Agreement, Precision, and Accuracy of Epifluorescence Microscopy Methods for Enumeration of Total Bacterial Numbers

Abstract

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.Bacterial abundance is an instrumental parameter in assessing the roles of bacteria in the environments (18,27,30,45). While a variety of techniques are available (1,30,53,60), staining bacterial cells with acridine orange (AO) (29) or 4Ј,6-diamidino-2-phenylindole (DAPI) (48) and counting them on black polycarbonate (PC) filters by epifluorescence microscopy have become the standard procedure for direct counting (9,18,30). The Live/Dead BacLight staining kit, which is widely accepted as a rapid measure of viability of individual cells, also provides a total count of bacteria (10). Currently, most studies reporting total bacterial numbers (TBNs) use one of the three staining methods described above. However, the basic question of which fluorochrome to use for a given samples still presents challenges, as comparative studies using two or more of these fluorochromes have often yielded conflicting results (10,17,20,34,37,40,49,52,54,57,58).A more perplexing question is whether TBN values based on different fluorochromes are interchangeable for a quantitative interpretation incorporating TBN data from different methods. A large-scale intersystem study, an analysis of long-term collection of longitudinal data, or a collaborative study by multiple laboratories often requires an amalgamated use of TBN values from different fluorochromes. Apart from the interc...

Evaluating the Reliability of Counting Bacteria Using Epifluorescence Microscopy

“…Similarly, previous investigations have reported the non-random distribution of bacterial cells on a filter following filtration of aquatic samples [5,46,47]. A similar study reported the homogeneous and heterogeneous distribution of food-contaminating bacteria in batches of infant milk powder after production or processing using colony forming units (CFU) counts [30].…”

“…Similarly, previous studies have reported a non-normal distribution of bacterial counts on nucleopore membrane filters [2,3,5,17,18,30]. Non-normal distribution of bacterial count data differentially affects the reliability and accuracy of the arithmetic mean depending on the number of randomly selected fields of view.…”

“…The arithmetic mean value of bacterial counts obtained under the most favorable conditions was considered as the true estimate of bacterial abundance [18]. However, it is highly challenging to obtain the variance of the true arithmetic mean value or any other measures of central tendencies, like the median or harmonic mean, in microscopic methods from samples that do not have a normal distribution [5]. To estimate the bias and variance of the arithmetic mean value of bacterial counts irrespective of the number of fields of view counted, a bootstrap method could be used to estimate these statistics because the assumption of normality is not necessary [19][20][21][22][23].…”

“…The technique of staining DNA using 4 6-diamidino-2-phenylindole (DAPI) and counting cells directly on a glass slide using an epifluorescence microscope has become the standard method of enumeration of bacteria [1][2][3][4][5][6][7]. This technique has been used for counting bacteria in different environmental samples including soils, brackish waters, pond and lake waters, seawater, marine intertidal sediments, natural biofilms and even oyster tissue homogenates (Table A1).…”

Novel Model for Multispecies Biofilms That Uses Rigid Gas-Permeable Lenses

“…Traditional nucleic acid stains, such as acridine orange, hexidium iodide, and Hoechst 33342, have been used to stain multiple species of planktonic and biofilm bacteria for visualization using standard fluorescent microscopy (21,46) and confocal laser scanning microscopy (10,27). However, generally these have been used as terminal analytical approaches for bacterial counts and/or viability (30,45) or to provide an evaluation of bacterial aggregation onto solid surfaces (10, 27) with minimal assessment for visualizing and maintaining cell viability. Our approach required that the fluorescent-labeled species continue to maintain sufficient fluorescent intensity and the ability to replicate over a 24-h interval required to actually "mature" into structured biofilms.…”