title: Propidium monoazide treatment to distinguish between live and dead methanogens in pure cultures and environmental samples.
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.
authors.affiliation: GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Telegrafenberg, 14473 Potsdam, Germany.
authors.forename: Janine
authors.initials: J
authors.lastname: Heise
authors.affiliation: GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Telegrafenberg, 14473 Potsdam, Germany.
authors.forename: Marcella
authors.initials: M
authors.lastname: Nega
authors.affiliation: GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Telegrafenberg, 14473 Potsdam, Germany.
authors.forename: Mashal
authors.initials: M
authors.lastname: Alawi
authors.affiliation: GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Telegrafenberg, 14473 Potsdam, Germany. Electronic address: Dirk.Wagner@gfz-potsdam.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Wagner
mesh_terms: D001386:Azides / Q000737:chemistry / Q000494:pharmacology*; D001431:Bacteriological Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D004783:Environmental Microbiology; D019605:Euryarchaeota / Q000145:classification* / Q000187:drug effects* / Q000235:genetics; D006812:Humic Substances / Q000032:analysis; D007364:Intercalating Agents / Q000737:chemistry; D050296:Microbial Viability; D008856:Microscopy, Fluorescence / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D011419:Propidium / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D012987:Soil / Q000737:chemistry; D012988:Soil Microbiology
chemical_list: D001386:Azides; D004269:DNA, Bacterial; D006812:Humic Substances; D007364:Intercalating Agents; D012987:Soil; C533957:propidium monoazide; D011419:Propidium
keywords: Extracellular DNA; LIVE/DEAD(®) BacLight™ kit; Live/dead differentiation; Membrane-compromised cells; Methanogenic archaea; Propidium monoazide (PMA); Viable cells