Department of Vaccinology and Applied Microbiology, HZI-Helmholtz Center for Infection Research, D-38124, Braunschweig, Germany.
Water Sci Technol. 2010;61(1):9-14. doi: 10.2166/wst.2010.773.
The assessment of the physiological state of the bacteria in drinking water is a critical issue, especially with respect to the presence of pathogenic bacteria. Though molecular methods can provide insight into the taxonomic composition of the drinking water microflora, the question if a bacterial species is alive or dead still needs to be addressed. To distinguish live and dead bacteria at the taxonomic level, we combined three methods: i) a staining procedure indicating membrane-injured cells (using SYTO9 and Propidium Iodide) that is considered to distinguish between live and dead cells, ii) Fluorescence Activated Cell Sorting (FACS) of the membrane injured and intact bacteria, and iii) molecular analyses of the RNA extracted from the bacteria before and after sorting to analyse the bacterial community at the species level. By staining and FACS analysis the drinking water bacteria could be separated according to their different membrane integrities, and RNA could be extracted from the live and dead sorted bacterial fractions. 16S rRNA based fingerprints revealed a diverse bacterial community in the drinking water samples with the majority being represented by 31 identified phylotypes. Most of the phylotypes referenced belonged to the phyla Proteobacteria (Alpha-, Beta-, Gamma-), Cyanobacteria and Bacteroidetes, and were mostly related to freshwater bacteria. 90% of the total phylotypes could be recovered after FAC-Sorting; 32% of the phylotypes occurred only in the "live" sorted fraction, 21% only in the "dead" sorted fraction, and 46% occurred in both fractions.
评估饮用水中细菌的生理状态是一个关键问题，特别是在存在致病菌的情况下。虽然分子方法可以深入了解饮用水微生物群的分类组成，但仍需要解决一个细菌是活的还是死的问题。为了在分类水平上区分活细菌和死细菌，我们结合了三种方法：i）一种指示膜损伤细胞的染色程序（使用 SYTO9 和碘化丙啶），用于区分活细胞和死细胞，ii）膜损伤和完整细菌的荧光激活细胞分选（FACS），以及 iii）从分选前后的细菌中提取 RNA 进行分子分析，以分析物种水平的细菌群落。通过染色和 FACS 分析，可以根据不同的膜完整性将饮用水细菌分离，并可以从活细菌和死细菌分选部分提取 RNA。基于 16S rRNA 的指纹图谱显示，饮用水样本中存在多样的细菌群落，其中大多数由 31 种已鉴定的生物型代表。参考的生物型大多数属于变形菌门（α-、β-、γ-）、蓝细菌门和拟杆菌门，主要与淡水细菌有关。90%的总生物型可以通过 FAC-Sorting 回收；32%的生物型仅出现在“活”分选部分，21%仅出现在“死”分选部分，46%出现在两个部分。
