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The use of pulsed electric fields (PEF) for the inactivation of micro-organisms is currently of considerable interest as an alternative method to the conventional thermal methods of food pasteurization presently in widespread use. Many parameters of PEF influence the effectiveness of inactivation, one of which is the pulse profile, which is the subject of the present study. In this study, a pulse-forming network was designed and constructed to allow direct comparison of the effectiveness of a rectangular "monophase" pulse (single polarity) with a rectangular "biphase" pulse (incorporating a rapid reversal of polarity for the second half of the pulse). The pulses of 30 kV/cm in electric field intensity, 2 μs in duration for monophase, and 1 μs for each phase for biphase, were applied to parallel-plate test cells. Three different species of bacteria were tested including Gram negative and positive cell types. The bacteria were suspended in peptone water solutions of varied peptone concentration and hence, conductivity. The results show that, contrary to previous findings, biphase pulsing did not provide superior inactivation levels compared with monophase pulsing, for all three bacteria tested. Increasing the conductivity of the suspension fluid produced a general trend toward similar levels of inactivation of biphase compared with monophase PEF treatment, but in no case was it found that biphase PEF treatment was superior to monophase PEF treatment.