Aims: Ethidium monoazide in combination with quantitative PCR (EMA-qPCR) has been considered as a promising method to enumerate viable cells; however, its efficacy can be significantly affected by disinfection conditions and various environments. In this study, thermal disinfection, osmotic pressure and acids with different pH values were systematically investigated to achieve the optimum conditions. Methods and Results: EMA treatment of pure cultures at low concentration (10 mu g ml(-1)) for 20 min resulted in effective differentiation between viable and nonviable bacteria and had no effect on viable cells. Heating at 85 degrees C for 35 min was the optimum condition that yields inactivated Escherichia coli (E. coli) cells that were not detected with EMA-qPCR. Performing EMA treatment in high-salt ion environment (sodium chloride concentration 4%) could weaken EMA inhibition effect. Both strong and weak acid solutions could react with EMA, change its absorption spectra and influence EMA inhibition effect. Because of the sublethal acidification injury, underestimation of cell counts were found using EMA-qPCR method, and 40-min incubation in Luria-Bertani medium could completely offset this error. Conclusion: Our results provided optimum EMA treatment, thermal disinfection and environment conditions for EMA-qPCR and demonstrated the feasibility of this method when enumerating viable cells under varied osmotic pressure and pH environment. Significance and Impact of the Study: Optimum EMA treatment, thermal disinfection and EMA-treated environment will be successfully applied in EMA-qPCR. Osmotic pressure and acid-induced injury can be detected by EMA-qPCR with optimization.