1887

Abstract

Oligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5′-end or enzymically at the 3′-end. Following specific hybridization of these probes to intracellular rRNA molecules, the hybrids were detected with anti-DIG F fragments labelled with fluorescent dyes. The antibody fragments penetrated through the bacterial cell periphery and specifically bound to their antigens. Probe-conferred and non-specific fluorescence per cell were quantified by flow cytometry and compared to values obtained with end-labelled fluorescent probes. The DIG reporter molecules could also be detected in whole fixed cells by antibodies labelled with either alkaline phosphatase or horseradish peroxidase. The penetration of the large antibody–enzyme complexes into the cells required lysozyme/EDTA treatment prior to the hybridization and has so far only been achieved for Gramnegative bacteria. This technique has the potential for significant signal amplification as compared to the fluorescently end-labelled oligonucleotides hitherto used for single cell identification in microbial ecology. Moreover, it can be used instead of fluorescent assays in natural samples showing autofluorescence.

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1991-12-01
2024-04-23
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References

  1. Amann R. I., Krumholz L., Stahl D. A. 1990a; Fluorescent-oligonucleotide probing of whole cells for determinative, phylo-genetic, and environmental studies in microbiology. Journal of Bacteriology 111:762–770
    [Google Scholar]
  2. Amann R. I., Binder B. J., Olson R. J., Chisholm S. W., Devereux R., Stahl D. A. 1990b; Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analysing mixed microbial populations. Applied and Environmental Microbiology 56:1919–1925
    [Google Scholar]
  3. Amann R., Springer N., Ludwig W., Gortz H.-D., Schleifer K.-H. 1991; Identification in situ and phylogeny of uncultured bacterial endosymbionts. Nature, London 351:161–164
    [Google Scholar]
  4. Bohlool B. B., Schmidt E. L. 1980; The immunofluorescence approach in microbial ecology. Advances in Microbial Ecology 4:203–241
    [Google Scholar]
  5. Brock T. D. 1987; The study of microorganisms in situ: progress and problems. Symposia of the Society for General Microbiology 41:1–17
    [Google Scholar]
  6. Colwell R. R., Brayton P. R., Grimes D. J., Roszak D. R., Huq S. A., Palmer L. M. 1985; Viable but nonculturable Vibrio cholerae and related pathogens in the environment: implications for the release of genetically engineered microorganisms. Biotechnology 3:817–820
    [Google Scholar]
  7. Currin C. A., Paerl H. W., Suba G. K., Alberte R. S. 1990; Immunofluorescence detection and characterization of N2-fixing microorganisms from aquatic environments. Limnology and Ocean-ography 35:59–71
    [Google Scholar]
  8. DeLong E. F., Wickham G. S., Pace N. R. 1989; Phylogenetic stains: ribosomal RNA-based probes for the identification of single microbial cells. Science 243:1360–1363
    [Google Scholar]
  9. Fliermans C. B., Bohlool B. B., Schmidt E. L. 1974; Autecological study of the chemoautotroph Nitrobacter by immunofluorescence. Applied Microbiology 30:676–684
    [Google Scholar]
  10. Giovannoni S. J., DeLong E. F., Olsen G. J., Pace N. R. 1988; Phylogenetic group-specific oligonucleotide probes for identifi-cation of single microbial cells. Journal of Bacteriology 170:720–726
    [Google Scholar]
  11. Giovannoni S. J., Britschgi T. B., Moyer C. L., Field K. G. 1990; Genetic diversity in Sargasso Sea bacterioplankton. Nature, London 345:60–63
    [Google Scholar]
  12. Heiles B. J., Genersch E., Kessler C., Neumann R., Eggers H. J. 1988; In situ hybridization with digoxigenin-labelled DNA of human papillomaviruses (HPV16/18) on HeLa and SiHa cells. Biotechniques 6:978–981
    [Google Scholar]
  13. Hopfl P., Ludwig W., Schleifer K.-H., Larsen N. 1989; The 23S ribosomal RN A higher-order structure of Pseudomonas cepacia and other prokaryotes. European Journal of Biochemistry 185:355–364
    [Google Scholar]
  14. Hsu S. M., Soban E. 1982; Color modification of diamino-benzidine (DAB) precipitation by metallic ions and its application for double immunochemistry. Journal of Histochemistry and Cytochemistry 30:1079–1082
    [Google Scholar]
  15. Jannasch H. W., Jones G. E. 1959; Bacterial populations in seawater as determined by different methods of enumeration. Limnology and Oceanography 4:128–139
    [Google Scholar]
  16. Langer P. R., Waldrop A. A., Ward D. C. 1981; Enzymatic synthesis of biotin-labelled polynucleotides: novel nucleic acid affinity probes. Proceedings of the National Academy of Sciences of the United States of America 786633–6637
    [Google Scholar]
  17. Leary J. J., Brigati D. J., Ward D. C. 1983; Rapid and sensitive colorimetric method for visualizing biotin-labelled DNA probes hybridized to DNA and RNA immobilized on nitrocellulose:bio-plots. Proceedings of the National Academy of Sciences of the United States of America 804045–4049
    [Google Scholar]
  18. Lewis D. L., Gattie D. K. 1991; The ecology of quiescent microbes. ASM News 57:27–32
    [Google Scholar]
  19. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  20. Olsen G. J., Lane D. J., Giovannoni S. J., Pace N. R., Stahl D. A. 1986; Microbial ecology and evolution: a ribosomal RNA approach. Annual Review of Microbiology 40:337–365
    [Google Scholar]
  21. Relman D. A., Loutit J. S., Schmidt T. M., Falkow S., Tompkins L. S. 1990; The agent of bacillary angiomatosis: an approach to the identification of uncultured pathogens. New England Journal of Medicine 323:1573–1580
    [Google Scholar]
  22. Saylor G. S., Layton A. C. 1990; Environmental application of nucleic acid hybridization. Annual Reviews of Microbiology 44:625–648
    [Google Scholar]
  23. Stahl D. A., Flesher B., Mansfield H. R., Montgomery L. 1988; Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Applied and Environmental Microbiology 54:1079–1084
    [Google Scholar]
  24. Stahl D. A., Amann R. I. 1991; Development and application of nucleic acid probes in bacterial systematics. In Sequencing and Hybridization Techniques in Bacterial Systematics205–248 Stackebrandt E., Goodfellow M. Chichester: John Wiley and Sons;
    [Google Scholar]
  25. Torsvik V., Goksoyr J., Daae F. L. 1990; High diversity in DNA of soil bacteria. Applied and Environmental Microbiology 56:782–787
    [Google Scholar]
  26. Tsien H. C., Bratina B. J., Tsuji K., Hanson R. S. 1990; Use of oligodeoxynucleotide signature probes for identification of physiological groups of methylotrophic bacteria. Applied and Environmental Microbiology 56:2858–2865
    [Google Scholar]
  27. Ward D., Weller R., Bateson M. M. 1990; 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature, London 345:63–65
    [Google Scholar]
  28. Weller R., Ward D. M. 1989; Selective recovery of 16S rRNA sequences from natural microbial communities in the form of cDNA. Applied and Environmental Microbiology 55:1818–1822
    [Google Scholar]
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