A flow-cytometric gram-staining technique for milk-associated bacteria
A flow-cytometric gram-staining technique for milk-associated bacteria
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DOI：
10.1128/aem.69.5.2857-2863.2003
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发表时间：
2003-05-01
影响因子：
4.4
通讯作者：
Jespersen, L
中科院分区：
文献类型：
--
作者：
Holm, C;Jespersen, L
本文介绍了一种革兰氏染色技术，该技术结合了两种荧光染料，俄勒冈州绿结合麦胚凝集素（WGA）和碘化己啶（HI）染色，然后用流式细胞仪检测。WGA染色革兰氏阳性菌，而HI结合到所有细菌的DNA后，通过EDTA透化，并在50 degreesC孵育15 min。对于WGA结合到革兰氏阳性菌，3 M氯化钾溶液被发现给最高的荧光强度。共12株代表散装罐牛奶中的一些主要细菌物种和这些的混合物进行染色，并通过流式细胞术进行分析。总的来说，染色方法显示革兰氏阳性和革兰氏阴性细菌群体之间的明显区分。对于7种革兰氏阳性菌和5种革兰氏阴性菌的静止期培养物，平均99%的细胞被正确解释。该方法仅受细菌生长期或条件（如在-18 ℃冷冻24 h）的轻微影响。对于任何这些条件，平均至少95%的细胞被正确解释。当静止期培养物在5 ℃下储存14天时，平均86%的细胞被正确解释。将革兰氏染色技术应用于接种金黄色葡萄球菌和大肠杆菌的散装罐乳的流式细胞术分析。这些结果表明，该技术适用于分析牛奶样品，而无需预培养。
A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50degreesC for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by How cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at -18degreesC for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5degreesC for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the How cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.