A rapid twofold dilution method for microbial enumeration and resuscitation of uninjured and sublethally injured bacteria

Aims: A rapid and simple method for enumerating uninjured and sublethally i njured bacterial cells, the twofold dilution method (2FD), was developed an d evaluated. Methods and Results: Following twofold serial dilution of samples in a 96 w ell microtiter plate, double strength selective broth or nonselective broth was added to each well. For resuscitation of heat-injured (55 degreesC for 10 min) coliforms, the selective broth was added to the wells after 3 h pr eresuscitation 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 un injured cells (P > 0.05) from those obtained using Petrifilm and standard p lating. Correlation of the scatterplot of spread plating and 2FD indicated a high level of agreement between these two methods (R-2 = 0.98 for total c ounts and R-2 = 0.96 for coliforms from mixed cultures; R-2 = 0.98 for tota l cell counts and R-2 = 0.94 for coliforms from faeces inoculated beef carc asses). Significance and Impact of the Study: The twofold dilution method recovered significantly higher numbers of heat-injured coliforms compared to convent ional plating methods (P < 0.05).