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Abstract
Monitoring the quality and safety of milk requires careful analysis of microbial and
somatic cell loading. Our aim was to demonstrate proof of the principle that flow
cytometry (FCM), coupled with fluorescence techniques for distinguishing between cell
types, could potentially be employed in a wide variety of biological assays relevant
to the dairy industry. To this end, we studied raw milk samples and ultraheat-treated
milk, into which known numbers of bacteria or mouse cells were inoculated. For bacterial
analyses, protein and lipids were removed, whereas only centrifugal lipid clearing
was needed for somatic cell analyses. Cleared samples were stained with fluorescent
dyes or with bacterial-specific fluorescent-labeled oligonucleotides and analyzed
by FCM. A fluoresceinated peptide nucleic acid probe enabled efficient enumeration
of bacteria in milk. Dual staining of samples with fluorescent dyes that indicate
live (5-cyanol-2,3-ditolyl tetrazolium chloride, CTC or SYTO 9) or damaged cells (oxonol
or propidium iodide, PI) enabled determination of viable bacteria in milk. Gram-positive
and -negative bacteria were distinguished using hexidium iodide and SYTO 13 in dual
staining of cleared milk samples. An FCM-based method gave a good correlation (r=0.88)
with total microscopic counts of somatic cells in raw milk. The FCM method also correlated
strongly (r=0.98) with the standard Fossomatic method for somatic cell detection.
We conclude that FCM, coupled with fluorescence staining techniques, offers potentially
diverse and rapid approaches to biological safety and quality testing in the dairy
industry. Potential application of flow cytometers to a broad range of assays for
milk biological quality should make this instrumentation more attractive and cost
effective to the dairy industry and indeed the broader food industry.