title: Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide.
abstract: BACKGROUND
  Viability staining with SYTO9 and propidium iodide (PI) is a frequently used tool in microbiological studies. However, data generated by such routinely used method are often not critically evaluated for their accuracy. In this study we aim to investigate the critical aspects of this staining method using Staphylococcus aureus and Pseudomonas aeruginosa as the model microorganisms for high throughput studies in microtiter plates. SYTO9 or PI was added alone or consecutively together to cells and the fluorescence intensities were measured using microplate reader and confocal laser scanning microscope.
  
  
  RESULTS
  We found that staining of S. aureus cells with SYTO9 alone resulted in equal signal intensity for both live and dead cells, whereas staining of P. aeruginosa cells led to 18-fold stronger signal strength for dead cells than for live ones. After counterstaining with PI, the dead P. aeruginosa cells still exhibited stronger SYTO9 signal than the live cells. We also observed that SYTO9 signal showed strong bleaching effect and decreased dramatically over time. PI intensity of the culture increased linearly with the increase of dead cell numbers, however, the maximum intensities were rather weak compared to SYTO9 and background values. Thus, slight inaccuracy in measurement of PI signal could have significant effect on the outcome.
  
  
  CONCLUSIONS
  When viability staining with SYTO9 and PI is performed, several factors need to be considered such as the bleaching effect of SYTO9, different binding affinity of SYTO9 to live and dead cells and background fluorescence.
authors.affiliation: Laboratory for Biointerfaces, Swiss Federal Laboratories for Materials Science and Technology (Empa), Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland. Philipp.Stiefel@empa.ch.
authors.forename: Philipp
authors.initials: P
authors.lastname: Stiefel
authors.affiliation: Laboratory for Biointerfaces, Swiss Federal Laboratories for Materials Science and Technology (Empa), Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland. Sabrina.Schmidt@empa.ch.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Schmidt-Emrich
authors.affiliation: Laboratory for Biointerfaces, Swiss Federal Laboratories for Materials Science and Technology (Empa), Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland. Katharina.Maniura@empa.ch.
authors.forename: Katharina
authors.initials: K
authors.lastname: Maniura-Weber
authors.affiliation: Laboratory for Biointerfaces, Swiss Federal Laboratories for Materials Science and Technology (Empa), Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland. Qun.Ren@empa.ch.
authors.forename: Qun
authors.initials: Q
authors.lastname: Ren
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry*; D050296:Microbial Viability; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000592:standards*; D009930:Organic Chemicals / Q000737:chemistry*; D038761:Photobleaching; D011419:Propidium / Q000737:chemistry*; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods; D013211:Staphylococcus aureus / Q000502:physiology*
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals; C103389:SYTO 9; D011419:Propidium