Flow cells were utilized to determine the effects of repetitive Syto9 staining on developing Acinetobacter sp. BD413 biofilm and to identify features describing reproducible biofilm architecture at 63× magnification. Syto9 is a general nucleic acid stain employed to visualize the entire microbial population of the biofilm and a component in the LIVE/DEAD® BacLight™ Bacterial Viability kits. CLSM images were quantified with the biofilm analysis software PHLIP to calculate six commonly used biofilm architecture characteristics. The characteristics biovolume and mean thickness were most reproducible when biofilms were grown in separate flow cells under controlled conditions, while roughness, porosity, total spreading and surface area to biovolume ratio exhibited inherent variability. Biovolume was more variable in separate flow cells than in channels of the same flow cell. However, even biofilms grown in channels of the same flow cell did not generate reproducible architectures based on the six characteristics. Results suggest difficulties in differentiating the effect of changes due to treatment from the natural variability of architecture development at the cellular level. Despite this high variability, biofilms only stained once developed into thicker structures containing more biomass than biofilms stained multiple times, suggesting that repeated staining with Syto9 affects architecture development. The application of Syto9 to monitor developing biofilms is not recommended.

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