步骤。
