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Appl Environ Microbiol. 1997 Aug; 63(8): 3268–3273.
PMCID: PMC168626

Improved sensitivity of whole-cell hybridization by the combination of horseradish peroxidase-labeled oligonucleotides and tyramide signal amplification.


The substrate fluorescein-tyramide was combined with oligonucleotide probes directly labeled with horseradish peroxidase to improve the sensitivity of in situ hybridization of whole fixed bacterial cells. Flow cytometry and quantitative microscopy of cells hybridized by this technique showed 10- to 20-fold signal amplifications relative to fluorescein-monolabeled probes. The application of the new technique to the detection of natural bacterial communities resulted in very bright signals; however, the number of detected cells was significantly lower than that detected with fluorescently monolabeled, rRNA-targeted oligonucleotide probes.

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Selected References

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  • Adams JC. Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem. 1992 Oct;40(10):1457–1463. [PubMed]
  • Amann R, Springer N, Ludwig W, Görtz HD, Schleifer KH. Identification in situ and phylogeny of uncultured bacterial endosymbionts. Nature. 1991 May 9;351(6322):161–164. [PubMed]
  • Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol. 1990 Jun;56(6):1919–1925. [PMC free article] [PubMed]
  • Amann RI, Krumholz L, Stahl DA. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol. 1990 Feb;172(2):762–770. [PMC free article] [PubMed]
  • Amann RI, Zarda B, Stahl DA, Schleifer KH. Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes. Appl Environ Microbiol. 1992 Sep;58(9):3007–3011. [PMC free article] [PubMed]
  • Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ. Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods. 1989 Dec 20;125(1-2):279–285. [PubMed]
  • Bobrow MN, Shaughnessy KJ, Litt GJ. Catalyzed reporter deposition, a novel method of signal amplification. II. Application to membrane immunoassays. J Immunol Methods. 1991 Mar 1;137(1):103–112. [PubMed]
  • Cary SC, Giovannoni SJ. Transovarial inheritance of endosymbiotic bacteria in clams inhabiting deep-sea hydrothermal vents and cold seeps. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5695–5699. [PMC free article] [PubMed]
  • Chao J, DeBiasio R, Zhu Z, Giuliano KA, Schmidt BF. Immunofluorescence signal amplification by the enzyme-catalyzed deposition of a fluorescent reporter substrate (CARD). Cytometry. 1996 Jan 1;23(1):48–53. [PubMed]
  • Hahn D, Amann RI, Zeyer J. Detection of mRNA in streptomyces cells by whole-cell hybridization with digoxigenin-labeled probes. Appl Environ Microbiol. 1993 Aug;59(8):2753–2757. [PMC free article] [PubMed]
  • Hodson RE, Dustman WA, Garg RP, Moran MA. In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities. Appl Environ Microbiol. 1995 Nov;61(11):4074–4082. [PMC free article] [PubMed]
  • Itoh T, Tomizawa J. Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H. Proc Natl Acad Sci U S A. 1980 May;77(5):2450–2454. [PMC free article] [PubMed]
  • Kerstens HM, Poddighe PJ, Hanselaar AG. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J Histochem Cytochem. 1995 Apr;43(4):347–352. [PubMed]
  • Polz MF, Distel DL, Zarda B, Amann R, Felbeck H, Ott JA, Cavanaugh CM. Phylogenetic analysis of a highly specific association between ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode. Appl Environ Microbiol. 1994 Dec;60(12):4461–4467. [PMC free article] [PubMed]
  • Porter J, Pickup R, Edwards C. Flow cytometric detection of specific genes in genetically modified bacteria using in situ polymerase chain reaction. FEMS Microbiol Lett. 1995 Dec 1;134(1):51–56. [PubMed]
  • Raap AK, van de Corput MP, Vervenne RA, van Gijlswijk RP, Tanke HJ, Wiegant J. Ultra-sensitive FISH using peroxidase-mediated deposition of biotin- or fluorochrome tyramides. Hum Mol Genet. 1995 Apr;4(4):529–534. [PubMed]
  • Roller C, Wagner M, Amann R, Ludwig W, Schleifer KH. In situ probing of gram-positive bacteria with high DNA G + C content using 23S rRNA-targeted oligonucleotides. Microbiology. 1994 Oct;140(Pt 10):2849–2858. [PubMed]
  • Trebesius K, Amann R, Ludwig W, Mühlegger K, Schleifer KH. Identification of Whole Fixed Bacterial Cells with Nonradioactive 23S rRNA-Targeted Polynucleotide Probes. Appl Environ Microbiol. 1994 Sep;60(9):3228–3235. [PMC free article] [PubMed]
  • Urdea MS, Warner BD, Running JA, Stempien M, Clyne J, Horn T. A comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res. 1988 Jun 10;16(11):4937–4956. [PMC free article] [PubMed]
  • Wagner M, Erhart R, Manz W, Amann R, Lemmer H, Wedi D, Schleifer KH. Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Appl Environ Microbiol. 1994 Mar;60(3):792–800. [PMC free article] [PubMed]
  • Wallner G, Amann R, Beisker W. Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry. 1993;14(2):136–143. [PubMed]
  • Wallner G, Erhart R, Amann R. Flow cytometric analysis of activated sludge with rRNA-targeted probes. Appl Environ Microbiol. 1995 May;61(5):1859–1866. [PMC free article] [PubMed]
  • Wessendorf MW, Brelje TC. Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas red, and cyanine 3.18. Histochemistry. 1992 Sep;98(2):81–85. [PubMed]
  • Yamaguchi N, Inaoka S, Tani K, Kenzaka T, Nasu M. Detection of specific bacterial cells with 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate and fast red TR in situ hybridization. Appl Environ Microbiol. 1996 Jan;62(1):275–278. [PMC free article] [PubMed]
  • Zarda B, Amann R, Wallner G, Schleifer KH. Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides. J Gen Microbiol. 1991 Dec;137(12):2823–2830. [PubMed]

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