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J Microbiol Methods. 2016 Feb;121:11-23. doi: 10.1016/j.mimet.2015.12.002. Epub 2015 Dec 4.

Propidium monoazide treatment to distinguish between live and dead methanogens in pure cultures and environmental samples.

Author information

1
GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Telegrafenberg, 14473 Potsdam, Germany.
2
GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Telegrafenberg, 14473 Potsdam, Germany. Electronic address: Dirk.Wagner@gfz-potsdam.de.

Abstract

In clinical trials investigating human health and in the analysis of microbial communities in cultures and natural environments, it is a substantial challenge to differentiate between living, potentially active communities and dead cells. The DNA-intercalating dye propidium monoazide (PMA) enables the selective masking of DNA from dead, membrane-compromised cells immediately before DNA extraction. In the present study, we evaluated for the first time a PMA treatment for methanogenic archaea in cultures and particle-rich environmental samples. Using microscopic analyses, we confirmed the applicability of the LIVE/DEAD(®) BacLight™ kit to methanogenic archaea and demonstrated the maintenance of intact cell membranes of methanogens in the presence of PMA. Although strain-specific differences in the efficiency of PMA treatment to methanogenic archaea were observed, we developed an optimal procedure using 130 μM PMA and 5min of photo-activation with blue LED light. The results showed that the effectiveness of the PMA treatment strongly depends on the texture of the sediment/soil: silt and clay-rich sediments represent a challenge at all concentrations, whereas successful suppression of DNA from dead cells with compromised membranes was possible for low particle loads of sandy soil (total suspended solids (TSS)≤200 mg mL(-1)). Conclusively, we present two strategies to overcome the problem of insufficient light activation of PMA caused by the turbidity effect (shielding) in particle-rich environmental samples by (i) dilution of the particle-rich sample and (ii) detachment of the cells and the free DNA from the sediment prior to a PMA treatment. Both strategies promise to be usable options for distinguishing living cells and free DNA in complex environmental samples.

KEYWORDS:

Extracellular DNA; LIVE/DEAD(®) BacLight™ kit; Live/dead differentiation; Membrane-compromised cells; Methanogenic archaea; Propidium monoazide (PMA); Viable cells

PMID:
26656002
DOI:
10.1016/j.mimet.2015.12.002
[Indexed for MEDLINE]

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