Flow Cytometry and Environmental Microbiology

Jonathan Porter1

1 University of Exeter, Exeter
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 11.2
DOI:  10.1002/0471142956.cy1102s27
Online Posting Date:  February, 2004
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This survey unit discusses many of the issues involved for flow cytometry in the field of microbiology, particularly the preparative procedures, which are far more stringent than many other procedures using larger cells. For instance, it is often necessary to filter laboratory agents multiple times to obtain the true particle‐free solutions needed for flow cytometry of microbes. It is difficult enough to recognize bacteria in cell extracts from soil, sediment, or sludge given the background of same‐size particles. This unit provides an excellent overview of a potentially large application area in flow cytometry and is written by one of the most respected scientists in the field.

Keywords: flow cytometry; cell sorting; environmental microbiology; viability; biomass

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Table of Contents

  • Applying Flow Cytometry and Cell Sorting to Environmental Microbiology
  • Environmental Monitoring of Microorganisms Using Flow Cytometry and Cell Sorting
  • Specific Detection of Microorganisms in Environmental Samples Using Flow Cytometry
  • Viability Assessment of Microorganisms Using Flow Cytometry
  • Linking Identity, Function, and Activity in Single Microbial Cells
  • Discussion and Future Prospects
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Abe, F. 1998. Hydrostatic pressure enhances vital staining with carboxyfluorescein or carboxydichlorofluorescein in Saccharomyces cerevisiae: Efficient detection of labeled yeasts by flow cytometry. Appl. Environ. Microbiol. 64:1139‐1142.
  Dr. Porter wishes to acknowledge the support of the Natural Environment Research Council, Swindon, United Kingdom.
   Amann, R.I., Ludwig, W., and Schleifer, K.‐H. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143‐169.
   Andersen, J.B., Sternberg, C., Poulsen, L.K., Bjorn, S.P., Givskov, M., and Molin, S. 1998. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64:2240‐2246.
   Avery, S.V., Harwood, J.L., and Lloyd, D. 1995. Quantification and characterization of phagocytosis in the soil amoeba Acanthamoeba castellanii by flow cytometry. Appl. Environ. Microbiol. 61:1124‐1132.
   Bernard, L., Schäfer, H., Joux, F., Courties, C. Muyzer, G., and Lebaron, P. 2000. Genetic diversity of total, active and culturable marine bacteria in coastal seawater. Aquat. Microb. Ecol. 23:1‐11.
   Blanchot, J., Andre, J.M., Navarette, C., and Neveux, J. 1997. Picophytoplankton dynamics in the equatorial Pacific: Diel cycling from flow cytometer observations. C.R. Acad. Sci. Ser. III Sci. Vie 320:925‐931.
   Bougrier, S., Hawkins, A.J.S., and Heral, M. 1997. Preingestive selection of different microalgal mixtures in Crassostrea gigas and Mytilus edulis analysed by flow cytometry. Aquaculture 150:123‐134.
   Breeuwer, P., Drocourt, J.‐L., Bunschoten, N., Zweitering, M.H., Rombouts, F.M., and Abee, T. 1995. Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluorescein diacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product. Appl. Environ. Microbiol. 61:1614‐1619.
   Campbell, L., Nolla, H.A., and Vaulot, D. 1994. The importance of Prochlorococcus to community structure in the central North Pacific ocean. Limnol. Oceanogr. 39:954‐961.
   Campbell, L., Liu, H.B., Nolla, H.A., and Vaulot, D. 1997. Annual variability of phytoplankton and bacteria in the subtropical North Pacific Ocean at Station ALOHA during the 1991‐1994 ENSO event. Deep‐Sea Res. Part I Oceanogr. Res. Pap. 44:167‐192.
   Chen, F., Gonzalez, J.M., Dustman, W.A., Moran, M.A., and Hodson, R.E. 1997. In situ reverse transcription, an approach to characterize genetic diversity and activities of prokaryotes. Appl. Environ. Microbiol. 63:4907‐4913.
   Chisholm, S.W., Olson, R.J., Zettler, E.R., Goeriche, R., Waterbury, J.B., and Welschmeyer, N.A. 1988. A novel free‐living prochlorophyte abundant in the oceanic euphotic zone. Nature 334:340‐343.
   Christensen, B.B., Sternberg, C., Andersen, J.B., Erberl, L., Moller, S., Givskov, M., and Molin, S. 1998. Establishment of new genetic traits in a microbial biofilm community. Appl. Environ. Microbiol. 64:2247‐2255.
   Currin, C.A., Paerl, H.W., Suba, G.K., and Alberte, R.S. 1990. Immunofluorescence detection and characterization of N2‐fixing microrganisms from aquatic environments. Limnol. Oceanogr. 35:59‐71.
   Davey, H.M. and Kell, D.B. 1996. Flow cytometry and cell sorting of heterogeneous microbial populations: The importance of single‐cell analyses. Microbiol. Rev. 60:641‐696.
   Deere, D., Porter, J., Pickup, R., and Edwards, C. 1996. Direct analysis of starved Aeromonas salmonicida. J. Fish Dis. 19:459‐467.
   DeLeo, P.C. and Baveye, P. 1996. Enumeration and biomass estimation of bacteria in aquifer microcosm studies by flow cytometry. Appl. Environ. Microbiol. 62:4580‐4586.
   Detmer, A.E. and Bathmann, U.V. 1997. Distribution patterns of autotrophic pico‐ and nano‐plankton and their relative contribution to algal biomass during spring in the Atlantic sector of the Southern Ocean. Deep Sea Res. Part II Top. Stud. Oceanogr. 44:299‐320.
   Diaper, J.P. and Edwards, C. 1994. Flow cytometric detection of viable bacteria from compost. FEMS Microbiol. Ecol. 14:213‐220.
   Dinsdale, M.G., Lloyd, D., and Jarvis, B. 1995. Yeast vitality during cider fermentation: Two approaches to the measurement of membrane potential. J. Inst. Brew. 101:453‐458.
   Dubelaar, G.B.J. and Gerritzen, P.L. 2000. CytoBuoy: A step forward towards using flow cytometry in operational oceanography. Sci. Mar. 64:255‐265.
   Dunn, A.K., Klimowicz, A.K., and Handelsman, J. 2003. Use of a promoter trap to identify Bacillus cereus genes regulated by tomato seed exudate and a rhizosphere resident, Pseudomonas aureofaciens. Appl. Environ. Microbiol. 69:1197‐1205.
   Fernandez‐Abalos, J.M., Fox, H., Pitt, C., Wells, B., and Doonan, J.H. 1998. Plant‐adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans. Mol. Microbiol. 27:121‐130.
   Forster, S., Snape, J.R., Lappin‐Scott, H.M., and Porter, J. 2002. Simultaneous fluorescent Gram staining and activity assessment of activated sludge bacteria. Appl. Environ. Microbiol. 68:4772‐4779.
   Gauci, M.R., Vesey, G., Narai, J., Veal, D., Williams, K.L., and Piper, J.A. 1996 Observation of single cell fluorescence in laser flow cytometry. Cytometry. 25:388‐393.
   Glockner, F.O., Fuchs, B.M., and Amann, R. 1999. Bacterioplankton compositions of lakes and oceans: A first comparison based on fluorescence in situ hybridization. Appl. Environ. Microbiol. 65:3721‐3726.
   Gray, N.D., Howarth, R., Pickup, R.W., Jones, J.G., and Head, I.M. 2000. Use of combined microautoradiography and fluoresence in situ hybridization to determine carbon metabolism in mixed natural communities of uncultured bacteria from the genus Achromatium. Appl. Environ. Microbiol. 66:4518‐4522.
   Hansen, L.H., Ferrari, B., Sørensen, A.H., Veal, D., and Sørensen, S.J. 2001. Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole‐cell biosensors and flow cytometry. Appl. Environ. Microbiol. 67: 239‐244.
   Head, I.M., Gray, N.D., Clarke, K.J., Pickup, R.W., and Jones, J.G. 1996. The phylogenetic position and ultrastructure of the uncultured bacterium Achromatium oxaliferum. Microbiology 142: 2341‐2354.
   Henningson, E.W., Krocova, Z., Sandstrom, G., and Forsman, M. 1998. Flow cytometric assessment of the survival ratio of Francisella tularensis in aerobiological samples. FEMS Microbiol. Ecol. 25:241‐249.
   Hodson, R.E., Dustman, W.A., Garg, R.P., and Moran, M.A. 1995. In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities. Appl. Environ. Microbiol. 61:4074‐4082.
   Jacobsen, C.N., Rasmussen, J., and Jakobsen, M. 1997. Viability staining and flow cytometric detection of Listeria monocytogenes. J. Microbiol. Methods 28:35‐43.
   Jernaes, M.W. and Steen, H.B. 1994. Staining of Escherichia coli for flow cytometry: Influx and efflux of ethidium bromide. Cytometry 17:302‐309.
   Jonker, R.R., Meulemans, J.T., Dubelaar, G.B.J., Wilkins, M.F., and Ringelberg, J. 1995. Flow cytometry—a powerful tool in analysis of biomass distributions in phytoplankton. Water Sci. Technol. 32:177‐182.
   Joux, F., Lebaron, P., and Troussellier, M. 1997. Succession of cellular states in a Salmonella typhimurium population during starvation in artificial seawater microcosms. FEMS Microbiol. Ecol. 22:65‐76.
   Kaprelyants, A.S. and Kell, D.B. 1993. Dormancy in stationary‐phase cultures of Micrococcus luteus: Flow cytometric analysis of starvation and resuscitation. Appl. Environ. Microbiol. 59:3187‐3196.
   Kaprelyants, A.S., Mukamolova, G.V., Davey, H.M., and Kell, D.B. 1996. Quantitative analysis of the physiological heterogeneity within starved cultures of Micrococcus luteus by flow cytometry and cell sorting. Appl. Environ. Microbiol. 62:1311‐1316.
   Kenter, U., Zimmermann, U., and Muller, H. 1996. Grazing rates of the freshwater ciliate Balanion planctonicum determined by flow cytometry. J. Plankton Res. 18:1047‐1053.
   Lange, J.L., Thorne, P.S., and Lynch, N. 1997. Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria. Appl. Environ. Microbiol. 63:1557‐1563.
   Langsrud, S. and Sundheim, G. 1996. Flow cytometry for rapid assessment of viability after exposure to a quaternary ammonium compound. J. Appl. Bacteriol. 81:411‐418.
   Lebaron, P., Catala, P., and Parthuisot, N. 1998a. Effectiveness of SYTOX green stain for bacterial viability assessment. Appl. Environ. Microbiol. 64:2697‐2700
   Lebaron, P., Parthuisot, N., and Catala, P. 1998b. Comparison of blue nucleic acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl. Environ. Microbiol. 64:1725‐1730.
   Lebaron, P., Servais, P., Baudoux, A.‐C., Bourrain, M., Courties, C., and Parthuisot, N. 2002. Variations of bacterial‐specific activity with cell size and nucleic acid content assessed by flow cytometry. Aquat. Microb. Ecol. 28:131‐140.
   Li, W.K.W. 1995. Composition of ultraphytoplankton in the Central North Atlantic. Mar. Ecol. Prog. Ser. 122:1‐8.
   Lloyd, D. 1993 Flow Cytometry in Microbiology. Springer‐Verlag, London.
   Manefield, M., Whiteley, A.S., Griffiths, R.I., and Bailey, M.J. 2002. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl. Environ. Microbiol. 68:5367‐5373.
   Marie, D., Vaulot, D., and Partensky, F. 1996. Application of the 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., Partensky, F., Jacquet, S., and Vaulot, D. 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., Brussaard, C.P.D., Thyrhaug, R., Bratbak, G., and Vaulot, D. 1999. Enumeration of marine viruses in culture and natural samples by flow cytometry. Appl. Environ. Microbiol. 65: 45‐52.
   Mason, D.J., Allman, R. Stark, J.M., and Lloyd, D. 1994. Rapid estimation of bacterial antibiotic susceptibility with flow cytometry. J. Microsc. 176:8‐16.
   Mason, D.J., Lopez‐Amoros, R., Allman, R., Stark, J.M., and Lloyd, D. 1995. The ability of membrane potential dyes and calcafluor white to distinguish between viable and non‐viable bacteria. J. Appl. Bacteriol. 78:309‐315.
   Monger, B.C. and Landry, M.R. 1993. Flow cytometric analysis of marine bacteria with Hoechst 33342. Appl. Environ. Microbiol. 59:905‐911.
   Montford, P. and Baleux, B. 1992. Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems. Cytometry 13:188‐192.
   Nebe‐von Caron, G. and Badley, R.A. 1995. Viability assessment of bacteria in mixed populations using flow cytometry. J. Microsc. 179:55‐66.
   Oerther, D.B., Pernthaler, J., Schramm, A., Amann, R. and Raskin, L. 2000. Monitoring precursor 16S rRNAs of Acinetobacter spp. in activated sludge wastewater treatment systems. Appl. Environ. Microbiol. 66:2154‐2165.
   Page, S. and Burns, R.G. 1991. Flow cytometry as a means of enumerating bacteria introduced into soil. Soil Biol. Biochem. 23:1025‐1028.
   Payne, M.J., Campbell, S., Patchett, R.A., and Kroll, R.G. 1992. The use of immobilized lectins in the separation of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. from pure cultures and foods. J. Appl. Bacteriol. 73:41‐52.
   Pernthaler, A., Pernthaler, J., Schattenhofer, M., and Amann, R. 2002. Identification of DNA‐synthesizing bacterial cells in coastal North Sea plankton. Appl. Environ. Microbiol. 68:5728‐5736.
   Porter, J., Edwards, C., Morgan, J.A.W., and Pickup, R.W. 1993. Rapid, automated separation of specific bacteria from lakewater and sewage using flow cytometry and cell sorting. Appl. Environ. Microbiol. 59:3327‐3333.
   Porter, J., Diaper, J., Edwards, C., and Pickup, R. 1995a. Direct measurements of natural planktonic bacterial community viability by flow cytometry. Appl. Environ. Microbiol. 61:2783‐2786.
   Porter, J., Edwards, C., and Pickup, R.W. 1995a. Rapid assessment of physiological status in Escherichia coli using fluorescent probes. J. Appl. Bacteriol. 79:399‐408.
   Porter, J., Pickup, R., and Edwards, C. 1995c. Flow cytometric detection of specific genes in genetically modified bacteria using in situ polymerase chain reaction. FEMS Microbiol. Lett. 134:51‐56.
   Porter, J., Pickup, R.W., Robinson, J., and Edwards, C. 1995d. Recovery of a bacterial sub‐population from sewage using immunofluorescent flow cytometry and cell sorting. FEMS Microbiol. Lett. 133:195‐199.
   Porter, J., Deere, D., Pickup, R., and Edwards, C. 1996. Fluorescent probes and flow cytometry: New insights into environmental bacteriology. Cytometry 23:91‐96.
   Porter, J., Deere, D., Hardman, M., Edwards, C., and Pickup, R. 1997a. Go with the flow—use of flow cytometry in environmental microbiology. FEMS Microbiol. Ecol. 24:93‐101
   Porter, J., Pickup, R.W., and Edwards, C. 1997b. Evaluation of flow cytometric methods for the detection and viability assessment of bacteria in soil. Soil Biol. Biochem. 29:91‐100.
   Porter, J., Robinson, J., Pickup, R., and Edwards, C. 1998. An evaluation of lectin‐mediated magnetic bead cell sorting for the targeted separation of enteric bacteria. J. Appl. Microbiol. 84:722‐732.
   Radajewski, S., Ineson, P., Parekh, N.R., and Murrell, J.C. 2000. Stable‐isotope probing as a tool in microbial ecology. Nature 403:646‐649.
   Resina‐Pelfort, O., Comas‐Riu, J., and Vives‐Rego, J. 2001. Effects of deflected droplet electrostatic cell sorting on the viability and exoproteolytic activity of bacterial cultures and marine bacterioplankton. System. Appl. Microbiol. 24:31‐36.
   Rice, J., Sleigh, M.A., Burkill, P.H., Tarran, G.A., O'Connor, C.D., and Zubkov, M.V. 1997. Flow cytometric analysis of characteristics of hybridisation of species‐specific fluorescent oligonucleotide probes to rRNA of marine nanoflagellates. Appl. Environ. Microbiol. 63:938‐944.
   Robertson, B.R. and Button, D.K. 1989. Characterizing aquatic bacteria according to population, cell size and apparent DNA content by flow cytometry. Cytometry 10:70‐76.
   Roth, B.L., Poot, M., Yue, S.T., and Millard, P.J. 1997. Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl. Environ. Microbiol. 63:2421‐2431.
   Servais, P., Agogue, H., Courties, C., Joux, F., and Lebaron, P. 2001. Are the actively respiring cells (CTC+) those responsible for bacterial production in aquatic environments? FEMS Microbiol. Ecol. 35:171‐179.
   Simon, N., LeBot, N., Marie, D., Partensky, F., and Vaulot, D. 1995. Fluorescent in situ hybridisation with ribosomal RNA targeted oligonucleotide probes to identify small phytoplankton by flow cytometry. Appl. Environ. Microbiol. 61:2506‐2513.
   Simon, N., Brenner, J., Edwardsen, B., and Medlin, L.K. 1997. The identification of Chysochromulina and Prymnesium (Haptophyta, Pymnesiophycae) using fluorescent or chemiluminescent oligonucleotide probes: A means for improving studies on toxic algae. Eur. J. Phycol. 32:393‐401.
   Tani, K., Kurokawa, K., and Nasu, M. 1998. Development of a direct in situ PCR method for detection of specific bacteria in natural environments. Appl. Environ. Microbiol. 64:1536‐1540.
   Tani, K., Muneta, M., Nakamura, K., Shibuya, K., and Nasu, M. 2002. Monitoring of Ralstonia eutropha KT1 in groundwater in an experimental bioaugmentation field by in situ PCR. Appl. Environ. Microbiol. 68:412‐416.
   Tombolini, R., Unge, A., Davey, M.E., de Bruijn, F.J., and Jansson, J.K. 1997. Flow cytometric and microscopic analysis of GFP‐tagged Pseudomonas fluorescens bacteria. FEMS Microbiol. Ecol. 22:17‐28.
   Thomas, J.C., Desrosiers, M., St.‐Pierre, Y., Lirette, P., Bisaillon, J.G., Beaudet, R., and Villemur, R. 1997. Quantitative flow cytometric detection of specific microorganisms in soil samples using rRNA targeted fluorescent probes and ethidium bromide. Cytometry 27:224‐232.
   Troussellier, M., Courties, C., and Zettelmaier, S. 1995. Flow cytometric analysis of coastal lagoon bacterioplankton and picophytoplankton: Fixation and storage effects. Estuarine Coastal Shelf Sci. 40:621‐633.
   Turner, K., Porter, J., Pickup, R.W., and Edwards, C. 2000. Changes in viability and macromolecular content of long‐term batch cultures of Salmonella typhimurium measured by flow cytometry. J. Appl. Microbiol. 89:90‐99.
   Tyndall, R.L., Hand, R.E. Jr., Mann, R.C., Evans, C., and Jernigan, R. 1985. Application of flow cytometry to detection and characterization of Legionella spp. Appl. Environ. Microbiol. 49:852‐857.
   Van der Waaij, L.A., Mesander, G., Limburg, P.C., and van der Waaij, D. 1994. Direct flow cytometry of anaerobic bacteria in human feces. Cytometry 16:270‐279.
   Van der Waaij, L.A., Limberg, P.C., Mesander, G., and Van der Waaij, D. 1996. In vivo IgA coating of anaerobic bacteria in human faeces. Gut 38:348‐354.
   Veldhuis, M.J.W., Cucci, T., and Sieracki, M.E. 1997. Cellular DNA content of marine phytoplankton using two new fluorochromes: Taxonomic and ecological implications. J. Phycol. 33:527‐541.
   Vesey, G., Hutton, P., Champion, A., Ashbolt, N., Williams, K.L., Warton, A., and Veal, D. 1994a. Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water. Cytometry 16:1‐6.
   Vesey, G., Narai, J., Ashbolt, N., Williams, K., and Veal, D. 1994b. Detection of specific microorganisms in environmental samples using flow cytometry. Methods Cell Biol. 42:489‐522.
   Vesey, G., Deere, D., Gauci, M.R., Williams, K.L., and Veal, D.A. 1997a. Evaluation of fluorochromes and excitation sources for immunofluorescence in water samples. Cytometry 29:147‐154.
   Vesey, G., Deere, D., Weir, C.J., Ashbolt, N., Williams, K.L., and Veal, D.A. 1997b. A simple method for evaluating Cryptosporidium specific antibodies used in monitoring environmental water samples. Lett. Appl. Microbiol. 25:316‐320.
   Vesey, G., Griffiths, K.R., Gauci, M.R., Deere, D., Williams, K.L., and Veal, D.A. 1997c. Simple and rapid measurement of Cryptosporidium excystation using flow cytometry. Int. J. Parasitol. 27:1353‐1359.
   Volsch, A., Nader, W.F., Geiss, H.K., Nebe, G., and Birr, C. 1990. Detection and analysis of two serotypes of ammonia‐oxidizing bacteria in sewage plants by flow cytometry. Appl. Environ. Microbiol. 56:2430‐2435.
   Votyakova, T.V., Kaprelyants, K.S., and Kell, D.B. 1994. Influence of viable cells on the resuscitation of dormant cells in Micrococcus luteus cultures held in an extended stationary phase: The population effect. Appl. Environ. Microbiol. 60:3284‐3291.
   Wallner, G., Amann, R., and Beisker, W. 1993. Optimizing fluorescent in situ hybridization with rRNA‐targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 14:136‐143.
   Wallner, G., Erhart, R., and Amann, R. 1995. Flow cytometric analysis of activated sludge with rRNA‐targeted probes. Appl. Environ. Microbiol. 61:1859‐1866.
   Wallner, G., Fuchs, B., Spring, S., Beisker, W., and Amann, R. 1997. Flow sorting of microorganisms for molecular analysis. Appl. Environ. Microbiol. 63:4223‐4231.
   Whiteley, A.S., Griffiths, R.I., and Bailey, M.J. 2003. Analysis of the microbial functional diversity within water‐stressed soil communities by flow cytometric analysis and CTC+ cell sorting. J. Microbiol. Meth. 54:257‐267.
   Wilkins, M.F., Boddy, L., Morris, C.W., and Jonker, R. 1996. A comparison of some neural and non‐neural methods for identification of phytoplankton from flow cytometry data. Comput. Appl. Biosci. 12:9‐18.
   Williams, S.C., Hong, Y., Danavall, D.C.A., Howard‐Jones, M.H., Gibson, D., Frischer, M.E., and Verity, P.G. 1998. Distinguishing between living and non‐living bacteria: Evaluation of the vital stain propidium iodide and its combined use with molecular probes in aquatic samples. J. Microbiol. Methods 32:225‐236.
   Yamaguchi, N. and Nasu, M. 1997. Flow cytometric analysis of bacterial respiratory and enzymatic activity in the natural aquatic environment. J. Appl. Microbiol. 83:43‐52.
   Yentsch, C.M. and Horan, P.K. (eds.). 1989. Cytometry in Aquatic Sciences. Cytometry (special issue) 10:497‐672.
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