Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques

Nature Reviews Microbiology volume 6, pages 339–348 (2008)Cite this article

Key Points

  • Specialized microorganisms catalyse central steps of global element cycling, such as nitrogen fixation or the mineralization of organic matter. There is an urgent need for the development of new methods for in situ microbial analysis, which originates from the restricted morphological diversity of prokaryotes and the limited usefulness of cultivation-based methods for quantifying species and genera at a spatial resolution that is relevant for microorganisms. Fluorescence in situ hybridization (FISH) enables reliable quantification of microbial populations in complex environmental samples.

  • FISH probes that target large taxonomic groups, such as the Bacteria, Archaea and Eukarya domains or the Alpha-, Beta- and Gammaproteobacteria classes are popular. Owing to their broad specificity, these probes can be used to analyse samples from many different environments that range from marine and freshwater environments to sediments and soils. They also facilitate an initial, rapid assessment of the dominance of certain taxa in particular environments. Most of these group-specific probes were published more than 10 years ago, when the ribosomal RNA (rRNA) database was less than 10% of its current size.

  • We address the question: which of these old probes are still valid? We checked the probes thoroughly against the comprehensive rRNA datasets of the SILVA project. The good news is that most probes can still be used for initial identification and quantification of microbial populations.

  • Failure to detect cells — that is, a false-negative FISH result — can be due to lack of cell permeabilization, low cellular ribosome content or low efficiency of probe binding based on the higher-order structure of the rRNA.

  • The new, more sensitive FISH assays have the greatest impact in oligotrophic environments, where the indigenous microbiota has low ribosome content, and in samples in which the background fluorescence hampers reliable quantification of less-frequent populations. With good microscopes, even populations of a relative abundance of 1 in 1,000 cells can be accurately quantified.

  • FISH enables studies of microorganisms in their natural contexts. Metagenomics cannot substitute for the information that can be gained by visualizing the identity and activity of single microbial cells in situ. Rather, it will make available huge sequence datasets that will help in improving existing probe sets and facilitate the development of new probes.

Abstract

The ribosomal-RNA (rRNA) approach to microbial evolution and ecology has become an integral part of environmental microbiology. Based on the patchy conservation of rRNA, oligonucleotide probes can be designed with specificities that range from the species level to the level of phyla or even domains. When these probes are labelled with fluorescent dyes or the enzyme horseradish peroxidase, they can be used to identify single microbial cells directly by fluorescence in situ hybridization. In this Review, we provide an update on the recent methodological improvements that have allowed more reliable quantification of microbial populations in situ in complex environmental samples, with a particular focus on the usefulness of group-specific probes in this era of ever-growing rRNA databases.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Basic steps of fluorescence in situ hybridization.
Figure 2: The principle of CARD–FISH (catalysed reported deposition–fluorescence in situ hybridization).
Figure 3: Current specificities of domain-specific probes.

Similar content being viewed by others

References

  1. Pedros-Alio, C. Marine microbial diversity: can it be determined? Trends Microbiol. 14, 257–263 (2006). An important paper on the extent of microbial diversity and the consequences of the ways it can be studied.

    Article  CAS  PubMed  Google Scholar 

  2. Sogin, M. L. et al. Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proc. Natl Acad. Sci. USA 103, 12115–12120 (2006). Showed that microbial diversity can be studied by high-throughput sequencing of rRNA tags.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Handelsman, J. Metagenomics: application of genomics to uncultured microorganisms. Microbiol. Mol. Biol. Rev. 68, 669–685 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Venter, J. C. et al. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304, 66–74 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. DeLong, E. F. et al. Community genomics among stratified microbial assemblages in the ocean's interior. Science 311, 496–503 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Rusch, D. B. et al. The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific. PLoS Biol. 5, e77 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Amann, R. I., Ludwig, W. & Schleifer, K. H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143–169 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Wagner, M., Horn, M. & Daims, H. Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes. Curr. Opin. Microbiol. 6, 302–309 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. DeLong, E. F., Wickham, G. S. & Pace, N. R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243, 1360–1363 (1989).

    Article  CAS  PubMed  Google Scholar 

  10. Amann, R. I., Krumholz, L. & Stahl, D. A. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172, 762–770 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Fox, G. E., Pechman, K. R. & Woese, C. R. Comparative cataloging of 16S ribosomal ribonucleic acid: molecular approach to procaryotic systematics. Int. J. Syst. Bacteriol. 27, 44–57 (1977).

    Article  CAS  Google Scholar 

  12. Woese, C. R. Bacterial evolution. Microbiol. Rev. 51, 221–271 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Handelsman, J., Rondon, M. R., Brady, S. F., Clardy, J. & Goodman, R. M. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem. Biol. 5, R245–R249 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Olsen, G. J., Lane, D. J., Giovannoni, S. J., Pace, N. R. & Stahl, D. A. Microbial ecology and evolution: a ribosomal rRNA approach. Annu. Rev. Microbiol. 40, 337–365 (1986).

    Article  CAS  PubMed  Google Scholar 

  15. Wallner, G., Fuchs, B., Spring, S., Beisker, W. & Amann, R. Flow sorting of microorganisms for molecular analysis. Appl. Environ. Microbiol. 63, 4223–4231 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Sekar, R., Fuchs, B. M., Amann, R. & Pernthaler, J. Flow sorting of marine bacterioplankton after fluorescence in situ hybridization. Appl. Environ. Microbiol. 70, 6210–6219 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Yilmaz, L. S. & Noguera, D. R. Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization. Appl. Environ. Microbiol. 70, 7126–7139 (2004). Outlined a mechanistic approach to the problem of hybridization efficiency in FISH.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Roller, C., Wagner, M., Amann, R., Ludwig, W. & Schleifer, K.-H. In situ probing of Gram-positive bacteria with high DNA G+C content using 23S rRNA-targeted oligonucleotides. Microbiology 140, 2849–2858 (1994).

    Article  CAS  PubMed  Google Scholar 

  19. Burggraf, S. et al. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes. Appl. Environ. Microbiol. 60, 3112–3119 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Pernthaler, A., Pernthaler, J. & Amann, R. in Molecular Microbial Ecology Manual (eds Kowalchuk, G., de Bruijn, F. J., Head, I. M., Akkermans, A. D. L. & van Elsas, J. D.) 711–726 (Kluwer Academic, Dordrecht, 2004).

    Google Scholar 

  21. Silverman, A. P. & Kool, E. T. Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA. Nucleic Acids Res. 33, 4978–4986 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bremer, H. & Dennis, P. P. in Escherichia coli and Salmonella: Cellular and Molecular Biology (eds Neidhart, F. C. et al.) 1553–1569 (ASM, Washington DC, 1996).

    Google Scholar 

  23. Glöckner, F. O. et al. An in situ hybridization protocol for detection and identification of planktonic bacteria. Syst. Appl. Microbiol. 19, 403–406 (1996).

    Article  Google Scholar 

  24. Morris, R. M. et al. SAR11 clade dominates ocean surface bacterioplankton communities. Nature 420, 806–810 (2002). FISH-based quantification of the “bugsthat rule the waves”.

    Article  CAS  PubMed  Google Scholar 

  25. Pernthaler, A., Preston, C. M., Pernthaler, J., DeLong, E. F. & Amann, R. Comparison of fluorescently labeled oligonucleotide and polynucleotide probes for the detection of pelagic marine bacteria and archaea. Appl. Environ. Microbiol. 68, 661–667 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Trebesius, K., Amann, R., Ludwig, W., Mühlegger, K. & Schleifer, K.-H. Identification of whole fixed bacterial cells with nonradioactive 23S rRNA-targeted polynucleotide probes. Appl. Environ. Microbiol. 60, 3228–3235 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Schönhuber, W., Fuchs, B., Juretschko, S. & Amann, R. Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification. Appl. Environ. Microbiol. 63, 3268–3273 (1997).

    PubMed  PubMed Central  Google Scholar 

  28. Pernthaler, A., Pernthaler, J. & Amann, R. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol. 68, 3094–3101 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ishii, K., Mussmann, M., MacGregor, B. J. & Amann, R. An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments. FEMS Microbiol. Ecol. 50, 203–212 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Pernthaler, A. & Amann, R. Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria. Appl. Environ. Microbiol. 70, 5426–5433 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zwirglmaier, K., Ludwig, W. & Schleifer, K.-H. Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization — RING –FISH. Mol. Microbiol. 51, 89–96 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Lanoil, B. D. & Giovannoni, S. Identification of bacterial cells by chromosomal painting. Appl. Environ. Microbiol. 63, 1118–1123 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Jensen, R. B. & Shapiro, L. The Caulobacter crescentus smc gene is required for cell cycle progression and chromosome segregation. Proc. Natl Acad. Sci. USA 96, 10661–10666 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Smolina, I., Lee, C. & Frank-Kamenetskii, M. Detection of low-copy-number genomic DNA sequences in individual bacterial cells by using peptide nucleic acid-assisted rolling-circle amplification and fluorescence in situ hybridization. Appl. Environ. Microbiol. 73, 2324–2328 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Maruyama, F., Kenzaka, T., Yamaguchi, N., Tani, K. & Nasu, M. Visualization and enumeration of bacteria carrying a specific gene sequence by in situ rolling circle amplification. Appl. Environ. Microbiol. 71, 7933–7940 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Fuchs, B. M. et al. Flow cytometric analysis of the in-situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 64, 4973–4982 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Behrens, S. et al. In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes. Appl. Environ. Microbiol. 69, 1748–1758 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Behrens, S., Fuchs, B. M., Mueller, F. & Amann, R. Is the in situ accessibility of the 16S rRNA of Escherichia coli for Cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30S ribosomal subunit? Appl. Environ. Microbiol. 69, 4935–4941 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Fuchs, B. M., Glöckner, F. O., Wulf, J. & Amann, R. Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 66, 3603–3607 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Yilmaz, L. S., Okten, H. E. & Noguera, D. R. Making all parts of the 16s rRNA of Escherichia coli accessible in situ to single DNA oligonucleotides. Appl. Environ. Microbiol. 72, 733–744 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Torimura, M. et al. Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base. Anal. Sci. 17, 155–160 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Behrens, S., Fuchs, B. M. & Amann, R. The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes. System. Appl. Microbiol. 27, 565–572 (2004).

    Article  CAS  Google Scholar 

  43. Pruesse, E. et al. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 35, 7188–7196 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Daims, H., Bruhl, A., Amann, R., Schleifer, K. -H. & Wagner, M. The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22, 434–444 (1999).

    Article  CAS  PubMed  Google Scholar 

  45. Manz, W., Amann, R., Ludwig, W., Wagner, M. & Schleifer, K.-H. Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15, 593–600 (1992).

    Article  Google Scholar 

  46. Buchholz-Cleven, B., Rattunde, B. & Straub, K. Screening for genetic diversity of isolates of anaerobic Fe(II)-oxidizing bacteria using DGGE and whole-cell hybridization. Syst. Appl. Microbiol. 20, 301–309 (1997).

    Article  Google Scholar 

  47. Yeates, C., Saunders, A. M., Crocetti, G. R. & Blackall, L. L. Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation. Microbiology 149, 1239–1247 (2003).

    Article  CAS  PubMed  Google Scholar 

  48. Siyambalapitiya, N. & Blackall, L. L. Discrepancies in the widely applied GAM42a fluorescence in situ hybridisation probe for Gammaproteobacteria. FEMS Microbiol. Lett. 242, 367–373 (2005).

    Article  CAS  PubMed  Google Scholar 

  49. Friedrich, U., Naismith, M. M., Altendorf, K. & Lipski, A. Community analysis of biofilters using fluorescence in situ hybridization including a new probe for the Xanthomonas branch of the class Proteobacteria. Appl. Environ. Microbiol. 65, 3547–3554 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Neef, A. Anwendung der in situ-Einzelzell-Identifizierung von Bakterien zur Populationsanalyse in komplexen mikrobiellen Biozönosen. Thesis, Univ. Munich (1997).

    Google Scholar 

  51. Manz, W., Amann, R., Ludwig, W., Vancanneyt, M. & Schleifer, K.-H. Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga– flavobacter–bacteroides in the natural environment. Microbiology 142, 1097–1106 (1996).

    Article  CAS  PubMed  Google Scholar 

  52. O'Sullivan, L. A., Weightman, A. J. & Fry, J. C. New degenerate Cytophaga–Flexibacter–Bacteroides-specific 16S ribosomal DNA-targeted oligonucleotide probes reveal high bacterial diversity in River Taff epilithon. Appl. Environ. Microbiol. 68, 201–210 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Neef, A., Amann, R., Schlesner, H. & Schleifer, K.-H. Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes. Microbiology 144, 3257–3266 (1998).

    Article  CAS  PubMed  Google Scholar 

  54. Musat, N. et al. Microbial community structure of sandy intertidal sediments in the North Sea, Sylt-Romo Basin, Wadden Sea. Syst. Appl. Microbiol. 29, 333–348 (2006).

    Article  PubMed  Google Scholar 

  55. Loy, A., Maixner, F., Wagner, M. & Horn, M. probeBase — an online resource for rRNA-targeted oligonucleotide probes: new features 2007. Nucleic Acids Res. 35, D800–D804 (2007). Comprehensive online resource for primer and probe sequences that provides various online tools for checking specificity and coverage.

    Article  CAS  PubMed  Google Scholar 

  56. Crocetti, G., Murto, M. & Bjornsson, L. An update and optimisation of oligonucleotide probes targeting methanogenic Archaea for use in fluorescence in situ hybridisation (FISH). J. Microbiol. Methods 65, 194–201 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Lücker, S. et al. Improved 16S rRNA-targeted probe set for analysis of sulfate-reducing bacteria by fluorescence in situ hybridization. J. Microbiol. Methods 69, 523–528 (2007).

    Article  CAS  PubMed  Google Scholar 

  58. Karner, M. B., DeLong, E. F. & Karl, D. M. Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature 409, 507–510 (2001).

    Article  CAS  PubMed  Google Scholar 

  59. Kuypers, M. M. M. et al. Massive nitrogen loss from the Benguela upwelling system through anaerobic ammonium oxidation. Proc. Natl Acad. Sci. USA 102, 6478–6483 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kuypers, M. M. M. et al. Anaerobic ammonium oxidation by anammox bacteria in the Black Sea. Nature 422, 608–611 (2003).

    Article  CAS  PubMed  Google Scholar 

  61. Niemann, H. et al. Novel microbial communities of the Haakon Mosby mud volcano and their role as a methane sink. Nature 443, 854–858 (2006). An interdisciplinary habitat study on a marine mud volcano that quantified and localized populations that catalyse either the aerobic or anaerobic oxidation of methane.

    Article  CAS  PubMed  Google Scholar 

  62. Knittel, K., Losekann, T., Boetius, A., Kort, R. & Amann, R. Diversity and distribution of methanotrophic archaea at cold seeps. Appl. Environ. Microbiol. 71, 467–479 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Lösekann, T. et al. Diversity and abundance of aerobic and anaerobic methane oxidizers at the Haakon Mosby mud volcano, Barents Sea. Appl. Environ. Microbiol. 73, 3348–3362 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Maixner, F. et al. Nitrite concentration influences the population structure of Nitrospira-like bacteria. Environ. Microbiol. 8, 1487–1495 (2006).

    Article  CAS  PubMed  Google Scholar 

  65. Daims, H., Lucker, S. & Wagner, M. daime, a novel image analysis program for microbial ecology and biofilm research. Environ. Microbiol. 8, 200–213 (2006).

    Article  CAS  PubMed  Google Scholar 

  66. Dubilier, N. et al. Endosymbiotic sulphate-reducing and sulphide-oxidizing bacteria in an oligochaete worm. Nature 411, 298–302 (2001).

    Article  CAS  PubMed  Google Scholar 

  67. Gieseke, A., Bjerrum, L., Wagner, M. & Amann, R. Structure and activity of multiple nitrifying bacterial populations co-existing in a biofilm. Environ. Microbiol. 5, 355–369 (2003).

    Article  CAS  PubMed  Google Scholar 

  68. Gieseke, A., Nielsen, J. L., Amann, R., Nielsen, P. H. & de Beer, D. In situ substrate conversion and assimilation by nitrifying bacteria in a model biofilm. Environ. Microbiol. 7, 1392–1404 (2005).

    Article  CAS  PubMed  Google Scholar 

  69. Tujula, N. A. et al. A CARD–FISH protocol for the identification and enumeration of epiphytic bacteria on marine algae. J. Microbiol. Methods 65, 604–607 (2006).

    Article  CAS  PubMed  Google Scholar 

  70. Woebken, D., Fuchs, B. M., Kuypers, M. M. M. & Amann, R. Potential interactions of particle-associated anammox bacteria with bacterial and archaeal partners in the Namibian upwelling system. Appl. Environ. Microbiol. 73, 4648–4657 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Pernthaler, A., Pernthaler, J., Schattenhofer, M. & Amann, R. Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton. Appl. Environ. Microbiol. 68, 5728–5736 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Oerther, D. B., Pernthaler, J., Schramm, A., Amann, R. & Raskin, L. Monitoring precursor 16S rRNAs of Acinetobacter spp. in activated sludge wastewater treatment systems. Appl. Environ. Microbiol. 66, 2154–2165 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Regan, J. M., Oldenburg, P. S., Park, H. D., Harrington, G. W. & Noguera, D. R. Simultaneous determination of bacterial viability and identity in biofilms using ethidium monoazide and fluorescent in situ hybridization. Water Sci. Technol. 47, 123–128 (2003).

    Article  CAS  PubMed  Google Scholar 

  74. Lee, N. et al. Combination of fluorescent in situ hybridization and microautoradiography — a new tool for structure–function analyses in microbial ecology. Appl. Environ. Microbiol. 65, 1289–1297 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Ouverney, C. C. & Fuhrman, J. A. Combined microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ. Appl. Environ. Microbiol. 65, 1746–1752 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Alonso, C. & Pernthaler, J. Incorporation of glucose under anoxic conditions by bacterioplankton from coastal North Sea surface waters. Appl. Environ. Microbiol. 71, 1709–1716 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Cottrell, M. T. & Kirchman, D. L. Contribution of major bacterial groups to bacterial biomass production (thymidine and leucine incorporation) in the Delaware estuary. Limnol. Oceanogr. 48, 168–178 (2003).

    Article  Google Scholar 

  78. Huang, W. E. et al. Raman–FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function. Environ. Microbiol. 9, 1878–1889 (2007). Showed that single microbial cells can be analysed for identity and function by a combination of stable-isotope Raman spectroscopy and FISH.

    Article  CAS  PubMed  Google Scholar 

  79. Orphan, V. J., House, C. H., Hinrichs, K. U., McKeegan, K. D. & Delong, D. F. Methane-consuming archaea revealed by directly coupled isotopic and phylogenetic analysis. Science 293, 484–487 (2001).

    Article  CAS  PubMed  Google Scholar 

  80. Lechene, C. P., Luyten, Y., McMahon, G. & Distel, D. L. Quantitative imaging of nitrogen fixation by individual bacteria within animal cells. Science 317, 1563–1566 (2007).

    Article  CAS  PubMed  Google Scholar 

  81. Kuypers, M. M. M. Sizing up the uncultivated majority. Science 317, 1510–1511 (2007).

    Article  CAS  PubMed  Google Scholar 

  82. Li, T. et al. Simultaneous analysis of microbial identity and function using NanoSIMS. Environ. Microbiol. 10, 580–588 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Pernthaler, J. & Amann, R. Fate of heterotrophic microbes in pelagic habitats: focus on populations. Microbiol. Mol. Biol. Rev. 69, 440–461 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Parkes, R. J. et al. Deep sub-seafloor prokaryotes stimulated at interfaces over geological time. Nature 436, 390–394 (2005).

    Article  CAS  PubMed  Google Scholar 

  85. Schippers, A. et al. Prokaryotic cells of the deep sub-seafloor biosphere identified as living bacteria. Nature 433, 861–864 (2005).

    Article  CAS  PubMed  Google Scholar 

  86. Biddle, J. F. et al. Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru. Proc. Natl Acad. Sci. USA 103, 3846–3851 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Glöckner, F. O., Fuchs, B. M. & Amann, R. Bacterioplankton composition in lakes and oceans: a first comparison based on fluorescence in situ hybridization. Appl. Environ. Microbiol. 65, 3721–3726 (1999).

    PubMed  PubMed Central  Google Scholar 

  88. Teske, A. et al. Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria. J. Bacteriol. 176, 6623–6630 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Jaspers, E. & Overmann, J. Ecological significance of microdiversity: identical 16S rRNA gene sequences can be found in bacteria with highly divergent genomes and ecophysiologies. Appl. Environ. Microbiol. 70, 4831–4839 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. West, N. J. et al. Closely related Prochlorococcus genotypes show remarkably different depth distributions in two oceanic regions as revealed by in situ hybridization using 16S rRNA-targeted oligonucleotides. Microbiology 147, 1731–1744 (2001).

    Article  CAS  PubMed  Google Scholar 

  91. Zwirglmaier, K. et al. Basin-scale distribution patterns of picocyanobacterial lineages in the Atlantic Ocean. Environ. Microbiol. 9, 1278–1290 (2007). Reported the basin-scale distribution patterns of cyanobacteria in the Atlantic Ocean.

    Article  CAS  PubMed  Google Scholar 

  92. Egholm, M. et al. PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules. Nature 365, 566–568 (1993).

    Article  CAS  PubMed  Google Scholar 

  93. Worden, A. Z., Chisholm, S. W. & Binder, B. J. In situ hybridization of Prochlorococcus and Synechococcus (marine cyanobacteria) spp. with rRNA-targeted peptide nucleic acid probes. Appl. Environ. Microbiol. 66, 284–289 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Obika, S. et al. Synthesis of 2′-O,4′-C-methyleneuridineand -cytidine. Novel bicyclic nucleosides having a fixed C3,-endo sugar puckering. Tetrahedron Lett. 38, 8735–8738 (1997).

    Article  CAS  Google Scholar 

  95. Koshkin, A. A. et al. LNA (locked nucleic acids): synthesis of the adenine, cytosine, guanine, 5′-methylcytosine, thymidine and uracil bicyclonucleoside monomers, oligomerisation, and unprecendented nucleic acid recognition. Tetrahedron 54, 3607–3630 (1998).

    Article  CAS  Google Scholar 

  96. Kubota, K., Ohashi, A., Imachi, H. & Harada, H. Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes. Appl. Environ. Microbiol. 72, 5311–5317 (2006). Showed that modified oligonucleotide probes which contain locked nucleotides have potential to improve in situ hybridization.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Schramm, A., Fuchs, B. M., Nielsen, J. L., Tonolla, M. & Stahl, D. A. Fluorescence in situ hybridization of 16S rRNA gene clones (Clone–FISH) for probe validation and screening of clone libraries. Environ. Microbiol. 4, 713–720 (2002).

    Article  CAS  PubMed  Google Scholar 

  98. Yilmaz, L. S. & Noguera, D. R. Development of thermodynamic models for simulating probe dissociation profiles in fluorescence in situ hybridization. Biotechnol. Bioeng. 96, 349–363 (2007).

    Article  CAS  PubMed  Google Scholar 

  99. Stahl, D. A. & Amann, R. in Nucleic Acid Techniques in Bacterial Systematics (eds Stackebrandt, E. & Goodfellow, M.) 205–248 (John Wiley & Sons, Chichester, 1991).

    Google Scholar 

  100. Amann, R. I. et al. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919–1925 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  101. Brosius, J., Dull, T. J., Sleeter, D. D. & Noller, H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J. Mol. Biol. 148, 107–127 (1981).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank the Max Planck Society and the Deutsche Forschungsgemeinschaft for funding. K. Knittel and F.O. Glöckner are acknowledged for helpful discussions and help with the SILVA rRNA databases.

Author information

Authors and Affiliations

  1. Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Celsiusstr. 1, Bremen, D-28359, Germany

    Rudolf Amann & Bernhard M. Fuchs

Authors
  1. Rudolf Amann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bernhard M. Fuchs
    View author publications

    You can also search for this author in PubMed Google Scholar

Supplementary information

Supplementary information S1 (figure)

Probe matching was carried out with the “weighted probe match” functionality of the probe match module of ARB1. (PDF 394 kb)

Supplementary information S2 (figure)

Probe matching was carried out with the “weighted probe match” functionality of the probe match module of ARB102. (PDF 476 kb)

Related links

Related links

DATABASES

Entrez Genome Project

Caulobacter crescentus

Escherichia coli

Metallosphaera sedula

Rhodopirellula baltica

Saccharomyces cerevisiae

SAR11

FURTHER INFORMATION

Rudolf Amann's homepage

Bernhard M. Fuchs's homepage

ARB project

NanoSIMS

probeBase

probeCheck

SILVA

Glossary

Microbial autecology

The science of identifying and quantifying distinct microorganisms and their activities.

Epifluorescence microscopy

The most common fluorescence-microscopy arrangement, in which excitation light is applied through the objective, which is also used for viewing the fluorescent specimen. The method excites and collects fluorescence throughout the cell, but has poor depth discrimination.

Flow cytometry

A technique that measures the fluorescence of individual cells as they pass through a laser beam in a water jet.

CARD–FISH

A method for increasing the sensitivity of FISH by using horseradish peroxidase-labelled oligonucleotide probes. The horseradish peroxidase catalyses the deposition of tyramine molecules, which results in fluorescent-signal amplification at the site of probe hybridization.

RING–FISH

A FISH method that allows single genes to be detected. High concentrations of a multiple-labelled polynucleotide probe and extended hybridization times are used. A network of probe molecules is formed, which is detected as a ring-shaped signal.

Rolling-circle amplification

A mode of DNA replication that uses a circular DNA molecule as a template to produce concatamers of linear DNA molecules.

MAR–FISH

A method in which microautoradiography — detection of the activity of individual cells by assaying the incorporation of a radiolabelled substrate — is combined with single-cell identification by FISH.

Raman microscopy

A microscopic method that allows Raman spectra within individual microbial cells to be recorded and can be used to detect the incorporation of substrate that has been labelled with stable isotope.

NanoSIMS

Nano-scale secondary-ion mass spectrometry. A high-resolution method that allows multi-isotope imaging.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Amann, R., Fuchs, B. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol 6, 339–348 (2008). https://doi.org/10.1038/nrmicro1888

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrmicro1888

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing