title: Potential for broad applications of flow cytometry and fluorescence techniques in microbiological and somatic cell analyses of milk.
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.
authors.affiliation: Centre for Fluorimetric Applications in Biotechnology, Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia. tgunasek@rna.bio.mq.edu.au
authors.forename: T S
authors.initials: TS
authors.lastname: Gunasekera
authors.forename: D A
authors.initials: DA
authors.lastname: Veal
authors.forename: P V
authors.initials: PV
authors.lastname: Attfield
mesh_terms: D000818:Animals; D001419:Bacteria / Q000302:isolation & purification*; D002417:Cattle; D002452:Cell Count; D015169:Colony Count, Microbial; D003257:Consumer Product Safety; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D005516:Food Microbiology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008414:Mastitis, Bovine / Q000378:metabolism; D008892:Milk / Q000166:cytology* / Q000382:microbiology* / Q000592:standards
chemical_list: D005456:Fluorescent Dyes