Heise Janine, Nega Marcella, Alawi Mashal, Wagner Dirk
GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Telegrafenberg, 14473 Potsdam, Germany.
GFZ German Research Centre for Geosciences, Helmholtz Centre Potsdam, Section Geomicrobiology, Telegrafenberg, 14473 Potsdam, Germany.
J Microbiol Methods. 2016 Feb;121:11-23. doi: 10.1016/j.mimet.2015.12.002. Epub 2015 Dec 4.
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.
在调查人类健康的临床试验以及对培养物和自然环境中的微生物群落进行分析时,区分活的、具有潜在活性的群落与死细胞是一项重大挑战。DNA嵌入染料单叠氮碘化丙啶(PMA)能够在DNA提取前立即选择性地屏蔽死的、细胞膜受损细胞的DNA。在本研究中,我们首次评估了PMA处理对培养物和富含颗粒的环境样品中产甲烷古菌的效果。通过显微镜分析,我们证实了LIVE/DEAD(®) BacLight™试剂盒对产甲烷古菌的适用性,并证明了在PMA存在的情况下产甲烷菌完整细胞膜的维持。尽管观察到PMA处理产甲烷古菌的效率存在菌株特异性差异,但我们开发了一种使用130 μM PMA和5分钟蓝光LED光光激活的优化程序。结果表明,PMA处理的有效性很大程度上取决于沉积物/土壤的质地:富含淤泥和粘土的沉积物在所有浓度下都是一个挑战,而对于低颗粒负荷的沙土(总悬浮固体(TSS)≤200 mg mL(-1)),成功抑制来自细胞膜受损的死细胞的DNA是可能的。总之,我们提出了两种策略来克服富含颗粒的环境样品中由浊度效应(屏蔽)导致的PMA光激活不足的问题,即(i)稀释富含颗粒的样品和(ii)在PMA处理之前将细胞和游离DNA从沉积物中分离出来。这两种策略有望成为区分复杂环境样品中活细胞和游离DNA的可用选项。
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