Singh A, Pyle B H, McFeters G A
Department of Microbiology, Montana State University, Bozeman.
J Microbiol Methods. 1989;10:91-101. doi: 10.1016/0167-7012(89)90005-5.
A direct viable counting method for enumerating viable bacteria was modified and made compatible with image analysis. A comparison was made between viable cell counts determined by the spread plate method and direct viable counts obtained using epifluorescence microscopy either manually or by automatic image analysis. Cultures of Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Yersinia enterocolitica and Pseudomonas aeruginosa were incubated at 35 degrees C in a dilute nutrient medium containing nalidixic acid. Filtered samples were stained for epifluorescence microscopy and analysed manually as well as by image analysis. Cells enlarged after incubation were considered viable. The viable cell counts determined using image analysis were higher than those obtained by either the direct manual count of viable cells or spread plate methods. The volume of sample filtered or the number of cells in the original sample did not influence the efficiency of the method. However, the optimal concentration of nalidixic acid (2.5-20 micrograms ml-1) and length of incubation (4-8 h) varied with the culture tested. The results of this study showed that under optimal conditions, the modification of the direct viable count method in combination with image analysis microscopy provided an efficient and quantitative technique for counting viable bacteria in a short time.
一种用于计数活菌的直接活菌计数方法被改进并使其与图像分析兼容。对通过平板涂布法测定的活菌数与使用落射荧光显微镜手动或通过自动图像分析获得的直接活菌数进行了比较。将大肠杆菌、鼠伤寒沙门氏菌、霍乱弧菌、小肠结肠炎耶尔森氏菌和铜绿假单胞菌的培养物在含有萘啶酸的稀释营养培养基中于35℃下培养。过滤后的样品进行落射荧光显微镜染色，并手动以及通过图像分析进行分析。培养后变大的细胞被视为活菌。使用图像分析测定的活菌数高于通过直接手动计数活菌或平板涂布法获得的活菌数。过滤的样品体积或原始样品中的细胞数不影响该方法的效率。然而，萘啶酸的最佳浓度（2.5 - 20微克/毫升）和培养时间（4 - 8小时）随所测试的培养物而变化。本研究结果表明，在最佳条件下，直接活菌计数方法与图像分析显微镜相结合提供了一种在短时间内计数活菌的高效定量技术。
