U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska


Date of this Version


Document Type



Letters in Applied Microbiology 2001, 33, 232-236


Aims: A rapid and simple method for enumerating uninjured and sublethally injured bacterial cells, the twofold dilution method (2FD), was developed and evaluated. Methods and Results: Following twofold serial dilution of samples in a 96 well microtiter plate, double strength selective broth or nonselective broth was added to each well. For resuscitation of heat-injured (55°C for 10 min) coliforms, the selective broth was added to the wells after 3 h preresuscitation time in buffered peptone water. The results of the 2FD were compared to plating methods for total and coliform plate counts from mixed cultures and beef carcass surface tissue samples. Conclusions: The 2FD method results were not significantly different for uninjured cells (P > 0.05) from those obtained using Petrifilm and standard plating. Correlation of the scatterplot of spread plating and 2FD indicated a high level of agreement between these two methods (R2 . 0.98 for total counts and R2 . 0.96 for coliforms from mixed cultures; R2 . 0.98 for total cell counts and R2 . 0.94 for coliforms from faeces inoculated beef carcasses). Significance and Impact of the Study: The twofold dilution method recovered significantly higher numbers of heat-injured coliforms compared to conventional plating methods (P < 0.05).