Evaluating the Time-Temperature Requirement for E. coli Destruction Using the Live/Dead Test

The EPA provides a time-temperature equation needed to meet class A standards for thermophilic anaerobic digestion, which should be sufficient to lower fecal coliform (FC) levels to below 1000 MPN/g dry solids. However, it has been observed that while FC levels meet those standards after digestion, there can be a sudden increase in FC concentration after centrifuge dewatering. Two sets of experiments using the Live/Dead test coupled with standard culturing methods on pure cultures of E. coli were conducted using thermal treatment at 55?C. The first trial used the Live/Dead with a microplate reader at times of 8, 16, 24, 48 and 144 hours. The second trial adopted the fluorescence microscopy for better quantitative analysis and with incubation times of up to 14 days. Both exponential and stationary phase E coli were compared side by side on the second trail as well. The results indicate that, thought no culturable E. coli can be recovered after 16 hr of heat treatment, intact cells can still be observed after 14 days based on the Live/Dead test. The stationary phase cells also showed stronger resistance to heat destruction than the exponential cells. Overall, the results imply a potential formation of intact but non-culturable cells after thermophilic treatment, and further work is needed in order to verify their culturability and implications to the actual pathogens.