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Chapter 32 : Cultivating Microorganisms from Dilute Aquatic Environments: Melding Traditional Methodology with New Cultivation Techniques and Molecular Methods

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Cultivating Microorganisms from Dilute Aquatic Environments: Melding Traditional Methodology with New Cultivation Techniques and Molecular Methods, Page 1 of 2

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Abstract:

In 1993, Don Button and colleagues broke new ground with their theoretical description and subsequent application of an extinction culturing method for aquatic microbial cells. Their approach was specifically tailored toward the isolation of oligotrophic cells by taking advantage of the unique ability those cells have to effectively compete at very low nutrient concentrations. The volume of water required for medium preparation and inoculation depends on the concentration of microbial cells in the raw sample and the design and goals of the particular cultivation experiment. As with any technique involving the cultivation or manipulation of live microorganisms, measures taken to minimize and eliminate the risk of contamination are crucial for a successful experiment. In seawater processing the entire collection container is acid and heat sterilized. The tangential flow filtration (TFF) unit and peristaltic pump are set up as directed by the manufacturer. Sterile seawater is stored at 4°C to minimize potential contamination and should be inspected for continued sterility via microscopy immediately prior to each use by following either the DAPI (4',6'-diamidino-2-phenylindole) staining protocol or a similar procedure. Coupled with rapid, sensitive, and specific screening methods such as fluorescence in situ hybridization (FISH), T-RFLP, or screening for the presence of specific functional genes via PCR, the high-throughput culturing (HTC) method allows scientists to tailor extensive cultivation efforts that target maximum overall diversity or focus their efforts on narrowly defined target microorganisms.

Citation: Becker J, Brandon M, Rappé M. 2007. Cultivating Microorganisms from Dilute Aquatic Environments: Melding Traditional Methodology with New Cultivation Techniques and Molecular Methods, p 399-406. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch32

Key Concept Ranking

Restriction Fragment Length Polymorphism
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Denaturing Gradient Gel Electrophoresis
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Microbial Ecology
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FIGURE 1

Flow diagram illustrating HTC procedures.

Citation: Becker J, Brandon M, Rappé M. 2007. Cultivating Microorganisms from Dilute Aquatic Environments: Melding Traditional Methodology with New Cultivation Techniques and Molecular Methods, p 399-406. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch32
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References

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