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Applied and Environmental Microbiology
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Microbial Ecology

Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology

Andreas Nocker, Priscilla Sossa-Fernandez, Mark D. Burr, Anne K. Camper
Andreas Nocker
1Center for Biofilm Engineering, Montana State University, Bozeman, Montana 59717
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  • For correspondence: anocker@erc.montana.edu
Priscilla Sossa-Fernandez
2Universidad de Antofagasta, Antofagasta, Chile
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Mark D. Burr
1Center for Biofilm Engineering, Montana State University, Bozeman, Montana 59717
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Anne K. Camper
1Center for Biofilm Engineering, Montana State University, Bozeman, Montana 59717
3Department of Civil Engineering, Montana State University, Bozeman, Montana 59717
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DOI: 10.1128/AEM.02987-06
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ABSTRACT

One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.

  • Copyright © 2007 American Society for Microbiology
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Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology
Andreas Nocker, Priscilla Sossa-Fernandez, Mark D. Burr, Anne K. Camper
Applied and Environmental Microbiology Aug 2007, 73 (16) 5111-5117; DOI: 10.1128/AEM.02987-06

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Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology
Andreas Nocker, Priscilla Sossa-Fernandez, Mark D. Burr, Anne K. Camper
Applied and Environmental Microbiology Aug 2007, 73 (16) 5111-5117; DOI: 10.1128/AEM.02987-06
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KEYWORDS

bacteria
Propidium
RNA, Ribosomal, 16S

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