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

Janine Heise, Marcella Nega, Mashal Alawi, Dirk Wagner

GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Telegrafenberg, 14473 Potsdam, Germany.

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