Yamaguchi Nobuyasu, Sasada Makoto, Yamanaka Mio, Nasu Masao
Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
Cytometry A. 2003 Jul;54(1):27-35. doi: 10.1002/cyto.a.10045.
Rapid and simple methods to detect viable pathogenic microbes in foods and drinks are required. Flow cytometry was used for the rapid detection of respiring Escherichia coli O157:H7 cells in apple juice, milk, and ground beef.
CTC (5-cyano-2,3-ditolyl tetrazolium chloride) was used to estimate the respiratory activity of bacteria. Fluorescein isothiocyanate (FITC)-labeled anti-E. coli O157:H7 direct antibody (FA) was used for the specific detection of target cells. Food samples were inoculated with starved E. coli O157:H7 and E. coli K-12 cells, and analyzed by both fluorescent microscopy and flow cytometry after double staining with FA and CTC.
Respiring E. coli O157:H7 cells in food samples showed strong fluorescence of both FA (green) and CTC (red); thus, they could be clearly and specifically distinguished from respiring E. coli K-12 or inactive cells. A good correlation was achieved in flow cytometric analysis between the numbers of inoculated viable E. coli O157:H7 and those detected in milk and apple juice. The detection threshold for this flow cytometry for E. coli O157:H7 in milk, apple juice, and ground beef was 10(3) cells/ml (milk and apple juice) or 10(3) cells/g (ground beef) of sample when the total bacterial number in the sample was 10(6) cells/ml.
Respiring E. coli O157:H7 in food samples can be detected specifically within a few hours. Flow cytometry with FA-CTC double staining can be used to examine food contamination with various pathogenic microbes demonstrating physiologic activity through the use of a suitable fluorescent antibody.
需要快速、简便的方法来检测食品和饮料中存活的致病微生物。流式细胞术用于快速检测苹果汁、牛奶和碎牛肉中具有呼吸活性的大肠杆菌O157:H7细胞。
使用CTC（5-氰基-2,3-二苯基四氮唑氯化物）评估细菌的呼吸活性。异硫氰酸荧光素（FITC）标记的抗大肠杆菌O157:H7直接抗体（FA）用于特异性检测靶细胞。将饥饿的大肠杆菌O157:H7和大肠杆菌K-12细胞接种到食品样品中，并用FA和CTC进行双重染色后，通过荧光显微镜和流式细胞术进行分析。
食品样品中具有呼吸活性的大肠杆菌O157:H7细胞显示出FA（绿色）和CTC（红色）的强荧光；因此，它们可以与具有呼吸活性的大肠杆菌K-12或无活性细胞清楚地、特异性地区分开来。在流式细胞术分析中，接种的存活大肠杆菌O157:H7数量与牛奶和苹果汁中检测到的数量之间具有良好的相关性。当样品中的总细菌数为10(6)个细胞/ml时，该流式细胞术对牛奶、苹果汁和碎牛肉中大肠杆菌O157:H7的检测阈值为10(3)个细胞/ml（牛奶和苹果汁）或10(3)个细胞/g（碎牛肉）。
食品样品中具有呼吸活性的大肠杆菌O157:H7可在数小时内被特异性检测到。通过使用合适的荧光抗体，FA-CTC双重染色的流式细胞术可用于检测各种具有生理活性的致病微生物对食品的污染。
