Abstract
Flow cytometry (FCM) is now becoming a routine tool for the enumeration and optical characterization of bacteria in marine environments. We investigated the effects of sample fixation and storage upon flow cytometric determination of marine bacteria. Fixed and unfixed seawater samples were analyzed by FCM immediately aboard ship and/or later in the laboratory, and the appearances of the fluorescence signals and bacterial counts of these samples were compared. Fixation and storage led to the formation of multiple peaks in fluorescence histograms; this was also seen in 22 out of 36 samples frozen in liquid nitrogen. Fixation did not, but storage did induce a decrease of bacterial counts: a rapid decrease during the first 3 days followed by a slower decline. The decline of cell numbers in stored samples was expressed by a regression model. Our studies indicate that precaution is necessary when interpreting the data from fixed and/or stored marine bacterial samples analyzed by FCM. The possibility that the procedure of fixation and storage leads to the appearance of high DNA and low DNA bacterial groups should be considered.
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Beveridge, T. J., F. M. R. Williams and J. J. Koval (1978): The effect of chemical fixatives on cell walls of Bacillus subtilis. Can. J. Microbiol., 24, 1439–1451.
Bullock, G. R. (1984): The current status of fixation for electron microscopy: a review. J. Microsc., 133, 1–15.
Button, D. K. and B. R. Robertson (2001): Determination of DNA content of aquatic bacteria by flow cytometry. Appl. Environ. Microbiol., 67, 1636–1645.
Cotner, J. B., M. L. Ogdahl and B. A. Biddanda (2001): Double-stranded DNA measurement in lakes with the fluorescent strain PicoGreen and the application to bacterial bioassays. Aquat. Microb. Ecol., 25, 65–74.
Davey, H. M. and D. B. Kell (1996): Flow cytometry and cell sorting of heterogeneous microbial populations: The importance of single-cell analyses. Microbiol. Rev., 60, 641–696.
Decamp, O. and N. Rajendran (1998): Bacterial loss and degradation of bacterial membrane in preserved seawater samples. Mar. Pollut. Bull., 36, 856–859.
del Giorgio, P. A., D. F. Bird, Y. T. Prairie and D. Planas (1996): Flow cytometric determination of bacterial abundance in lake plankton with the green nucleic acid stain SYTO 13. Limnol. Oceanogr., 41, 783–789.
Fry, J. C. and A. R. Davies (1985): An assessment of methods for measuring volumes of planktonic bacteria, with particular reference to television image analysis. J. Appl. Bacteriol., 58, 105–112.
Gasol, J. M. and P. A. del Giorgio (2000): Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities. Sci. Mar., 64, 197–224.
Gasol, J. M. and X. A. Moran (1999): Effects of filtration on bacterial activity and picoplankton community structure as assessed by flow cytometry. Aquat. Microb. Ecol., 16, 251–264.
Gasol, J. M., U. L. Zweifel, F. Peters, J. A. Fuhrman and A. Hagstrom (1999): Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria. Appl. Environ. Microbiol., 65, 4475–4483.
Gasol, J. M., M. Comerma, J. C. Garcia, J. Armengol, E. O. Casamayor, P. Kojecka and K. Simek (2002): A transplant experiment to identify the factors controlling bacterial abundance, activity, production, and community composition in a eutrophic canyon-shaped reservoir. Limnol. Oceanogr., 47, 62–77.
Guindulain, T. and J. Vives-Rego (2002): Involvement of RNA and DNA in the staining of Escherichia coli by SYTO 13. Lett. Appl. Microbiol., 34, 182–188.
Guindulain, T., J. Comas and J. Vies-Rego (1997): Use of nucleic acid dyes SYTO-13, TOTO-1, and YOYO-1 in the study of Escherichia coli and marine prokaryotic populations by flow cytometry. Appl. Environ. Microbiol., 63, 4608–4611.
Gundersen, K., G. Bratbak and M. Heldal (1996): Factors influencing the loss of bacteria in preserved seawater samples. Mar. Ecol. Prog. Ser., 137, 305–310.
Haselkorn, R. and P. Doty (1961): The reaction of formaldehyde with polynucleotides. J. Biol. Chem., 236, 2738–2745.
Jellett, J. F., W. K. W. Li, P. M. Dickie, A. Boraie and P. E. Kepkay (1996): Metabolic activity of bacterioplankton communities assessed by flow cytometry and single carbon substrate utilization. Mar. Ecol. Prog. Ser., 136, 213–225.
Jochem, F. J. (2001): Morphology and DNA content of bacterioplankton in the northern Gulf of Mexico: Analysis by epifluorescence microscopy and flow cytometry. Cytometry, 25, 179–194.
Kepner, R. L. and J. R. Pratt (1994): Use of fluorochromes for direct enumeration of total bacteria in environmental samples: Past and present. Microbiol. Rev., 58, 603–615.
Kogure, K., U. Simidu and N. Taga (1979): A tentative direct microscopic method for counting living marine bacteria. Can. J. Microbiol., 25, 415–420.
Lebaron, P., N. Parthuisot and P. Catala (1998): Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl. Environ. Microbiol., 64, 1725–1730.
Lebaron, P., P. Servais and H. Agogue (2001): Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems? Appl. Environ. Microbiol., 67, 1775–1782.
Li, W. K. W., J. F. Jellett and P. M. Dickie (1995): DNA distributions in planktonic bacteria stained with TOTO or TO-PRO. Limnol. Oceanogr., 40, 1485–1495.
Marie, D., D. Vaulot and F. Partensky (1996): Application of novel nucleic acid dyes YOYO-1, YO-PRO-1, and PicoGreen for flow cytometric analysis of marine prokaryotes. Appl. Environ. Microbiol., 62, 1649–1655.
Marie, D., F. Partensky, S. Jacquet and D. Vaulot (1997): Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I. Appl. Environ. Microbiol., 63, 186–193.
Marie, D., F. Partensky, D. Vaulot and C. Brussaard (1999): Enumeration of phytoplankton, bacteria and viruses in marine samples. p. 11.11.1–11.11.15. In Current Protocols in Cytometry, ed. by J. P. Robinson, John Wiley & Sons, Inc., New York.
Nagata, T., T. Someya, T. Konda, M. Yamamoto, K. Morikawa, M. Fukui, N. Kuroda, K. Takahashi, S. Oh, M. Mori, S. Arai and K. Kato (1989): Intercalibration of the acridine orange direct count method of aquatic bacteria. Bull. Japan. Soc. Microb. Ecol., 4, 89–99.
Noble, R. T. and J. A. Fuhrman (1998): Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquat. Microb. Ecol., 14, 113–118.
Pirker, H., C. Pausz, K. E. Stoderegger and G. J. Herndl (2005): Simultaneous measurement of metabolic activity and membrane integrity in marine bacterioplankton determined by confocal laser-scanning microscopy. Aquat. Microb. Ecol., 39, 225–233.
Polacow, I., L. Cabasso and H. J. Li (1976): Histone redistribution and conformational effect on chromatin induced by formaldehyde. Biochemistry, 15, 4559–4565.
Porter, K. G. and Y. S. Feig (1980): The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr., 25, 943–948.
Servais, P., E. O. Casamayor, C. Courties, P. Catala, N. Parthuisot and P. Lebaron (2003): Activity and diversity of bacterial cells with high and low nucleic acid content. Aquat. Microb. Ecol., 33, 41–51.
Shibata, A., Y. Goto, H. Saito, T. Kikuchi, T. Toda and S. Taguchi (2006): Comparison of SYBR Green I and SYBR Gold stains for enumerating bacteria and viruses by epifluorescence microscopy. Aquat. Microb. Ecol., 43, 223–231.
Suganuma, A. and H. Morioka (1979): Morphological changes in membrane systems of Staphylococci after different fixation procedures. J. Electron. Microsc., 28, 29–35.
Troussellier, M., C. Courties and S. Zettelmaier (1995): Flow cytometric analysis of coastal lagoon bacterioplankton and picophytoplankton: fixation and storage effects. Estuar. Coast. Shelf Sci., 40, 621–633.
Troussellier, M., C. Courties, P. Lebaron and P. Servais (1999): Flow cytometric discrimination of bacterial populations in seawater based on SYTO 13 staining of nucleic acids. FEMS Microbiol. Ecol., 29, 319–330.
Turley, C. M. and D. J. Hughes (1992): Effects of storage on direct estimates of bacterial numbers of preserved seawater samples. Deep-Sea Res., 39, 375–394.
Vaque, D., E. O. Casamayor and J. M. Gasol (2001): Dynamics of whole community bacterial production and grazing losses in seawater incubations as related to the changes in the proportions of bacteria with different DNA content. Aquat. Microb. Ecol., 25, 163–177.
Vitzthum, F., G. Geiger, H. Bisswanger, H. Brunner and J. Bernhagen (1999): A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system. Anal. Biochem., 276, 59–64.
Vosjan, J. H. and G. J. van Noort (1998): Enumerating nucleoid-visible marine bacterioplankton: bacterial abundance determined after storage of formalin fixed samples agrees with isopropanol rinsing method. Aquat. Microb. Ecol., 14, 149–154.
Weinbauer, M. G., C. Beckmann and M. G. Hofle (1998): Utility of green fluorescent nucleic acid dyes and aluminum oxide membrane filters for rapid epifluorescence enumeration of soil and sediment bacteria. Appl. Environ. Microbiol., 64, 5000–5003.
Yanada, M., T. Yokokawa, C. W. Lee, H. Tanaka, I. Kudo and Y. Maita (2000): Seasonal variation of two different heterotrophic bacterial assemblages in subarctic coastal seawater. Mar. Ecol. Prog. Ser., 204, 289–292.
Zubkov, M. V., B. M. Fuchs, P. H. Burkill and R. Amann (2001): Comparison of cellular and biomass specific activities of dominant bacterioplankton groups in stratified waters of the Celtic Sea. Appl. Environ. Microbiol., 67, 5210–5218.
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Kamiya, E., Izumiyama, S., Nishimura, M. et al. Effects of fixation and storage on flow cytometric analysis of marine bacteria. J Oceanogr 63, 101–112 (2007). https://doi.org/10.1007/s10872-007-0008-7
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DOI: https://doi.org/10.1007/s10872-007-0008-7