Gunasekera T S, Veal D A, Attfield P V
Centre for Fluorimetric Applications in Biotechnology, Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.
Int J Food Microbiol. 2003 Aug 25;85(3):269-79. doi: 10.1016/s0168-1605(02)00546-9.
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.
监测牛奶的质量和安全性需要仔细分析微生物和体细胞载量。我们的目的是证明这样一个原理：流式细胞术（FCM）结合用于区分细胞类型的荧光技术，有可能应用于与乳制品行业相关的各种生物学检测中。为此，我们研究了生鲜乳样品和超高温处理乳，向其中接种了已知数量的细菌或小鼠细胞。对于细菌分析，去除了蛋白质和脂质，而体细胞分析仅需进行离心脂质清除。将处理后的样品用荧光染料或细菌特异性荧光标记寡核苷酸染色，然后通过流式细胞术进行分析。一种荧光肽核酸探针能够有效地对牛奶中的细菌进行计数。用指示活细胞（5-氰基-2,3-二甲基四唑氯化物，CTC或SYTO 9）或受损细胞（恶嗪或碘化丙啶，PI）的荧光染料对样品进行双重染色，可以测定牛奶中的活菌数。在对处理后的牛奶样品进行双重染色时，使用碘化己锭和SYTO 13区分革兰氏阳性菌和阴性菌。基于流式细胞术的方法与生鲜乳中体细胞的总显微镜计数具有良好的相关性（r = 0.88）。流式细胞术方法与体细胞检测的标准Fossomatic方法也具有很强的相关性（r = 0.98）。我们得出结论，流式细胞术结合荧光染色技术，为乳制品行业的生物安全和质量检测提供了潜在的多样化和快速的方法。流式细胞仪在广泛的牛奶生物质量检测中的潜在应用，应会使这种仪器对乳制品行业乃至更广泛的食品行业更具吸引力且更具成本效益。
