Real-time monitoring of cell viability and cell density on the basis of a three dimensional optical reflectance method (3D-ORM): investigation of the effect of sub-lethal and lethal injuries

  • Alison Brognaux
  • Jörg Bugge
  • Friedel H. Schwartz
  • Philippe Thonart
  • Samuel Telek
  • Frank Delvigne
Fermentation, Cell Culture and Bioengineering


Cell density and cell viability have been followed on-line by using a three-dimensional optical reflectance method (3D-ORM) probe. This method has allowed to highlight the differences between a well-mixed and a scale-down bioreactor configured in order to reproduce mixing deficiencies during a fed-batch culture of Escherichia coli. These differences have been observed both for the obscuration factor (OBF) and the coincidence probability delivered by the probe. These parameters are correlated to flow cytometry measurement based on the PI-uptake test and cell density based on optical density measurement. This first set of results has pointed out the fact that the 3D-ORM probe is sensitive to sub-lethal injuries encountered by microbial cells in process-related conditions. The effect of lethal injuries has been further investigated on the basis of additional experiments involving heat stress and a sharp increase of the OBF has been observed indicating that cells are effectively injured by the increase of temperature. However, further improvement of the probe are needed in order to give access to single-cell measurements.


Scale-down Flow cytometry Membrane permeability Viability In-situ probing 



Coincidence probability


Focused beam reflectance method


Forward scatter


Green fluorescent protein


Multi scattering analysis


Obscuration factor


Optical reflectance method


Process analytical technology


Propidium iodide


Time of flight



Alison Brognaux is supported by a FRIA PhD grant and gratefully acknowledges the Belgian Fund for Scientific Research (FNRS) for financial support.


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Copyright information

© Society for Industrial Microbiology and Biotechnology 2013

Authors and Affiliations

  • Alison Brognaux
    • 1
  • Jörg Bugge
    • 2
  • Friedel H. Schwartz
    • 2
  • Philippe Thonart
    • 1
  • Samuel Telek
    • 1
  • Frank Delvigne
    • 1
  1. 1.Gembloux Agro-Bio Tech, Unité de Bio-industries/CWBIUniversité de LiègeGemblouxBelgium
  2. 2.Sequip S + E GmbHDüsseldorfGermany

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