In situ cultivation of previously uncultivable microorganisms using the ichip
This protocol details the construction and use of the ichip, a platform developed to isolate previously uncultivable microorganisms from a range of environmental samples, by enabling exposure to natural growth factors through in situ culture. Most microbial species remain uncultivated, and modifying...
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| Published in: | Nature protocols Vol. 12; no. 10; pp. 2232 - 2242 |
|---|---|
| Main Authors: | , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
London
Nature Publishing Group UK
01.10.2017
Nature Publishing Group |
| Subjects: | |
| ISSN: | 1754-2189, 1750-2799, 1750-2799 |
| Online Access: | Get full text |
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| Abstract | This protocol details the construction and use of the ichip, a platform developed to isolate previously uncultivable microorganisms from a range of environmental samples, by enabling exposure to natural growth factors through
in situ
culture.
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 ∼2–3 h. After
in situ
incubation, retrieval of the ichip and processing of its contents will take ∼1–4 h, depending on which specific procedures are used. |
|---|---|
| AbstractList | 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]2-3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take [similar]1-4 h, depending on which specific procedures are used. 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 ∼2-3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take ∼1-4 h, depending on which specific procedures are used. 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 ∼2-3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take ∼1-4 h, depending on which specific procedures are used.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 ∼2-3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take ∼1-4 h, depending on which specific procedures are used. Not provided. This protocol details the construction and use of the ichip, a platform developed to isolate previously uncultivable microorganisms from a range of environmental samples, by enabling exposure to natural growth factors through in situ culture.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 ∼2–3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take ∼1–4 h, depending on which specific procedures are used. 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 â^¼2-3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take â^¼1-4 h, depending on which specific procedures are used. 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]2-3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take [similar]1-4 h, depending on which specific procedures are used. Keywords: Microbial, cultivation, uncultivated species, ichip, drug discovery, growth factors, microbe, microorganism, isolation chip, in situ cultivation, high-throughput culture, culture, bacterial culture, microbial culture, soil, soil sample, seawater, environmental microbiology, antimicrobial, antimicrobial drug, antimicrobial drug discovery, in situ culture, axenic culture, single-cell culture This protocol details the construction and use of the ichip, a platform developed to isolate previously uncultivable microorganisms from a range of environmental samples, by enabling exposure to natural growth factors through in situ culture. 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 ∼2–3 h. After in situ incubation, retrieval of the ichip and processing of its contents will take ∼1–4 h, depending on which specific procedures are used. |
| Audience | Academic |
| Author | Spoering, Amy L Berdy, Brittany Epstein, Slava S Ling, Losee L |
| Author_xml | – sequence: 1 givenname: Brittany surname: Berdy fullname: Berdy, Brittany organization: Department of Biology, Northeastern University – sequence: 2 givenname: Amy L surname: Spoering fullname: Spoering, Amy L organization: NovoBiotic Pharmaceuticals – sequence: 3 givenname: Losee L surname: Ling fullname: Ling, Losee L organization: NovoBiotic Pharmaceuticals – sequence: 4 givenname: Slava S surname: Epstein fullname: Epstein, Slava S email: s.epstein@neu.edu organization: Department of Biology, Northeastern University, NovoBiotic Pharmaceuticals, LiDakSum Marine Biopharmaceutical Research Center, Ningbo University |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/29532802$$D View this record in MEDLINE/PubMed https://www.osti.gov/biblio/1539795$$D View this record in Osti.gov |
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| ContentType | Journal Article |
| Copyright | Springer Nature Limited 2017 COPYRIGHT 2017 Nature Publishing Group Copyright Nature Publishing Group Oct 2017 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2017. |
| Copyright_xml | – notice: Springer Nature Limited 2017 – notice: COPYRIGHT 2017 Nature Publishing Group – notice: Copyright Nature Publishing Group Oct 2017 – notice: Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2017. |
| CorporateAuthor | Northeastern Univ., Boston, MA (United States) |
| CorporateAuthor_xml | – name: Northeastern Univ., Boston, MA (United States) |
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| DOI | 10.1038/nprot.2017.074 |
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| Title | In situ cultivation of previously uncultivable microorganisms using the ichip |
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