In situ cultivation of previously uncultivable microorganisms using the ichip
In situ cultivation of previously uncultivable microorganisms using the ichip
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DOI：
10.1038/nprot.2017.074
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发表时间：
2017-10-01
期刊：
影响因子：
14.8
通讯作者：
Epstein, Slava S.
中科院分区：
文献类型：
--
作者：
Berdy, Brittany;Spoering, Amy L.;Epstein, Slava S.
大多数微生物物种仍未培养，修改人工营养介质只会增加可培育性。我们认为，培养需求未知的物种的另一种方法是使用自然产生的生长因子组合。为了实现这一点，我们将从不同环境样本中提取的细胞放入扩散室，然后再返回自然孵化，从而将培养转移到微生物的自然栖息地。通过将小室缩小，每个小室只放入一到几个细胞，我们就可以在无菌培养中一步培养和分离微生物。我们把这个培育平台称为‘隔离芯片’，或‘iChip’。根据这项研究，这个平台已经被证明可以将微生物回收率从5倍提高到300倍。此外，它还提供了一组独特的微生物，这些微生物是标准培养方法无法获得的。在这里，我们以构建和应用ICHIPS用于土壤细菌的环境培养为例，提供了一个简单的方案；该方案包括：(I)制备iChip；(Ii)收集环境样本；(Iii)连续稀释细胞并将它们加载到iChip中；(Iv)将iChip返回环境中进行培养；(V)回收iChip并收获生长的材料；以及(Vi)驯化iChip衍生的菌落，以便在实验室中生长。IChip的完全组装和部署是一个相对简单的过程，根据经验，大约需要2-3小时。在原位孵化之后，iChip的取回和内容物的处理将类似于1-4小时，这取决于使用的特定程序。
Most microbial species remain uncultivated, and modifying artificial nutrient media brings only an incremental increase in cultivability. We reasoned that an alternative way to cultivate species with unknown requirements is to use naturally occurring combinations of growth factors. To achieve this, we moved cultivation into the microbes' natural habitat by placing cells taken from varying environmental samples into diffusion chambers, which are then returned to nature for incubation. By miniaturizing the chambers and placing only one to several cells into each chamber, we can grow and isolate microorganisms in axenic culture in one step. We call this cultivation platform the `isolation chip', or 'ichip'. This platform has been shown to increase microbial recovery from 5- to 300-fold, depending on the study. Furthermore, it provides access to a unique set of microbes that are inaccessible by standard cultivation. Here we provide a simple protocol for building and applying ichips for environmental cultivation of soil bacteria as an example; the protocol consists of (i) preparing the ichip; (ii) collecting an environmental sample; (iii) serially diluting cells and loading them into the ichip; (iv) returning the ichip to the environment for incubation; (v) retrieving the ichip and harvesting grown material; and (vi) domestication of the ichip-derived colonies for growth in the laboratory. The ichip's full assembly and deployment is a relatively simple procedure that, with experience, takes similar to 2-3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take similar to 1-4 h, depending on which specific procedures are used.