Doll Katharina, Jongsthaphongpun Katrin L, Stumpp Nico S, Winkel Andreas, Stiesch Meike
Department of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
Department of Prosthetic Dentistry and Biomedical Materials Science, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
J Microbiol Methods. 2016 Nov;130:61-68. doi: 10.1016/j.mimet.2016.07.016. Epub 2016 Jul 19.
Biofilm-associated infections pose severe problems in modern implant medicine. Screening for new implant materials with antibacterial properties requires reliable quantification of colonizing bacteria. There are many different methods to quantify biofilms on solid surfaces in vitro, employing different (bio-)chemical/microbiological reference parameters. It is therefore difficult to compare studies with different quantification techniques. Here, we have evaluated commonly used microscopic, microbiologic and biochemical methods to quantify bacterial biofilms, in order to clarify their comparability and applicability. Two bacterial species frequently involved in biofilm-associated infections, Staphylococcus aureus and Aggregatibacter actinomycetemcomitans, were used as model organisms; their initial adhesion and biofilm formation on titanium and on antibacterial copper were analyzed using the following methods: LIVE/DEAD fluorescence staining and confocal laser-scanning microscopy, ultrasonic or a newly developed enzymatic detachment followed by standard plate counting (CFU method), a resazurin-based assay, the BacTiter-Glo™ assay and crystal violet staining. The methods differed greatly in complexity, reliability and the applicability to initial adhesion and biofilm formation. To screen biofilm formation on a multitude of surfaces, the resazurin-based and the BacTiterGlo™ assay are well suited. LIVE/DEAD staining and confocal laser-scanning microscopy can be applied for a more detailed analysis of both, initial adhesion and biofilm formation. When using the CFU method for screening purposes, the introduced enzymatic detachment procedure is to be favored over ultrasonic detachment. There is not one single method, which is suitable for all purposes. The appropriate biofilm quantification method has to be chosen on the basis of the specific scientific question.
生物膜相关感染在现代植入医学中引发了严重问题。筛选具有抗菌特性的新型植入材料需要对定殖细菌进行可靠的定量分析。体外定量固体表面生物膜的方法有很多种，采用不同的（生物）化学/微生物学参考参数。因此，很难比较采用不同定量技术的研究。在此，我们评估了常用的显微镜、微生物学和生物化学方法来定量细菌生物膜，以阐明它们的可比性和适用性。两种经常参与生物膜相关感染的细菌物种，金黄色葡萄球菌和伴放线聚集杆菌，被用作模式生物；使用以下方法分析它们在钛和抗菌铜上的初始粘附和生物膜形成：活/死荧光染色和共聚焦激光扫描显微镜、超声或新开发的酶解，然后进行标准平板计数（CFU法）、基于刃天青的测定、BacTiter-Glo™测定和结晶紫染色。这些方法在复杂性、可靠性以及对初始粘附和生物膜形成的适用性方面差异很大。为了筛选多种表面上的生物膜形成，基于刃天青的测定和BacTiterGlo™测定非常适用。活/死染色和共聚焦激光扫描显微镜可用于对初始粘附和生物膜形成进行更详细的分析。当使用CFU法进行筛选时，引入的酶解程序比超声解更可取。没有一种方法适用于所有目的。必须根据具体的科学问题选择合适的生物膜定量方法。
