Changes in growth kinetics and metabolic activity of microorganisms under the presence of a moderate electric field (MEF) have been hypothesized as being due to temporary permeabilization of cell membranes. We investigated herein the effects of frequency and growth stage on cell membrane permeabilization of Lactobacillus acidophilus OSU 133 during MEF fermentation. Cells were stained with two fluorescent nucleic acid stains: the green, nonselective, cell membrane permeable SYTO 9, and the red, cell membrane impermeable propidium iodide (PI). Fluorescence exhibition post-treatment was assessed using fluorescence microscopy. Total plate counting was done to determine whether or not the permeabilized population represented live cells. Fermentation treatments investigated were conventional (control) and MEF (2 V/cm, 45, 60, 1,000, 10,000 Hz) at 30 degrees C. Studies were conducted at 45 Hz for lag, exponential, and stationary phases of growth. Low frequency MEF treated cells exhibited significantly greater numbers of red cell counts than conventional treatments; further, no significant differences existed in viable counts between MEF and conventional treatments, suggesting that the red counts represent permeabilized live cells. MEF treatments at the early stage of bacterial growth at 45 Hz exhibited the maximum permeabilization followed by treatments at 60 Hz. MEF treated samples at frequencies higher than 60 Hz did not exhibit redfluorescence. Cells at lag phase showed the greatest susceptibility to permeabilization followed by those at exponential phase. No evidence of electroporation was observed during the stationary phase. To our knowledge, these observations provide the first evidence that cell membrane permeabilization occurs under the presence of electric fields as low as those under MEF. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 85-94, 2009