Resuscitation of Stressed Fecal Coliforms and Their Subsequent Detection by Radiometric and Impedance Techniques

Abstract

Approximately 99% of cells in a heat-stressed Escherichia coli culture were injured and were able to grow on trypticase soy agar (TSA) but not on Violet Red Bile Agar (VRBA). Employing a surface-overlay or pour-overlay method, complete recovery of this thermally-stressed population required incubation of the TSA plates at 35 C for 6 h before overlaying with VRBA and incubating at 45 C. While the surface-overlay technique provides a more accurate index of injury than the pour-overlay method, there appears to be little difference in plating procedures with respect to recovery of injured cells. Heat-stressed cells were inactivated by the bile salts mixture in the conventional EC broth and by incubation temperatures of 42–45 C. Most of the heat-stressed cells were able to recover in 3 h at 35–37 C, without any evidence of replication, in EC broth minus the bile salts mixture (EC-B), adjusted to pH 6.1–6.5. Under similar conditions freeze-stressed cells recovered without multiplication within 1 h and the replication of an unstressed population of E. coli was evident in 2 h but not in 1 h of incubation. Both radiometry and impedance show promise as rapid (17–18 h) screening techniques for determining if a cooked food meets the microbial criterion of 0 fecal coliforms/g. A resuscitation period of 3 h was essential for reliable detection of thermally-stressed fecal coliforms by either radiometry or impedance. An impedance based-MPN procedure (18 h or less) compared favorably with a TSA/VRBA pour-overlay method (24 h) and a conventional 3-tube most probable number technique (48–72 h) for enumerating freeze-injured and uninjured fecal coliforms.