Abstract
Various methods have been reported to quantify total biofilm or different components of biofilm; however, these methods are often confusedly used, leading to discrepancies and misleading results. In this study, different methods for quantification of biofilm, including those for total biomass, total amount of bacterial cells, viable cell number, and amount of extracellular polymeric substances, were systematically compared in microtiter plates. To evaluate which method is suitable for assessment of biofilm removal and for bacterial killing, biofilm samples were treated with various cleaners possessing removing and/or killing capacities. It was found that most of the methods tested in this study in general exhibited high reproducibility and repeatability. Crystal Violet staining was a simple but reliable method for total biomass quantification. Total bacteria cell numbers could be reliably quantified by the fluorescent DNA-binding dye Acridine Orange. Viable cells could be quantified by either an ATP-based assay or a proliferation assay. Both of these viability methods showed a broad detection range and led to precise measurement. For quantification of proteins in the biofilm, staining with fluorescein isothiocyanate was most suitable. Furthermore, it was revealed that a combination of different methods is required to determine if a cleaner kills or removes biofilm.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7396-9) contains supplementary material, which is available to authorized users.
Highlights
Under natural conditions, most bacteria occur in the form of a biofilm
It was observed that P. aeruginosa formed the biofilm mainly at the liquid/air interface on the walls (Fig. S2a), whereas the S. aureus biofilm mainly adhered to the bottom and in the corners (Fig. S2b)
Incubation for 40 min was chosen for treatment as it was found to give reproducible results
Summary
Most bacteria occur in the form of a biofilm. They adhere to surfaces embedded in a self-produced layer of extracellular polymeric substances (EPS) (Flemming and Wingender 2001; Sutherland 2001). The EPS protect bacteria against environmental influences such as UV irradiation, antibiotics, and disinfection, which make them much more tolerant to these influences compared to planktonic bacterial cells (Cochran et al 2000; Elasri and Miller 1999; Stewart and Costerton 2001). The unique structure of biofilm makes it difficult to be removed. Particular precautions have to be taken in the field of health care. Hospital-acquired infections, which often arise from incomplete removal of biofilm from instruments and device surfaces, account for a substantial part of health problems and costs (Zimlichman et al 2013)
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