Quantifying implant-associated biofilms: Comparison of microscopic, microbiologic and biochemical methods

• No single biofilm quantification method is suitable for all purposes.
• For rapid high-throughput screening resazurin and BacTiter-Glo™ are suited.
• LIVE/DEAD staining and CLSM is suited for detail analysis.
• Choose the appropriate method on basis of the specific scientific question.

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.