Assessment of Physiological Conditions in E. Coli Fermentations by Epifluorescent Microscopy and Image Analysis So ´nia Carneiro Centre of Biological Engineering, IBB-Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal Anto ´nio L. Amaral Centre of Biological Engineering, IBB-Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal Departamento de Engenharia Quı ´mica e Biolo ´gica, Instituto Superior de Engenharia de Coimbra, Instituto Polite ´cnico de Coimbra, Rua Pedro Nunes, Quinta da Nora, 3030-199 Coimbra, Portugal Ana C. A. Veloso Centre of Biological Engineering, IBB-Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal CIMO - Escola Superior Agra ´ria de Braganc ¸a, Instituto Polite ´cnico de Braganc ¸a, Campus de Santa Apolo ´nia, Apartado 1172, 5301-855 Braganc ¸a, Portugal Departamento de Engenharia Quı ´mica e Biolo ´gica, Instituto Superior de Engenharia de Coimbra, Instituto Polite ´cnico de Coimbra, Rua Pedro Nunes, Quinta da Nora, 3030-199 Coimbra, Portugal Teresa Dias CIMO - Escola Superior Agra ´ria de Braganc ¸a, Instituto Polite ´cnico de Braganc ¸a, Campus de Santa Apolo ´nia, Apartado 1172, 5301-855 Braganc ¸a, Portugal Anto ´nio M. Peres CIMO - Escola Superior Agra ´ria de Braganc ¸a, Instituto Polite ´cnico de Braganc ¸a, Campus de Santa Apolo ´nia, Apartado 1172, 5301-855 Braganc ¸a, Portugal LSRE-Escola Superior Agra ´ria de Braganc ¸a, Instituto Polite ´cnico de Braganc ¸a, Campus de Santa Apolo ´nia, Apartado 1172, 5301-855 Braganc ¸a, Portugal Euge ´nio C. Ferreira and Isabel Rocha Centre of Biological Engineering, IBB-Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal DOI 10.1002/bp.134 Published online June 3, 2009 in Wiley InterScience (www.interscience.wiley.com). The development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influ- ence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Esche- richia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both pro- tein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fer- mentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes. V V C 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 882–891, 2009 Keywords: Recombinant E. coli fermentations, epifluorescent microscopy, physiology assessment, image analysis Introduction The cultivation of microbial strains expressing recombi- nant proteins has become an increasingly important Additional Supporting Information may be found in the online ver- sion of this article. Correspondence concerning this article should be addressed to I. Rocha at irocha@deb.uminho.pt. 882 V V C 2009 American Institute of Chemical Engineers