A Method for Viability Testing of Pectobacterium carotovorum in Postharvest Processing by Means of Flow Cytometry

Abstract

Rapid detection methods such as flow cytometric analysis enable the detection of phytopathogenic and human pathogenic bacteria and hence, the monitoring and optimisation of inactivation processes. The aim of this study was to develop a method for viability testing of the soft-rot-causing Gram-negative bacteria Pectobacterium carotovorum spp. carotovorum by flow cytometry based on a combination of carboxyfluorescein diacetate (cFDA) and propidium iodide. Due to the cell membrane composition of Gram-negative bacteria, the uptake ability of cFDA indicating esterase activity is limited. In this study, an adequate dye concentration (0.83 mM cFDA) and incubation time (45 min) at 37 °C for bacteria suspensions with a theoretical optical density (OD620) above 5 were defined, which enables a reliable determination of esterase activity of Gram-negative bacteria. This developed staining procedure was successfully applied to monitor inactivation treatments. It was shown that the test bacteria (Escherichia coli and P. carotovorum) lose their culturability due to the applied thermal treatment, but physiological activities were still detectable using flow cytometry. The remaining physiological activities may still result in product spoilage and may also cause human diseases. For example, E. coli cells still showed esterase activity after 10 min of thermal treatment at 70 °C. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria.