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首页> 外文期刊>Applied Microbiology >Agreement, Precision, and Accuracy of Epifluorescence Microscopy Methods for Enumeration of Total Bacterial Numbers
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Agreement, Precision, and Accuracy of Epifluorescence Microscopy Methods for Enumeration of Total Bacterial Numbers

机译：枚举总细菌数的落射荧光显微镜方法的一致性，精确度和准确性

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To assess interchangeability of estimates of bacterial abundance by different epifluorescence microscopy methods, total bacterial numbers (TBNs) determined by most widely accepted protocols were statistically compared. Bacteria in a set of distinctive samples were stained with acridine orange (AO), 4′-6-diamidino-2-phenylindole (DAPI), and BacLight and enumerated by visual counting (VC) and supervised image analysis (IA). Model II regression and Bland-Altman analysis proved general agreements between IA and VC methods, although IA counts tended to be lower than VC counts by 7% on a logarithmic scale. Distributions of cells and latex beads on polycarbonate filters were best fitted to negative binomial models rather than to Poisson or log-normal models. The fitted models revealed higher precisions of TBNs by the IA method than those by the VC method. In pairwise comparisons of the staining methods, TBNs by AO and BacLight staining showed good agreement with each other, but DAPI staining had tendencies of underestimation. Although precisions of the three staining methods were comparable to one another (intraclass correlation coefficients, 0.97 to 0.98), accuracy of the DAPI staining method was rebutted by disproportionateness of TBNs between pairs of samples that carried 2-fold different volumes of identical cell suspensions. It was concluded that the TBN values estimated by AO and BacLight staining are relatively accurate and interchangeable for quantitative interpretation and that IA provides better precision than does VC. As a prudent measure, it is suggested to avoid use of DAPI staining for comparative studies investigating accuracy of novel cell-counting methods.
机译：为了评估通过不同的落射荧光显微镜方法估计细菌丰度的互换性，对通过最广泛接受的方案确定的细菌总数（TBN）进行了统计比较。一组独特样品中的细菌用4啶橙（AO），4'-6-二D基-2-苯基吲哚（DAPI）和BacLight染色，并通过视觉计数（VC）和监督图像分析（IA）进行计数。模型II回归和Bland-Altman分析证明了IA和VC方法之间的总体一致性，尽管IA数量往往比VC数量低7％。聚碳酸酯过滤器上的细胞和乳胶珠的分布最适合负二项式模型，而不是泊松或对数正态模型。拟合模型显示，IA方法比VC方法具有更高的TBN精度。在染色方法的成对比较中，通过AO和BacLight染色获得的TBN彼此显示出良好的一致性，但DAPI染色则存在被低估的趋势。尽管这三种染色方法的精度可以相互比较（类内相关系数为0.97至0.98），但DAPI染色方法的准确性却被TBN的不相称性所驳斥，该对TBN在成对的样品中携带了2倍不同体积的相同细胞悬液。结论是，通过AO和BacLight染色估计的TBN值相对准确且可互换用于定量解释，并且IA提供的精度优于VC。作为一种审慎的措施，建议在研究新型细胞计数方法准确性的比较研究中避免使用DAPI染色。

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