Abstract
Traditional techniques for assessment of microbial numbers and activity generally lack the specificity required for risk assessment following environmental release of genetically engineered microbial inocula. Immunological and molecular-based techniques, such as DNA probing and genetic tagging, were initially used to determine the presence or absence of microorganisms in environmental samples. Increasingly they are being developed for quantification of populations of specific organisms, either indigenous or introduced, in the environment. In addition, they are being used to quantify the activity of particular organisms or groups of organisms, greatly extending the range of techniques available to the microbial ecologist. This article reviews the use of traditional techniques for the quantification of microbial population size and activity and the application of molecular techniques, including DNA probing, genetic marking, use of fluorescent probes, and quantitative PCR, in combination with advanced cell detection techniques such as confocal laser scanning microscopy and flow cytometry.
Similar content being viewed by others
References
Gustafsson, K. and Jansson, J. K. (1993) Ecological risk assessment of the deliberate release of genetically modified microorganisms.Ambio. 22, 236–242.
Jansson, J. K. (1995) Tracking genetically engineered microorganisms in nature.Curr. Opinion Biotech. 6, 275–283.
Torsvik, V., Goksøyr, J., and Daae, F. L. (1990) High diversity in DNA of soil bacteria.Appl. Environ. Microbiol. 56, 782–787.
Bloem, J., Veninga, M., and Shepherd, J. (1995) Fully automatic determination of soil bacterium numbers, cell volumes, and frequencies of dividing cells by confocal laser scanning microscopy and image analysis.Appl. Environ. Microbiol. 61, 926–936.
Torrella, F. and Morita, R. Y. (1981) Microcultural study of bacterial size changes and micro colony and ultramicrocolony formation by heterotrophic bacteria in seawater.Appl. Environ. Microbiol. 41, 518–527.
Nybroe, O., Christoffersen, K., and Riemann, B. (1992) Survival ofBacillus licheniformis in seawater model-ecosystems.Appl. Environ. Microbiol. 58, 252–259.
Schloter, M., Borlinghaus, R., Bode, W., and Hartmann, A. (1993) Direct identification and localization ofAzospirillum in the rhizosphere of wheat using fluorescence-labelled monoclonal antibodies and confocal scanning laser microscopy.Microscopy 171, 173–177.
DeLong, E. F., Wickham, G. S., and Pace, N. R. (1989) Phylogenetic strains: ribosomal RNA-based probes for the identification of single cells.Science 243, 1360–1363.
Manz, W., Szewzyk, U., Ericsson, P., Amann, R., Schieffer, K.-H., and Stenström, T.-A. (1993)In situ identification of bacteria in drinking water and adjoining biofilms by hybridization with 16S and 23S rRNA-directed fluorescent oligonucleotide probes.Appl. Environ. Microbiol. 59, 2293–2298.
Assmus, B., Hutzler, P., Kirchhof, G., Amann, R., Lawrence, J. K., and Hartmann, A. (1995)In situ localization ofAzospirillum brasilense in the rhizosphere of wheat with fluorescently labeled rRNA-targeted oligonucleotide probes and scanning confocal laser microscopy.Appl. Environ. Microbiol. 61, 1013–1019.
Amann, K. I., Binder, B. J., Olson, R. J., Chisholm, S. W., Devereux, R., and Stahl, D. A. (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.Appl. Environ. Microbiol. 56, 1919–1925.
Spring, S., Amann, K., Ludwig, W., Schleifer, K.-H., and Petersen, N. (1992) Phylogenetic diversity and identification of nonculturable magnetotactic bacteria.System. Appl. Microbiol. 15, 116–122.
Stephen, J. R., McCaig, A. E., Smith, Z., Prosser, J. I., and Embley, T. M. (1997) Molecular diversity of soil and marine 16S rDNA sequences related to β-subgroup ammonia oxidising bacteria.Appl. Environ. Microbiol., submitted.
Amann, R. I., Ludwig, W., and Schleifer, K. H. (1995) Phylogenetic identification andin situ detection of individual cells without cultivation.Microbiol. Rev. 59, 143–169.
Boye, M., Ahl, T., and Molin, S. (1995) Application of a strain-specific rRNA oligonucleotide probe targetingPseudomonas fluorescens Ag1 in a mesocosm study of bacterial release into the environment.Appl. Environ. Microbiol. 61, 1384–1390.
Hahn, D., Amann, R. I., Ludwig, W., Akkermans, A. D. L., and Schleifer, K.-H. (1992) Detection of micro-organisms in soil afterin situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides.J. Gen. Microbiol. 138, 879–887.
Prosser, J. I. (1994) Molecular marker systems for the detection of genetically modified microorganisms in the environment.Microbiology 140, 5–17.
Jansson, J. K. (1995) Tracking genetically engineered microorganisms in nature.Curr. Opinion Biotech. 6, 275–283.
Silcock, D., Waterhouse, K. N., Glover, L. A., Prosser, J. I., and Killham, K. (1992) Detection of a single genetically modified bacterial cell in soil by using charge coupled device-enhanced microscopy.Appl. Environ. Microbiol. 58, 2444–2448.
Viles, C. J. and Sieracki, M. E. (1992) Measurement of marine picoplankton cell size using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.Appl. Environ. Microbiol. 58, 584–592.
Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier, M. J. (1992) Primary structure of theAequorea victoria green-fluorescent protein.Gene 111, 229–233.
Chalfie, M., Tu, Y., Euskircher, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression.Science 263, 802–805.
Heim, K., Prasher, D. C., and Tsien, R. Y. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein.Proc. Natl. Acad. Sci. USA 91, 12,501–12,504.
Crameri, A., Whitehorn, E. A., Tate, E. and Stemmer, W. P. C. (1996) Improved green fluorescent protem by molecular evolution using DNA shuffling.Nat. Biotech. 14, 315–319.
Helm, R. and Tsien, K. Y. (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer.Curr. Biol. 6, 178–182.
Tombolini, K., Unge, A., Davey, M. E., de Bruijn, F. J., and Jansson, J. K. (1997) Flow cytometric and microscopic analysis ofgfp-tagged,Pseudomonas fluorescens bacteria.FEMS Microbiol. Ecol. 22, 17–28.
Caldwell, D. E., Korber, D. R., and Lawrence, J. R., (1992) confocal laser microscopy and digital image analysis in microbial ecology.Adv. Microb. Ecol. 12, 1–67.
Lawrence, J. R., Korber, D. K., Hoyle, B. D., Costerton, J. W., and Caldwell, D. E. (1991) Optical sectioning of microbial biofilms.J. Bacteriol. 173, 6558–6567.
Ghiorse, W. C., Miller, D. N., Sandoli, R. L., and Siering, P. L. (1996) Applications of laser scanning microscopy for analysis of aquatic microhabitats.Microsc. Res. Tech. 33, 73–86.
Porter, J., Diaper, J., Edwards, C., and Pickup, K. (1995) Direct measurements of natural planktonic bacterial community viability by flow-cytometry.Appl. Environ. Microbiol. 61, 2783–2786.
Page, S. and Burns, K. G. (1991) Flow cytometry as a means of enumerating bacteria introduced into soil.Soil Biol. Biochem. 23, 1025–1028.
Christensen, H., Olsen, R. A., and Bakken, L. K. (1995) Flow cytometric measurements of cell volume and DNA content during culture of indigenous soil bacteria.Microbial. Ecol. 29, 49–62.
Wallner, G., Erhart, K., and Amann, K (1995) Flow cytometric analysis of activated sludge with rRNA-targeted probes.Appl. Environ. Microbiol. 61, 1859–1866.
Porter, J., Edwards, C., Morgan, J. A. W., and Pickup, R. (1993) Rapid automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting.Appl. Environ. Microbiol. 59, 3327–3333.
Roszak, D. B. and Colwell, R. R. (1987) Survival strategies of bacteria in the natural environment.Microbiol. Rev. 51, 365–379.
Drahos, D., Hemming, B. C., and McPherson, S. (1986) Tracking recombinant organisms in the environment: β-galactosidase as a selectable non-antibiotic marker for fluorescent pseudomonads.Bio/Technology 4, 439–444.
Hofte, M., Mergeay, M., and Verstraete, W. (1990) Marking theRhizopseudomonas strain 7NSK2 with a Mu d(lac) element for ecological studies.Appl. Environ. Microbiol. 56, 1046–1052.
Ryder, M. H., Pankhurst, C. E., Rovira, A. D., Correll, K. L., and Keller, K. M. O. (1994) Detection of introduced bacteria in the rhizosphere using maker genes and DNA probes, inMolecular ecology of the Rhizosphere (O'Gara, F., Dowling, B., and Boesten, B., eds.), VCH Publishers, Weinheim, Germany, pp. 29–47.
Winstanley, C., Morgan, J. A. W., Pickup, R. W., Jones, J. G., and Saunders, J. K. (1989) Differential regulation of Lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system.Appl. Environ. Microbiol. 55, 770–777.
Wipat, A., Wellington, E. M. H., and Saunders, V. A. (1991)Streptomyces marker plasmids for monitoring survival and spread of streptomycetes in soil.Appl. Environ. Microbiol. 57, 3322–3330.
Morgan, J. A. W., Winstanley, C., Pickup, K. W., Jones, J. G., and Saunders, J. K. (1989) Direct phenotypic and genotypic detection of a recombinant pseudomonad population released into lake water.Appl. Environ. Microbiol. 55, 2537–2544.
Grant, F. A., Glover, L. A., Killham, K., and Prosser, J. I. (1991), Luminescence-based viable cell enumeration ofErwinia carotovora in the soil.Soil. Biol. Biochem. 23, 1021–1024.
Cebolla, A., Vazquez, M. E., and Palomares, A. J. (1995) Expression vectors for the use of eukaryotic luciferases as bacterial markers with different colors of luminescence.Appl. Environ. Microbiol. 61, 660–668.
Thompson, I. P., Lilley, A. K., Ellis, R. J., Bramwell, P. A., and Bailey, M. J. (1995) Survival, colonization and dispersal of genetically modifiedPseudomonas fluorescens SBW25 in the phytosphere of field grown sugar beet.Bio/Technology 13, 1493–1497.
Cebolla, A., Ruiz-Berraquero, F., and Palomares, A. J. (1993) Stable tagging ofRhizobium meliloti with the firefly luciferase gene for environmental monitoring.Appl. Environ. Microbiol. 59, 2511–2519.
Flemming, C. A., Leung, K. T., Lee, H., Trevors, J. T., and Greer, C. W. (1994) Survival oflux-lac-marked biosurfactant-producingPseudomonas aeruginosa UG2L in soil monitored by nonselective plating and PCR.Appl. Environ. Microbiol. 60, 1606–1616.
Nybroe, O. (1995) Assessment of metabolic-activity of single bacterial-cells—new developments in microcolony and dehydrogenase assaysFEMS Microbiol. Ecol. 17, 77–83.
Binnerup, S. J., Jensen, D. F., Thordalchristensen, H., and Sorensen, J. (1993) Detection of viable, but non-culturablePseudomonas fluorescens, DF57 in soil using a microcolony epifluorescence technique.FEMS Microbiol. Ecol. 12, 97–105.
Belser, L. W. and Schmidt, E. L. (1978) Diversity in the ammonia oxidizing nitrifier population of a soil.Appl. Environ. Microbiol. 36, 584–588.
Fredrickson J. K., Bezdicek, D. F., and Brockman, F. J. (1988) Enumeration of Tn5 mutant bacteria in soil by using a most-probable-number DNA hybridization procedure and antibiotic-resistance.Appl. Environ. Microbiol. 54, 446–453.
Diaper, J. P. and Edwards, C. (1994) Survival ofStaphylococcus aureus in lakewater monitored by flow cytometry.Microbiology 140, 35–42.
Jepras, K. I., Carter, J., Pearson, S. C., Paul, F. E., and Wilkinson, M. J. (1995) Development of a robust flow cytometric assay for determining numbers of viable bacteria.Appl. Environ. Microbiol. 61, 2696–2701.
Webster, J. J., Hampton, G. J., Wilson, J. T., Ghiorse, W. C., and Leach, F. R. (1985) Determination of microbial cell numbers in subsurface samples.Ground Water 23, 17–25.
Kogure, K., Simidu, U., and Taga, N. (1979) A tentative direct microscopic method for counting living marine bacteria.Can. J Microbiol. 25, 415–420.
Xu, H. S., Roberts, N., Singleton, F. L., Attwell, K. W., Grimes, D. J., and Colwell, K. R. (1982) Survival and viability of nonculturableEscherichia coli andVibrio cholerae in the estuarine and marine environment.Microbial Ecol. 8, 313–323.
Heijnen, C. E., Page, S., and van Elsas, J. D. (1995) Metabolic activity ofFlavobacterium strain P25 during starvation and after introduction into bulk soil and the rhizosphere of wheat.FEMS Microbiol. Ecol. 129–140.
Poulsen, L. K., Ballard, G., and Stahl, D. A. (1993) Use of rRNA fluorescencein situ hybridization for measuring the activity of single cells in young and established biofilms.Appl. Environ. Microbiol. 59, 1354–1360.
Brock, M. L. and Brock, T. D. (1968) The application of microautoradiographic techniques to ecological studies.Mitteilungen der Iunternationale vereinigung fur theoretische und angewandte Limnologie 15, 1–29.
Fliermans, C. B. and Schmidt, E. L. (1975) Autoradiography and immunofluorecence combined for auto ecological study of single cell activity withNitrobacter.Appl. Microbiol. 30, 674–684.
Rattray, E. A. S., Prosser, J. I., Kiliham, K., and Glover, L. A. (1990) Luminescence-based nonextractive techniques forin situ detection ofEscherichia coli in soil.Appl. Environ. Microbiol. 56, 3368–3374.
Möller, A., Gustafsson, K., and Jansson, J. K. (1994) Specific monitoring by PCR amplification and bioluminescence of firefly luciferase gene-tagged bacteria added to environmental samples.FEMS Microbiol. Ecol. 15, 193–206.
Meikle, A., Killham, K., Prosser, J. I., and Glover, L. A. (1992) Luminometric measurement of population activity of genetically modifiedPseudomonas fluorescens in the soil.FEMS Microbiol. Lett. 99, 217–220.
Rattray, E. A. S., Prosser, J. I., Glover, L. A., and Killham, K. (1995) Characterization of rhizosphere colonization by luminescentEnterobacter clocae at the population and single-cell levels.Appl. Environ. Microbiol. 61, 2950–2957.
Meikle, A., Amin-Hanjani, S., Glover, L. A., Killham, K., and Prosser, J. I. (1995) The effect of matric potential on survival and activity of aPseudomonas fluorescens inoculum in soil.Soil Biol. Biochem. 27, 881–892.
Duncan, S., Glover, L. A., Killham, K., and Prosser, J. I. (1994) Luminescence-based detection of activity of starved and viable but nonculturable bacteria.Appl. Environ. Microbiol. 60, 1308–1316.
Lindow, S. E. (1995) The use of reporter genes in the study of microbial ecology.Mol. Ecol. 4, 555–566.
Bloem, J. (1995) Fluorescent staining of microbes for direct counts. inMolecular Microbial Ecology Manual (Akkermans, A. D. L., van Elsas, J. D., and de Bruijn, F. J., eds.), Kluwer Academic, Dordrecht, The Netherlands, pp. 1–12.
Christensen, H., Bakken, L.R., and Olsen, R. A. (1993) Soil bacterial DNA and biovolume profiles measured by flow-cytometry.FEMS Microbiol. Ecol. 102, 129–140.
Meikle, A., Glover, L. A., Killham, K., and Prosser, J. I. (1994) Potential luminescence as an indicator of activation of genetically modifiedPseudomonas fluorescens in liquid culture and in soil.Soil Biol. Biochem. 26, 747–755.
Möller, A., Norrby, A.-M., Gustafsson, K., and Jansson, J. K. (1995) Luminometry and PCR-based monitoring of gene-tagged cyanobacteria in Baltic Sea microcosms.FEMS Microbiol. Lett. 129, 43–50.
Zaat, S. A. J., Slegtenhorst-Eegdeman, K., Tommassen, J., Geli, V., Wijffelman, C. A., and Lugtenberg, B. J. J. (1994) Construction ofphoE-caa, a novel PCR-and immunologically detectable marker gene forPseudomonas putida.Appl. Environ. Microbiol. 60, 3965–3973.
Hwang, I. and Farrand, S. K. (1994) A novel gene tag for identifying microorganisms released into the environment.Appl. Environ. Microbiol. 60, 913–920.
van Elsas, J. D., Van Overbeek, L. S., and Fouchier, R. (1991) A specific marker,pat, for studying the fate of introduced bacteria and their DNA in soil using a combination of detection techniques.Pl. Soil 138, 49–60.
Lebarron, P. and Joux, F. (1994) Flow cytometric analysis of the cellular DNA content ofSalmonella typhimurium andAlteromonas haloplanktis during starvation and recovery in seawater.Appl. Environ. Microbiol. 60, 4345–4350.
Porter, J., Pickup, K., and Edwards, C. (1995) Flow cytometric detection of specific genes in genetically modified bacteria usingin situ polymerase chain reaction.FEMS Microbiol. Lett. 134, 51–56.
Torsvik, V. (1980) Isolation of bacterial DNA from soil.Soil Biol. Biochem. 12, 15–21.
Holben, W. E., Jansson, J. K., Chelm, B. K., and Tiedje, J. M. (1988) DNA probe method for the detection of specific microorganisms in the soil bacterial community.Appl. Environ. Microbiol. 54, 703–711.
Ogram, A., Sayler, G. S., and Barkay, T. (1988) DNA extraction and purification from sediments.J. Microb. Methods 7, 57–66.
Trevors, J. T. and van Elsas, J. D. (1995)Nucleic Acids in the Environment, Springer Lab Manual, Springer-Verlagg Berlin, Germany.
Bej, A. K., DiCesare, J. L., Haff, L., and Atlas, R. M. (1991) Detection ofEscherichia coli andShigella spp. in water by using the polymerase chain reaction and gene probes foruid.Appl. Environ. Microbiol. 57, 1013–1017.
Bej, A. K., Mahbubani, M. H., and Atlas, R. M. (1991) Detection of viableLegionella pneumaphilia in water by polymerase chain reaction and gene probe methods.Appl. Environ. Microbiol. 57, 597–600.
Recorbet, G., Picard, C., Normand, P., and Simonet, P. (1993) Kinetics of the persistence of chromosomal DNA from genetically engineeredEscherichia coli introduced into soil.Appl. Environ. Microbiol. 59, 4289–4294.
Rosado, A. S., Seldin, L., Wolters, A. C., and van Elsas, J. D. (1996) Quantitative 16S rDNA-targeted polymerase chain reaction and oligonucleotide hybridization for the detection ofPaenibacillus azotofixans in soil and the wheat rhizosphere.FEMS Microbiol. Ecol. 19, 153–164.
Jansson, J. K. and Leser, T. (1996) Quantitative PCR of environmental samples, inMolecular Microbial Ecology Manual (Akkermans A. D. L., van Elsas, J. D., and de Bruijn, F. J., eds.), Kluwer Academic, Dordrecht, The Netherlands, pp. 2.7.4.1–19.
Leser, T. D. (1995) Quantitation ofPseudomonas sp. strain B13 (FRI) in the marine environment by competitive polymerase chain reaction.J. Microbiol. Methods 22, 249–262.
Möller, A. and Jansson, J. K. (1997) Quantification of genetically-tagged cyanobacteria in Baltic Sea sediment by competitive PCR.BioTechniques 22, 512–518.
Simon, L. Lévesque, C., and Lelonde, M. (1992) Rapid quantification by PCR of endomycorrhizal flingi colonising roots.PCR Methods Applic. 2, 76–80.
Jansson, J. K., Holben, W. E., and Tiedje, J. M. (1989) Detection in soil of a deletion in an engineered DNA sequence by using DNA probes.Appl. Environ. Microbiol. 55, 3022–3025.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Jansson, J.K., Prosser, J.I. Quantification of the presence and activity of specific microorganisms in nature. Mol Biotechnol 7, 103–120 (1997). https://doi.org/10.1007/BF02761746
Issue Date:
DOI: https://doi.org/10.1007/BF02761746