Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Ferrari, Belinda C.; Gillings, Michael R.

doi:10.1128/aem.02407-08

Cited by 37 publications

(25 citation statements)

References 34 publications

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“…The fact that the overwhelming part of microbial diversity remains uncultivated and unexplored presents exciting opportunities for basic and applied discoveries. The renewed interest in microbial cultivation (1,5,7,8,14,15,33,42) has led to the development of several innovative approaches to bring new species into culture. Most of these approaches share one basic strategy: to mimic the environment of target organisms.…”

Section: Discussionmentioning

confidence: 99%

Use of Ichip for High-Throughput In Situ Cultivation of “Uncultivable” Microbial Species

Nichols

Cahoon

Trakhtenberg

et al. 2010

Appl Environ Microbiol

620

513

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One of the oldest unresolved microbiological phenomena is why only a small fraction of the diverse microbiological population grows on artificial media. The "uncultivable" microbial majority arguably represents our planet's largest unexplored pool of biological and chemical novelty. Previously we showed that species from this pool could be grown inside diffusion chambers incubated in situ, likely because diffusion provides microorganisms with their naturally occurring growth factors. Here we utilize this approach and develop a novel high-throughput platform for parallel cultivation and isolation of previously uncultivated microbial species from a variety of environments. We have designed and tested an isolation chip (ichip) composed of several hundred miniature diffusion chambers, each inoculated with a single environmental cell. We show that microbial recovery in the ichip exceeds manyfold that afforded by standard cultivation, and the grown species are of significant phylogenetic novelty. The new method allows access to a large and diverse array of previously inaccessible microorganisms and is well suited for both fundamental and applied research.It has been known for over a century that the overwhelming majority of microbial species do not grow on synthetic media in vitro and remain unexplored (13,32,37,39,40,43). The rRNA and metagenomics approaches demonstrated a spectacular diversity of these uncultivated species (11, 21, 25-27, 30, 36). Accessing this "missing" microbial diversity is of significant interest for both basic and applied sciences and has been recognized as one of the principal challenges for microbiology today (12,29,41). In recent years, technical advances in cultivation methodologies have recovered a diverse set of ecologically relevant species (1,3,5,7,15,20,24,28,33,42). However, by and large the gap between microbial diversity in nature and that in culture collections remains unchanged, and most microbial phyla still have no cultivable representatives (25,29). Earlier, we developed a novel method of in situ cultivation of environmental microorganisms inside diffusion chambers (15). The rationale for such an approach was that diffusion would provide cells inside the chamber with naturally occurring growth components and enable those species that grew in nature at the time of the experiment to also grow inside the diffusion chambers. Expectedly, this method yields a rate of microbial recovery many times larger than those of standard techniques. Even so, this method is laborious and does not allow an efficient, high-throughput isolation of microbial species en masse. This limits the method's applicability, for example, in the drug discovery effort. Here we transform this methodology into a high-throughput technology platform for massively parallel cultivation of "uncultivable" species. Capitalizing on earlier microfluidics methods developed for microbial storage and screening (4, 16), we have designed and tested an isolation chip, or ichip for short, which consists of hundreds of miniature ...

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Section: Discussionmentioning

confidence: 99%

Use of Ichip for High-Throughput In Situ Cultivation of “Uncultivable” Microbial Species

Nichols

Cahoon

Trakhtenberg

et al. 2010

Appl Environ Microbiol

620

513

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“…In a similar study, Ferrari and coworkers had cultured microcolonies of rare soil bacteria by growing them on filters suspended on soil slurry in the absence of added nutrients (22). Micromanipulation was used to grow one isolate in pure culture, and a follow-up study showed that these microcolonies could be reliably micromanipulated for downstream cultivation (23).…”

Section: Approaches To Culturing the Missing Bacterial Diversitymentioning

confidence: 99%

Growing Unculturable Bacteria

2012

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The bacteria that can be grown in the laboratory are only a small fraction of the total diversity that exists in nature. At all levels of bacterial phylogeny, uncultured clades that do not grow on standard media are playing critical roles in cycling carbon, nitrogen, and other elements, synthesizing novel natural products, and impacting the surrounding organisms and environment. While molecular techniques, such as metagenomic sequencing, can provide some information independent of our ability to culture these organisms, it is essentially impossible to learn new gene and pathway functions from pure sequence data. A true understanding of the physiology of these bacteria and their roles in ecology, host health, and natural product production requires their cultivation in the laboratory. Recent advances in growing these species include coculture with other bacteria, recreating the environment in the laboratory, and combining these approaches with microcultivation technology to increase throughput and access rare species. These studies are unraveling the molecular mechanisms of unculturability and are identifying growth factors that promote the growth of previously unculturable organisms. This minireview summarizes the recent discoveries in this area and discusses the potential future of the field. What is an unculturable bacterium? While at first glance, there appears to be a contradiction in the title of this review, in this context, "unculturable" indicates that current laboratory culturing techniques are unable to grow a given bacterium in the laboratory. That all organisms must be growing in their natural environment is axiomatic; that many we cannot currently grow will be cultured in the future is certain. Therefore, "unculturable" does not mean "can never be cultured" but, rather, signifies that we lack critical information on their biology, and this presents both challenges and opportunities. These opportunities are the chance to learn the molecular principles behind this recalcitrant growth, allowing us to add that information to our repertoire of microbiological techniques and gaining access to previously hidden metabolic diversity that will provide new natural products and reveal factors that can contribute to both ecological balance and host health. This review examines the recent approaches that microbiologists are employing to convert currently unculturable bacteria into cultured isolates in the laboratory while concurrently beginning to discover the mechanisms behind their apparent unculturability.

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“…Some media that are traditionally used for soil microbiological studies returned low viable counts and did not result in the isolation of members of rarely isolated groups. Like the Soil extract agar as used by [2] and [5]. Newly developed media in contrast resulted in high viable counts and in the isolation of members of rarely isolated group regardless of the inoculums size [8].…”

Section: Discussionmentioning

confidence: 99%

“…The effect of growth media and incubation time were investigated in viable counts as described by [5], [9]and [10]. Some media that are traditionally used for soil microbiological studies returned low viable counts and did not result in the isolation of members of rarely isolated groups.…”

Section: Discussionmentioning

confidence: 99%

Effect of Growth Media and Incubation Time on the Culturability of Soil Bacteria

Chikere¹,

Udochukwu²

2014

IOSRJPBS

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Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Cited by 37 publications

References 34 publications

Use of Ichip for High-Throughput In Situ Cultivation of “Uncultivable” Microbial Species

Use of Ichip for High-Throughput In Situ Cultivation of “Uncultivable” Microbial Species

Growing Unculturable Bacteria

Effect of Growth Media and Incubation Time on the Culturability of Soil Bacteria

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