Rapid enumeration of four indicator microorganisms in ground beef using a freeze-dried four-culture method

A recently reported, miniaturized method can simultaneously enumerate 4 critical indicator microorganisms in 24 h on a single 96-well microtiter plate, and is a convenient monitoring system for ensuring food plant hygiene. However, the utility of this method is limited by the necessity of plates being freshly prepared prior to each use. This study was undertaken to develop a method that would permit these plates to be prepared and stored in a stable form that could be conveniently used later. A microtiter plate filled with media dedicated to enumerating 4 specific classes of bacteria was freeze-dried. A given series was dedicated to enumerating either, total mesophilic bacteria, Gram-negative bacteria, coliform bacteria, or Escherichia coli. Freeze-dried plates were reconstituted simply by adding 100 μL of sterile water to each well. After adding samples, two-fold serial dilutions were performed, and the plate was incubated for 24 h at 32°C. Growth of 4 indicator microorganisms was detected in each series using metabolic indicators. The numerical estimates that these procedures yielded correlated very closely with numbers gained from conventional spread-plating methods (r² > 0.90). Additionally, the estimates obtained from liquid media microtiter plates and freeze-dried media plates showed a significantly strong relationship (r² > 0.92). Analysis of commercial ground beef showed a highly associated relationship (r² > 0.94) between the liquid four-culture and freeze-dried four-culture plate methods. The greatly enhanced facility of using premanufactured, freeze-dried microtiter plates would make this a most convenient way to perform microbial food quality analysis.