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Button DK, Schut F, Quang P, Martin R, Robertson BR.. Viability and isolation of marine bacteria by dilution culture: theory, procedures and initial results. Appl Environ Microbiol 59: 881-891

Institute of Marine Science, University of Alaska Fairbanks, Fairbanks, Alaska 99775.
Applied and Environmental Microbiology (Impact Factor: 3.95). 03/1993; 59(3):881-91.
Source: PubMed

ABSTRACT Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 10 or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60% and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 10 cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms.

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    • "Hence, in an effort to study the abundant freshwater Actinobacteria in the laboratory we cultivated a freshwater community with drastically reduced complexity (Garcia et al 2014). This stable mixed culture was obtained using triple filtered water from Lake Grosse Fuchskuhle as media and a dilution method (Button et al 1993) in a high-throughput screening approach (Garcia et al 2014). Initial screening revealed that the mixed culture contained and Testing Laboratory (RTL) for shotgun metagenomic sequencing and 16S rRNA gene amplicon sequencing, respectively. "
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    ABSTRACT: Microorganisms are usually studied either in highly complex natural communities or in isolation as monoclonal model populations that we manage to grow in the laboratory. Here, we uncover the biology of some of the most common and yet uncultured bacteria in freshwater environments using a mixed culture from Lake Grosse Fuchskuhle. From a single shotgun metagenome of a freshwater mixed culture of low complexity, we recovered four high quality metagenome-assembled genomes (MAGs) for metabolic reconstruction. This analysis revealed the metabolic interconnectedness and niche partitioning of these naturally dominant bacteria. In particular, vitamin- and amino acid- biosynthetic pathways were distributed unequally with a member of Crenarchaeota most likely being the sole producer of vitamin B12 in the mixed culture. Using coverage based partitioning of the genes recovered from a single MAG intra-population metabolic complementarity was revealed pointing to 'social' interactions for the common good of populations dominating freshwater plankton. As such our MAGs highlight the power of mixed cultures to extract naturally occurring 'interactomes' and to overcome our inability to isolate and grow the microbes dominating in nature. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Molecular Ecology 07/2015; DOI:10.1111/mec.13319 · 6.49 Impact Factor
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    • "The conventional dilution-to-extinction method utilizes serial dilution to isolate single bacterial cells into test tubes or wells on microtiter plates (Button et al., 1993; Rappé et al., 2002; Schut et al., 1993; Sizova et al., 2012). The same principle can be applied to microfluidic devices to load single cells into microscale reaction chambers (Boedicker et al., 2008). "
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    ABSTRACT: Abstract Populations of bacterial cells that grow under the same conditions and/or environments are often considered to be uniform and thus can be described by ensemble average values of their physiologic, phenotypic, genotypic or other parameters. However, recent evidence suggests that cell-to-cell differences at the gene expression level could be an order of magnitude greater than previously thought even for isogenic bacterial populations. Such gene expression or transcriptional-level heterogeneity determines not only the fate of individual bacterial cells in a population but could also affect the ultimate fate of the population itself. Although techniques for single-cell gene expression measurement in eukaryotic cells have been successfully implemented for a decade or so, they have only recently become available for single bacterial cells. This is due to the difficulty of efficient lysis of most bacterial cells, as well as short half-life time (low stability) of bacterial mRNA. In this article, we review the recent progress and challenges associated with analyzing gene expression levels in single bacterial cells using various semi-quantitative and quantitative methods. In addition, a review of the recent progress in applying microfluidic devices to isolate single bacterial cells for gene expression analysis is also included.
    Critical Reviews in Biotechnology 04/2014; DOI:10.3109/07388551.2014.899556 · 7.84 Impact Factor
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    • "Different techniques were previously developed to isolate aquatic bacteria such as the dilution culture (Button et al., 1993) and the filtration acclimatization method (Hahn et al., 2004). In an attempt to isolate acI members, we tried several variations and combinations of these methods. "
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    ABSTRACT: Actinobacteria of the acI lineage are often the numerically dominant bacterial phylum in surface freshwaters, where they can account for > 50% of total bacteria. Despite their abundance, there are no described isolates. In an effort to obtain enrichment of these ubiquitous freshwater Actinobacteria, diluted freshwater samples from Lake Grosse Fuchskuhle, Germany, were incubated in 96-well culture plates. With this method, a successful enrichment containing high abundances of a member of the lineage acI was established. Phylogenetic classification showed that the acI Actinobacteria of the enrichment belonged to the acI-B2 tribe, which seems to prefer acidic lakes. This enrichment grows to low cell densities and thus the oligotrophic nature of acI-B2 was confirmed.
    Environmental Microbiology Reports 02/2014; 6(1):21-7. DOI:10.1111/1758-2229.12104 · 3.26 Impact Factor
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