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Applied and Environmental Microbiology, December 2005, p. 8714-8720, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8714-8720.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Microcolony Cultivation on a Soil Substrate Membrane System Selects for Previously Uncultured Soil Bacteria

Belinda C. Ferrari,1* Svend J. Binnerup,2 and Michael Gillings1

Department of Biological Sciences, Division of Environmental and Life Sciences, Macquarie University, Sydney, Australia,1 National Environmental Research Institute, Department of Environmental Chemistry and Microbiology, Roskilde, Denmark2

Received 9 February 2005/ Accepted 28 July 2005

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously "unculturable" organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.

* Corresponding author. Mailing address: Department of Biological Sciences, Division of Environmental and Life Sciences, Macquarie University, Sydney 2109, NSW, Australia. Phone: 61 2 9850 9252. Fax: 61 2 9850 8253. E-mail: bferrari{at}rna.bio.mq.edu.au.

Applied and Environmental Microbiology, December 2005, p. 8714-8720, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8714-8720.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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