Macroscale versus microscale methods for physiological analysis of biofilms formed in 96-well microtiter plates

J Microbiol Methods. 2013 Dec;95(3):342-9. doi: 10.1016/j.mimet.2013.10.002. Epub 2013 Oct 16.


Microtiter plates with 96 wells have become one of the preferred platforms for biofilm studies mainly because they enable high-throughput assays. In this work, macroscale and microscale methods were used to study the impact of hydrodynamic conditions on the physiology and location of Escherichia coli JM109(DE3) biofilms formed in microtiter plates. Biofilms were formed in shaking and static conditions, and two macroscale parameters were assayed: the total amount of biofilm was measured by the crystal violet assay and the metabolic activity was determined by the resazurin assay. From the macroscale point of view, there were no statistically significant differences between the biofilms formed in static and shaking conditions. However, at a microscale level, the differences between both conditions were revealed using scanning electron microscopy (SEM). It was observed that biofilm morphology and spatial distribution along the wall were different in these conditions. Simulation of the hydrodynamic conditions inside the wells at a microscale was performed by computational fluid dynamics (CFD). These simulations showed that the shear strain rate was unevenly distributed on the walls during shaking conditions and that regions of higher shear strain rate were obtained closer to the air/liquid interface. Additionally, it was shown that wall regions subjected to higher shear strain rates were associated with the formation of biofilms containing cells of smaller size. Conversely, regions with lower shear strain rate were prone to have a more uniform spatial distribution of adhered cells of larger size. The results presented on this work highlight the wealth of information that may be gathered by complementing macroscale approaches with a microscale analysis of the experiments.

Keywords: Biofilm heterogeneity; Computational fluid dynamics; Crystal violet; Microtiter plate; Resazurin; Scanning electron microscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteriological Techniques / methods*
  • Biofilms / growth & development*
  • Escherichia coli / cytology
  • Escherichia coli / physiology*
  • Gentian Violet / metabolism
  • Hydrodynamics*
  • Microscopy, Electron, Scanning
  • Oxazines / metabolism
  • Staining and Labeling
  • Xanthenes / metabolism


  • Oxazines
  • Xanthenes
  • resazurin
  • Gentian Violet