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首页> 外文期刊>Applied and Environmental Microbiology >Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ
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Combined Microautoradiography–16S rRNA Probe Technique for Determination of Radioisotope Uptake by Specific Microbial Cell Types In Situ

机译：结合微放射自显影技术– 16S rRNA探针技术原位确定特定微生物细胞类型的放射性同位素吸收

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摘要

We propose a novel method for studying the function of specific microbial groups in situ. Since natural microbial communities are dynamic both in composition and in activities, we argue that the microbial “black box” should not be regarded as homogeneous. Our technique breaks down this black box with group-specific fluorescent 16S rRNA probes and simultaneously determines 3H-substrate uptake by each of the subgroups present via microautoradiography (MAR). Total direct counting, fluorescent in situ hybridization, and MAR are combined on a single slide to determine (i) the percentages of different subgroups in a community, (ii) the percentage of total cells in a community that take up a radioactively labeled substance, and (iii) the distribution of uptake within each subgroup. The method was verified with pure cultures. In addition, in situ uptake by members of the α subdivision of the class Proteobacteria(α-Proteobacteria) and of the Cytophaga-Flavobacteriumgroup obtained off the California coast and labeled with fluorescent oligonucleotide probes for these subgroups showed that not only do these organisms account for a large portion of the picoplankton community in the sample examined (～60% of the universal probe-labeled cells and ～50% of the total direct counts), but they also are significant in the uptake of dissolved amino acids in situ. Nearly 90% of the total cells and 80% of the cells belonging to the α-Proteobacteria and Cytophaga-Flavobacterium groups were detectable as active organisms in amino acid uptake tests. We suggest a name for our triple-labeling technique, substrate-tracking autoradiographic fluorescent in situ hybridization (STARFISH), which should aid in the “dissection” of microbial communities by type and function.
机译：我们提出了一种新的方法来研究原位特定微生物群的功能。由于天然微生物群落在组成和活动方面都是动态的，因此我们认为微生物“黑匣子”不应被视为同质的。我们的技术使用特定于组的荧光16S rRNA探针打破了这个黑匣子，并同时通过微放射自显影（MAR）确定了每个亚组对3H底物的摄取。总直接计数，荧光原位杂交和MAR组合在一张幻灯片上，以确定（i）社区中不同亚组的百分比，（ii）社区中吸收放射性标记物质的总细胞百分比， （iii）每个亚组内摄取的分布。该方法已通过纯培养物验证。另外，从加利福尼亚海岸获得的并在加利福尼亚海岸获得的Proteobacteria（α-Proteobacteria）类和Cytophaga-Flavobacteriumgroup的α细分成员的原位摄取并用荧光寡核苷酸探针标记这些亚类，这表明这些生物不仅造成了在所研究的样本中，微微浮游生物群落的很大一部分（约60％的通用探针标记细胞和约50％的总直接计数），但它们在原位摄取溶解氨基酸方面也很重要。在氨基酸摄取测试中，可检测到约90％的总细胞和80％的属于α-变形杆菌和细胞吞噬黄杆菌属的细胞是活性生物。我们建议使用我们的三重标记技术，即底物跟踪放射自显影荧光原位杂交（STARFISH），该技术应有助于按类型和功能对微生物群落进行“解剖”。

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