Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry
Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry
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DOI：
10.1128/aem.02750-06
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发表时间：
2007-05-01
影响因子：
4.4
通讯作者：
Egli, Thomas
中科院分区：
文献类型：
--
作者：
Berney, Michael;Hammes, Frederik;Egli, Thomas
市售的LIVE/DEAD BacLight试剂盒在微生物学各个领域的研究人员中越来越受欢迎。它与流式细胞术结合使用带来了关于如何解释LIVE/DEAD染色结果的新问题。中间状态，通常难以检测与落射荧光显微镜，是一种常见的现象时，该测定用于流式细胞术，仍然缺乏理由。这里显示，碘化丙啶与绿色荧光总核酸染色剂组合应用于大肠杆菌、沙门氏菌鼠伤寒血清型、福氏志贺菌和淡水细菌群落的UVA照射细胞导致清晰且独特的流式细胞术染色模式。革兰氏阴性菌E.在大肠杆菌以及两种肠道病原体中，该模式可能与中间细胞状态的存在有关，该中间细胞状态的特征在于细菌外膜受到的特异性损伤程度。这一假设得到以下事实的支持：EDTA处理的未辐照细胞表现出相同的染色特性。相反，在缺乏外膜的革兰氏阳性粪肠球菌中没有观察到这种模式。我们的观察为LIVE/DEAD染色增加了一个新的方面，到目前为止，人们认为LIVE/DEAD染色仅依赖于细胞质膜的通透性。
The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with How cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.