Abstract
Cultivation of microorganisms is necessary for testing hypotheses generated from cultivation-independent microbial community analysis and modern “omics” techniques, yet many of the organisms identified using these methods resist cultivation with enrichment-, selection-, or solid medium-based approaches. Success in isolating some of the “most wanted” microorganisms has come from dilution-to-extinction methodology in a high-throughput format which effectively isolates cells upon inoculation and allows for competition-free growth in sterilized natural milieu or defined media. This methodology has been revolutionary to studies of marine microorganisms, where many of the most abundant taxa are also very small, slowly growing cells that are adapted to low nutrient concentrations and frequently have complicated nutrient requirements that are not easily predicted from geochemical data. Coupling dilution-to-extinction inoculation with large numbers of wells and monitoring with highly sensitive flow cytometry provides a means to bring many important and elusive microbial taxa into the laboratory and thus support experimental investigation of their physiological capabilities.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Staley JT, Konopka A (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39:321–346
Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276:734–740
Giovannoni SJ, Rappe MS, Vergin KL, Adair NL (1996) 16S rRNA genes reveal stratified open ocean bacterioplankton populations related to the Green Non-Sulfur bacteria. Proc Natl Acad Sci USA 93:7979–7984
Morris RM, Rappé MS, Connon SA et al (2002) SAR11 clade dominates ocean surface bacterioplankton communities. Nature 420:806–810
Tyson GW, Chapman J, Hugenholtz P et al (2004) Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature 428:37–43
Peng X, Jayakumar A, Ward BB (2013) Community composition of ammonia-oxidizing archaea from surface and anoxic depths of oceanic oxygen minimum zones. Front Microbiol 4:177
Wrighton KC, Thomas BC, Sharon I et al (2012) Fermentation, hydrogen, and sulfur metabolism in multiple uncultivated bacterial phyla. Science 337:1661–1665
Steindler L, Schwalbach MS, Smith DP, Chan F, Giovannoni SJ (2011) Energy starved candidatus pelagibacter ubique substitutes light-mediated ATP production for endogenous carbon respiration. PLOS One 6:e19725
Sun J, Steindler L, Thrash JC et al (2011) One carbon metabolism in SAR11 pelagic marine bacteria. PLOS One 6:e23973
Schut F, Prins RA, Gottschal JC (1997) Oligotrophy and pelagic marine bacteria: facts and fiction. Aquat Microb Ecol 12:177–202
Button DK, Schut F, Quang P, Martin R, Robertson BR (1993) Viability and isolation of marine bacteria by dilution culture: theory, procedures, and initial results. Appl Environ Microbiol 59:881–891
Giovannoni S, Stingl U (2007) The importance of culturing bacterioplankton in the ‘omics’ age. Nat Rev Micro 5:820–826
Button DK (1998) Nutrient uptake by microorganisms according to kinetic parameters from theory as related to cytoarchitecture. Microbiol Mol Biol Rev 62:636–645
Button DK, Robertson BR, Lepp PW, Schmidt TM (1998) A small, dilute-cytoplasm, high-affinity, novel bacterium isolated by extinction culture and having kinetic constants compatible with growth at ambient concentrations of dissolved nutrients in seawater. Appl Environ Microbiol 64:4467–4476
Nelson CE, Carlson CA (2012) Tracking differential incorporation of dissolved organic carbon types among diverse lineages of Sargasso Sea bacterioplankton. Environ Microbiol 14:1500–1516
Connon SA, Giovannoni SJ (2002) High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates. Appl Environ Microbiol 68:3878–3885
Stingl U, Tripp HJ, Giovannoni SJ (2007) Improvements of high-throughput culturing yielded novel SAR11 strains and other abundant marine bacteria from the Oregon coast and the Bermuda Atlantic Time Series study site. ISME J 1:361–371
Marshall KT, Morris RM (2013) Isolation of an aerobic sulfur oxidizer from the SUP05/Arctic96BD-19 clade. ISME J 7:452–455
Chisholm SW, Frankel SL, Goericke R et al (1992) Prochlorococcus marinus nov. gen. nov. sp.: an oxyphototrophic marine prokaryote containing divinyl chlorophyll a and b. Arch Microbiol 157:297–300
Song J, Oh H-M, Cho J-C (2009) Improved culturability of SAR11 strains in dilution-to-extinction culturing from the East Sea, West Pacific Ocean. FEMS Microbiol Lett 295:141–147
Rappé MS (2013) Stabilizing the foundation of the house that ‘omics builds: the evolving value of cultured isolates to marine microbiology. Curr Opin Microbiol 2013:1–7
Rappé MS, Connon SA, Vergin KL, Giovannoni SJ (2002) Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature 418:630–633
Carini P, Steindler L, Beszteri S, Giovannoni SJ (2013) Nutrient requirements for growth of the extreme oligotroph ‘Candidatus Pelagibacter ubique’ HTCC1062 on a defined medium. ISME J 7:592–602
Wright JJ, Konwar KM, Hallam SJ (2012) Microbial ecology of expanding oxygen minimum zones. Nat Rev Micro 10:381–394
Giovannoni SJ, Connon SA (2002) State of Oregon acting by and through the State Board of Higher Education on behalf of Oregon State University (Corvallis, US), assignee. High-throughput microbial culturing. US patent 6,951,714
Zobell CE, Anderson DQ (1936) Observations on the multiplication of bacteria in different volumes of stored sea water and the influence of oxygen tension and solid surfaces. Biol Bull 71:324–342
Dagg M, Benner R, Lohrenz S, Lawrence D (2004) Transformation of dissolved and particulate materials on continental shelves influenced by large rivers: plume processes. Cont Shelf Res 24:833–858
Rabalais NN, Turner RE, Justic D, Dortch Q, Wiseman JWJ, Sen Gupta BK (1996) Nutrient changes in the Mississippi River and system responses on the adjacent continental shelf. Estuaries Coasts 19:386–407
Solis RS, Powell GL (1999) Hydrography, mixing characteristics, and residence times of gulf of Mexico estuaries. In: Bianchi TS, Pennock JR, Twilley RR (eds) Biogeochemistry of gulf of mexico estuaries. Wiley, Hoboken
Thrash JC, Ahmadi S, Torok T, Coates JD (2010) Magnetospirillum bellicus sp. nov., a novel dissimilatory perchlorate-reducing alphaproteobacterium isolated from a bioelectrical reactor. Appl Environ Microbiol 76:4730–4737
Dowdle PR, Oremland RS (1998) Microbial oxidation of elemental selenium in soil slurries and bacterial cultures. Environ Sci Technol 32:3749–3755
Jackson CR, Randolph KC, Osborn SL, Tyler HL (2013) Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables. BMC Microbiol 13:274
Raina JB, Tapiolas D, Willis BL, Bourne DG (2009) Coral-associated bacteria and their role in the biogeochemical cycling of sulfur. Appl Environ Microbiol 75:3492–3501
Klindworth A, Pruesse E, Schweer T et al (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucl Acids Res 41:e1
Tanaka T, Kawasaki K, Daimon S et al (2014) A hidden pitfall in the preparation of agar media undermines microorganism cultivability. Appl Environ Microbiol 80:7659–7666
Buerger S, Spoering A, Gavrish E, Leslin C, Ling L, Epstein SS (2012) Microbial scout hypothesis, stochastic exit from dormancy, and the nature of slow growers. Appl Environ Microbiol 78:3221–3228
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer-Verlag Berlin Heidelberg
About this protocol
Cite this protocol
Thrash, J.C., Weckhorst, J.L., Pitre, D.M. (2015). Cultivating Fastidious Microbes. In: McGenity, T., Timmis, K., Nogales , B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_67
Download citation
DOI: https://doi.org/10.1007/8623_2015_67
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-662-45178-6
Online ISBN: 978-3-662-45179-3
eBook Packages: Springer Protocols