Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water
Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water
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DOI：
10.1128/aem.70.9.5343-5348.2004
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发表时间：
2004-09-01
影响因子：
4.4
通讯作者：
McFeters, GA
中科院分区：
文献类型：
--
作者：
Lisle, JT;Hamilton, MA;McFeters, GA
细菌丰度的直接总数是评估生态和工业应用中的水的生物量和细菌质量的核心。已经确定了几个因素，有助于在使用荧光显微镜时的细菌丰度计数的变化，其中最重要的是保留足够数量的细胞每个过滤器，以确保在所得数据的统计置信度的可接受的水平。先前的研究已经评估了导致这种差异的总直接计数方法的组分，试图保持每个过滤器的细菌细胞丰度值约为106个细胞过滤器。在本研究中，我们确定了每个过滤器细菌细胞数量的下限，在该下限下丰度估计的统计可靠性不再可以接受。我们的研究结果表明，当每个过滤器的细菌细胞数量逐渐减少到105以下时，显微镜方法越来越高估真正的细菌丰度（范围为15.0至99.3%）。固相细胞计数器仅略微高估了真实细菌丰度，并且在每个过滤器的相同细菌丰度范围内（范围，8.9至12.5%）更一致。固相细胞仪方法进行细菌总数直接计数的偏差较小，并进行显着优于任何显微镜方法。还发现在三个单独过滤器上计数5个视野的显微镜计数数据在统计学上等同于在单个过滤器上计数20个视野的数据。
Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 106 cells filter. In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 105, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.