A rapid twofold dilution method for microbial enumeration and resuscitation of uninjured and sublethally injured bacteria.
OBJECTIVE
A rapid and simple method for enumerating uninjured and sublethally injured bacterial cells, the twofold dilution method (2FD), was developed and evaluated.


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 degrees 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 (R(2)=0.98 for total counts and R(2)=0.96 for coliforms from mixed cultures; R(2)=0.98 for total cell counts and R(2)=0.94 for coliforms from faeces inoculated beef carcasses).


CONCLUSIONS
The twofold dilution method recovered significantly higher numbers of heat-injured coliforms compared to conventional plating methods (P < 0.05).
Kang
D H
DH
United States Department of Agriculture, Agricultural Research Service, Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, NE 68933-0166, USA.
Siragusa
G R
GR
D000818:Animals; D002417:Cattle; D015169:Colony Count, Microbial / Q000379:methods*; D003470:Culture Media; D004755:Enterobacteriaceae / Q000254:growth & development*; D004926:Escherichia coli / Q000254:growth & development; D005243:Feces / Q000382:microbiology; D006358:Hot Temperature; D008460:Meat / Q000382:microbiology
D003470:Culture Media