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Applied and Environmental Microbiology, August 2007, p. 5111-5117, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.02987-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Andreas Nocker,1* Priscilla Sossa-Fernandez,2 Mark D. Burr,1 and Anne K. Camper1,3

Center for Biofilm Engineering, Montana State University, Bozeman, Montana 59717,1 Universidad de Antofagasta, Antofagasta, Chile,2 Department of Civil Engineering, Montana State University, Bozeman, Montana 597173

Received 22 December 2006/ Accepted 14 June 2007

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.

* Corresponding author. Mailing address: Montana State University, Center for Biofilm Engineering, 366 EPS Building, P.O. Box 173980, Bozeman, MT 59717-3980. Phone: (406) 994-1849. Fax: (406) 994-6098. E-mail: anocker{at}erc.montana.edu

{triangledown} Published ahead of print on 22 June 2007.

Applied and Environmental Microbiology, August 2007, p. 5111-5117, Vol. 73, No. 16
0099-2240/07/$08.00+0     doi:10.1128/AEM.02987-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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