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. 1996 Apr;62(4):1416–1423. doi: 10.1128/aem.62.4.1416-1423.1996

Development and field application of a quantitative method for examining natural assemblages of protists with oligonucleotide probes.

E L Lim 1, D A Caron 1, E F Delong 1
PMCID: PMC167908  PMID: 8919803

Abstract

A fluorescent in situ hybridization method that uses rRNA-targeted oligonucleotide probes for counting protists in cultures and environmental water samples is described. Filtration, hybridization, and enumeration of fixed cells with biotinylated eukaryote-specific probes and fluorescein isothiocyanate-conjugated avidin were performed directly on 0.4-microns-pore-size polycarbonate filters of Transwell cell culture inserts (Costar Corp., Cambridge, Mass.). Counts of various species of cultured protists by this probe hybridization method were not significantly different from counts obtained by the 4',6-diamidino-2-phenylindole (DAPI) and acridine orange (AO) staining methods. However, counts of total nanoplankton (TNAN) based on probe hybridizations in several field samples and in samples collected from a mesocosm experiment were frequently higher than TNAN counts obtained by staining with DAPI or AO. On the basis of these results, 25 to 70% of the TNAN determined with probes were not detectable by DAPI or AO staining. The underestimation of TNAN abundances in samples stained with DAPI or AO was attributed to the existence of small nanoplanktonic cells which could be detected with probes but not DAPI or AO and the difficulty associated with distinguishing DAPI- or AO-stained protists attached to or embedded in aggregates. We conclude from samples examined in this study that enumeration of TNAN with oligonucleotide probes provides estimates of natural TNAN abundances that are at least as high as (and in some cases higher than) counts obtained with commonly employed fluorochrome stains. The quantitative in situ hybridization method we have described here enables the direct enumeration of free-living protists in water samples with oligonucleotide probes. When combined with species-specific probes, this method will enable quantitative studies of the abundance and distribution of specific protistan taxa.

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Selected References

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  1. Amann R. I., Binder B. J., Olson R. J., Chisholm S. W., Devereux R., Stahl D. A. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol. 1990 Jun;56(6):1919–1925. doi: 10.1128/aem.56.6.1919-1925.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amann R. I., Krumholz L., Stahl D. A. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol. 1990 Feb;172(2):762–770. doi: 10.1128/jb.172.2.762-770.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amann R. I., Ludwig W., Schleifer K. H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995 Mar;59(1):143–169. doi: 10.1128/mr.59.1.143-169.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DeLong E. F., Wickham G. S., Pace N. R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 1989 Mar 10;243(4896):1360–1363. doi: 10.1126/science.2466341. [DOI] [PubMed] [Google Scholar]
  5. DiChristina T. J., DeLong E. F. Design and application of rRNA-targeted oligonucleotide probes for the dissimilatory iron- and manganese-reducing bacterium Shewanella putrefaciens. Appl Environ Microbiol. 1993 Dec;59(12):4152–4160. doi: 10.1128/aem.59.12.4152-4160.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Giovannoni S. J., DeLong E. F., Olsen G. J., Pace N. R. Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol. 1988 Feb;170(2):720–726. doi: 10.1128/jb.170.2.720-726.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hicks R. E., Amann R. I., Stahl D. A. Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences. Appl Environ Microbiol. 1992 Jul;58(7):2158–2163. doi: 10.1128/aem.58.7.2158-2163.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hobbie J. E., Daley R. J., Jasper S. Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl Environ Microbiol. 1977 May;33(5):1225–1228. doi: 10.1128/aem.33.5.1225-1228.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lim E. L., Amaral L. A., Caron D. A., DeLong E. F. Application of rRNA-based probes for observing marine nanoplanktonic protists. Appl Environ Microbiol. 1993 May;59(5):1647–1655. doi: 10.1128/aem.59.5.1647-1655.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Manz W., Amann R., Szewzyk R., Szewzyk U., Stenström T. A., Hutzler P., Schleifer K. H. In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. Microbiology. 1995 Jan;141(Pt 1):29–39. doi: 10.1099/00221287-141-1-29. [DOI] [PubMed] [Google Scholar]
  11. Raskin L., Poulsen L. K., Noguera D. R., Rittmann B. E., Stahl D. A. Quantification of methanogenic groups in anaerobic biological reactors by oligonucleotide probe hybridization. Appl Environ Microbiol. 1994 Apr;60(4):1241–1248. doi: 10.1128/aem.60.4.1241-1248.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sherr B. F., Sherr E. B., Fallon R. D. Use of monodispersed, fluorescently labeled bacteria to estimate in situ protozoan bacterivory. Appl Environ Microbiol. 1987 May;53(5):958–965. doi: 10.1128/aem.53.5.958-965.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Simon N., LeBot N., Marie D., Partensky F., Vaulot D. Fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes to identify small phytoplankton by flow cytometry. Appl Environ Microbiol. 1995 Jul;61(7):2506–2513. doi: 10.1128/aem.61.7.2506-2513.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sogin M. L., Gunderson J. H. Structural diversity of eukaryotic small subunit ribosomal RNAs. Evolutionary implications. Ann N Y Acad Sci. 1987;503:125–139. doi: 10.1111/j.1749-6632.1987.tb40603.x. [DOI] [PubMed] [Google Scholar]

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