Enumeration of Viable Counts in Raw Milk Using the Automated Fluorescence Microscopic Method

Abstract

An automated fluorescence microscopic method developed for the measurement of viable bacterial counts was applied to the detection and enumeration of viable bacteria in milk. In the automated analysis of viable bacteria, bacterial cells were recovered from milk after the treatments with EDTA and Triton X-100, stained with 6-CFDA, and the stained cells were counted with the automated microscopic method. Salmonella Enteritidis expressing the enhanced green fluorescent protein (S. Enteritidis-EGFP) was used to elucidate the relationship between the fluorescent bacterial counts by the automated microscopic method and the viable counts by the conventional plating method. A high correlation coefficient (r²=0.97) was obtained for the relationship between the counts by the microscopic method and those by the plating method. When S. Enteritidis-EGFP was recovered from sterile milk after treatments with EDTA and Triton X-100, the correlation coefficient was calculated to be r²=0.91. Viable counts by the plating method and the counts of 6-CFDA stained bacterial cells by the automated microscopic method were determined for various raw milk samples. Thirty-seven raw milk samples were measured and the correlation coefficient was calculated to be r²=0.73.