title: Is biofilm removal properly assessed? Comparison of different quantification methods in a 96-well plate system.
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.
authors.affiliation: Laboratory for Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland.
authors.forename: Philipp
authors.initials: P
authors.lastname: Stiefel
authors.affiliation: Borer Chemie AG, Gewerbestrasse 13, 4528, Zuchwil, Switzerland.
authors.forename: Urs
authors.initials: U
authors.lastname: Rosenberg
authors.affiliation: Laboratory for Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland.
authors.forename: Jana
authors.initials: J
authors.lastname: Schneider
authors.affiliation: Borer Chemie AG, Gewerbestrasse 13, 4528, Zuchwil, Switzerland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Mauerhofer
authors.affiliation: Laboratory for Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland.
authors.forename: Katharina
authors.initials: K
authors.lastname: Maniura-Weber
authors.affiliation: Laboratory for Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland. qun.ren@empa.ch.
authors.forename: Qun
authors.initials: Q
authors.lastname: Ren
mesh_terms: D001419:Bacteria / Q000187:drug effects*; D058491:Bacterial Load; D001431:Bacteriological Techniques / Q000379:methods*; D018441:Biofilms / Q000187:drug effects*; D018533:Biomass; D004203:Disinfection / Q000379:methods*; D050296:Microbial Viability / Q000187:drug effects; D015203:Reproducibility of Results
keywords: Biofilm quantification; Biofilm removal; Enzymatic cleaner; Method comparison; Microtiter plate