Pulsed electric fields cause sublethal injury in Escherichia coli D. Garcı ´a, N. Go ´ mez, S. Condo ´ n, J. Raso, and R. Pag  an Tecnologı´a de los Alimentos, Facultad de Veterinaria C/Miguel Servet, 177, 50013, Zaragoza, Spain 2002/307: received 11 October 2002 and accepted 28 November 2002 ABSTRACT D. GARCI ´ A, N. GO ´ MEZ, S. CONDO ´ N, J. RASO, AND R. PAGA ´ N . 2003. Aims: The objective was to investigate the occurrence of sublethal injury in Escherichia coli by pulsed electric fields (PEF) at different pH values. Methods and Results: The occurrence of sublethal injury in PEF-treated E. coli cells depended on the pH of the treatment medium. Whereas a slight sublethal injury was detected at pH 7, 99®95% of survivors were injured when cells were treated at pH 4 for 400 ls at 19 kV. The PEF-injured cells were progressively inactivated by a subsequent holding at pH 4. Conclusions: PEF cause sublethal injury in E. coli. The measurement of sublethal injury using a selective medium plating technique allowed prediction of the number of cells that would be inactivated by subsequent storage in acidic conditions. Significance and Impact of the Study: This work could be useful for improving food preservation by PEF technology and contributes to the knowledge of the mechanism of microbial inactivation by PEF. Keywords: Escherichia coli, pulsed electric fields, pH, sublethal injury. INTRODUCTION Pulsed electric fields (PEF) are one of the most relevant non-thermal processes for food preservation because of their potential to inactivate microorganisms without altering organoleptic and nutritional properties of foods (Barbosa- C anovas et al. 1999). Knowledge of the mechanisms of microbial inactivation is required to design effective PEF treatments that, alone or in combination with other physical treatments or antimi- crobial agents, could form alternatives to traditional heat preservation. Microbial inactivation by PEF is believed to be caused by the effects of PEF on the cell envelopes. PEF can cause formation of pores affecting the integrity and functionality of the membrane. These pores can be reversible or irreversible, depending on the degree of membrane damage (Weaver and Chizmadzhev 1996). A proportion of cells would become leaky during PEF but reseal to a greater or lesser extent after it. Therefore, microorganisms that survive PEF treatments may be sublethally injured. Since sublethal injury is supposed to be related to the higher sensitivity of survivors to stress conditions after treatment, from a practical point of view the occurrence of sublethally injured cells would prove valuable in developing appropriate combination processes. Moreover, under suit- able conditions, sublethally injured cells might be repaired, therefore their occurrence is a very important aspect to be taken into account in food safety. A few cells being able to repair damage after PEF treatment could result in infective concentrations. Most published data (Simpson et al. 1999; Russell et al. 2000; Dutreux et al. 2000a, 2000b; Ulmer et al. 2002) has not demonstrated the occurrence of sublethal injury after PEF treatments using a selective medium plating technique. Only Liang et al. (2002) and Unal et al. (2001) have observed sublethally injured cells when PEF were combined with Correspondence to: Dr Rafael Pag an Tom as, Tecnologı´a de los Alimentos, Facultad de Veterinaria, C/Miguel Servet, 177, 50013, Zaragoza, Spain. Tel.: 34-976- 761581, Fax: 34-976-761590 (e-mail: pagan@posta.unizar.es). ª 2003 The Society for Applied Microbiology Letters in Applied Microbiology 2003, 36, 140–144