During microbial bioprocesses development, it is crucial monitoring cell stress response as such information determines the process efficiency. Multi-parameter flow cytometry coupled with fluorescent stains [PI and DiOC(6)(3) (3.3'-dihexylocarbocyanine iodide)] was used to monitor bacterial batch cultures. It was observed that the most significant differences in the cell physiological responses of these microorganisms were observed at the early stationary phase. While Bacillus licheniformis batch culture displayed 91% of healthy cells, 1.3% of cells with depolarised cytoplasmic membrane and 8% of cells with both depolarised and permeabilised cytoplasmic membrane as the culture reached this phase. Lactobacillus rhamnosus; batch culture displayed 66% of healthy cells, 29% of cells with depolarised cytoplasmic membrane and 5% of depolarised and permeabilised cytoplasmic membrane, at that stage. However, at the late stationary phase, both bacterial cultures showed a high proportion of polarised healthy cells, suggesting that the microorganisms could be using storage materials as a survival strategy. It was found that the double staining protocol using PI and DiOC(6)(3) could be used to monitor cell physiological response of both bacterial batch cultures and provided important physiological information at the individual cell level that is difficult to obtain in any other way, which can be used to enhance bioprocesses efficiency. (C) 2009 Elsevier Inc. All rights reserved.