Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems

Abstract

Flow cytometry was used to count bac-of this study showed that flow cytometry terial cells from diverse origins: one was a reliable technique for counting a strain of E. coli, one sample of lake water, mixture of bacteria in samples from and 18 samples of estuary water. To verify the accuracy and the precision of this technique, total bacteria counts made by flow cytometry were compared with counts by direct observation using epifluorescence microscopy. The results aquatic ecosystems.Key terms: Bacteria, enumeration, flow cytometry, epifluorescence microscopy, aquatic ecosystems Bacterial abundances in water samples are usually estimated by the indirect method of growing the bacterial cells in culture medium. The selective specificity of culture media can permit both the counting and the qualification of a certain population of bacteria (e.g., Escherichia coli) among a bacterial community. Even the least specific culture media is still selective with respect to total bacterial cell numbers in a sample. For example, a nutrient agar allows only the growth of heterotrophic bacteria. However, the selective pressure of the culture media (e.g., the presence of 2-3-5 triphenyl tetrazolium chloride towards E . coli) can prohibit the development of a certain number of cells that are stressed but not dead, and that should develop in the medium. Thus there is a n underestimation of bacterial abundance. A second, more direct method to estimate bacterial populations is the technique of epifluorescence microscopy. Coupled with a fluorochrome dye to stain cells, epifluorescence microscopy allows the enumeration of all bacterial cells in a water sample. This direct counting method is especially important in bacterial ecology for determining total bacterial numbers in aquatic ecosystems. However, this method is both time consuming and tedious if one is to have a high level of precision in the estimation of bacterial abundances (12). The technique of flow cytometry permits the quantification of physical and biochemical aspects of individual cells. This technique has been verified for the analysis of reference bacterial populations using fluorochrome dyes (ethydium bromide and mithramycin) (10, 11) and for the detection of pathogenic bacteria in milk, blood, and cooling tower waters using fluorescent antibodies and fluorochrome dyes (1,3,13). Recently, flow cytometry has been used to characterize bacterial communities in aquatic ecosystems according to cell size and relative DNA content (6).

Flow cytometric determination of bacterial abundance in lake plankton with the green nucleic acid stain SYTO 13

“…Cytometric determinations of bacterial abundance were considerably more precise than epifluorescence counts, both among replicates of the same sample and among replicate water samples, in agreement with previous reports (Montfort and Baleux 1992;Monger and Landry 1993;Karl 1994). The average C.V. of bacterial abundance from triplicate epifluorescence counts of the same water sample was 14.5%, within the range reported by others (Kepner and Pratt 1994), whereas the average C.V. for triplicate or quadruplicate cytometric counts of the same water sample was 2.7%.…”

“…1978 Amended: 8 December 1995 abundance in lake plankton with Flow cytometry has become a common procedure in aquatic ecology, particularly in studies of autotrophic organisms (Yentsch and Horan 1989;Troussellier et al 1993). Surprisingly, there has been no parallel increase in the number of papers using flow cytometry to study planktonic bacteria, despite several demonstrations that bacterioplankton abundance, cell size, and DNA contents and even grazing rates on bacteria can be accurately estimated by flow cytometry (Troussellier et al 1995;Monger and Landry 1993;Button and Robertson 1993;Montfort and Baleux 1992). Flow cytometry has not superseded epifluorescence microscopy for several reasons.…”

Evaluation of Laser In Situ Scattering Instrument for Measuring Concentration of Phytoplankton, Purple Sulfur Bacteria, and Suspended Inorganic Sediments in Lakes

“…Numerous instruments have traditionally been used from long ago to measure the size distribution of suspended particles (both organic and inorganic) in aquatic systems, such as Coulter-counters (Jonasz and Prandke 1986;Tsai et al 1987;Stramski and Mobley 1997;Huisman 1999), microscopes (Monfort and Baleaux 1992), flow cytometers (Cavender-Bares et al 1998;Robertson et al 1998;Cristina et al 2001), laboratory laser particle-size analyzers (Edelvang and Austen 1997), etc. However, when these instruments are used, a previous handling and treatment of samples is always needed, which can damage the suspended aggregates modifying the size distribution and thus the results (Alldredge et al 1990;Del Giorgio et al 1996).…”

Quantitative assessment of toxic and nontoxic Microcystis colonies in natural environments using fluorescence in situ hybridization and flow cytometry

“…Flow cytometry (FCM) is useful for the rapid identification and enumeration of bacteria from aquatic environments. Individual cells are objectively identified on the basis of light scattering and fluorescence emitted from fluorochromes bound to cells [31]. Here, we describe the adaptation of techniques developed for aquatic microbiology, FISH and FCM, for the identification and enumeration of toxic and nontoxic Microcystis, which gives rise to green fluorescence on labeled Microcystis cells and red fluorescence due to the photosynthetic pigments (Figure 3).…”