Development of a miniaturized four-culture method for the rapid enumeration of four bacterial groups in ground beef

Aims: A simplified and rapid method was developed to identify and enumerate total mesophilic microbial load, gram-negative bacteria, coliforms, and Escherichia coli in ground beef, using a single module. Methods and Results: A 96-well microtiter plate was divided into four sections of two lanes (24 wells), with each section dedicated to each of the four critical bacterial groups. Double strength of four different selective broths (100 μ1) was added to each well and an aliquot of sample (100 μl) from the first well was transferred to the next to achieve a twofold serial dilution in each series (24 wells). Initial bacterial load was calculated using the final consecutive positive reaction exhibiting color change or fluorescence. To validate the method, ground beef (n = 32), inoculated with a three-strain mixture of E. coli, together with 30 samples of commercial ground beef, were assayed by the four-culture method and conventional plating. For all media tested, the coefficients of determinations (r²) between the results derived from the four-culture method and those of conventional plating method ranged from 0.83 to 0.92. Conclusions: The high degree of association between the two methods indicates that the four-culture method could be easily applied to the enumeration of four critical bacterial groups in ground beef. Significance and Impact of the Study: This method will facilitate sanitation monitoring at meat processing plants by shortening time required for results, reducing consumable material costs, simplifying procedures, reducing the space required, and cutting the cost of start up equipment.