This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pernthaler, A.
Right arrow Articles by Amann, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pernthaler, A.
Right arrow Articles by Amann, R.
Agricola
Right arrow Articles by Pernthaler, A.
Right arrow Articles by Amann, R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 2004, p. 5426-5433, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5426-5433.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Simultaneous Fluorescence In Situ Hybridization of mRNA and rRNA in Environmental Bacteria

Annelie Pernthaler* and Rudolf Amann

Max-Planck-Institut für Marine Mikrobiologie, Bremen, Germany

Received 23 January 2004/ Accepted 20 May 2004

We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.

* Corresponding author. Mailing address: Max-Planck-Institut für Marine Mikrobiologie, Celsiusstraße 1, D-28359 Bremen, Germany. Phone: 49 421 2028934. Fax: 49 421 2028580. E-mail: aperntha{at}mpi-bremen.de.

Applied and Environmental Microbiology, September 2004, p. 5426-5433, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5426-5433.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hoshino, T., Yilmaz, L. S., Noguera, D. R., Daims, H., Wagner, M. (2008). Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH. Appl. Environ. Microbiol. 74: 5068-5077 [Abstract] [Full Text]  
  • De Long, S. K., Kinney, K. A., Kirisits, M. J. (2008). Prokaryotic Suppression Subtractive Hybridization PCR cDNA Subtraction, a Targeted Method To Identify Differentially Expressed Genes. Appl. Environ. Microbiol. 74: 225-232 [Abstract] [Full Text]  
  • Smolina, I., Lee, C., Frank-Kamenetskii, M. (2007). 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 [Abstract] [Full Text]  
  • Maruyama, F., Tani, K., Kenzaka, T., Yamaguchi, N., Nasu, M. (2006). Quantitative Determination of Free-DNA Uptake in River Bacteria at the Single-Cell Level by In Situ Rolling-Circle Amplification. Appl. Environ. Microbiol. 72: 6248-6256 [Abstract] [Full Text]  
  • Kalyuzhnaya, M. G., Zabinsky, R., Bowerman, S., Baker, D. R., Lidstrom, M. E., Chistoserdova, L. (2006). Fluorescence In Situ Hybridization-Flow Cytometry-Cell Sorting-Based Method for Separation and Enrichment of Type I and Type II Methanotroph Populations.. Appl. Environ. Microbiol. 72: 4293-4301 [Abstract] [Full Text]  
  • Maruyama, F., Kenzaka, T., Yamaguchi, N., Tani, K., Nasu, M. (2005). Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification. Appl. Environ. Microbiol. 71: 7933-7940 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»