100A-20 |
Development of miniaturized method for the simultaneous enumeration of four classifications of bacteria |
M. J. COSTELLO and D. H. Kang. Department of Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6376 Food plant sanitation will be improved with a simplified method for enumerating bacteria that requires little investment in equipment, consumable materials or training. Conventional plating methods are both cumbersome and expensive in both materials and labor. This project developed a procedure that identifies and enumerates total mesophilic microbial load, gram negative bacteria, coliforms, and Escherichia coli in 24 h, using a single module. Equipment requirements are an incubator capable of holding 32C, an 8-tip multi-channel pipettor, and an ultraviolet lamp. 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 classifications. To each microtiter well was added a 100 microliter aliquot of selective broth media, with metabolic indicators added. Hamburger was blended with 9 volumes of a pH 7.2 phosphate buffer. The first well in each series was inoculated with 100 microliter of homogenate. A 100 microliter aliquot from that first well was transferred to the next to achieve a two-fold dilution. The process was repeated with each succeeding well, achieving a repetitive two-fold dilution until all 24 wells in each series were inoculated. The plates were covered and placed in an incubator. Initial bacterial load was calculated using the last well in a series exhibiting color change or fluorescence. For comparison, the bacterial load of hamburger was enumerated using traditional agar plating methods. The bacterial numbers derived from the microtiter plate method exhibited a high correlation with numbers measured directly using standard agar methods. For most bacterial counts, the coefficient of determination (R2) exceeded 0.9. 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.
Session 100A, Food Microbiology: General II
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