Microfluidic fluorescence in situ hybridization and flow cytometry (µFlowFISH). Lab Chip

Biotechnology and Bioengineering Department, Sandia National Laboratories, PO Box 969, MS 9291, Livermore, CA 94550, USA.
Lab on a Chip (Impact Factor: 6.12). 08/2011; 11(16):2673-9. DOI: 10.1039/c1lc20151d
Source: PubMed


We describe an integrated microfluidic device (μFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(vi) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The μFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed, and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp.strain RCH2 that are involved in Cr(vi) reduction and immobilization. Combined labeling and detection efficiencies of 74-97% were observed in experiments with simple mixtures of cultured cells, confirming specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of μFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford site. We were able to monitor the numbers of Pseudomonas sp. with only 100-200 cells loaded into the microchip. The μFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome.

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    • "In fact, most microorganisms are recalcitrant to laboratory cultivation and it is estimated that 95–99 % of known microbial species cannot be isolated in the laboratory (and instead are identified by novel 16SrDNA sequences obtained from environmental samples; Amann et al. 1995; Hugenholtz et al. 1998; Whitman et al. 1998). Though recent advances in cultivation techniques have helped overcome some of these limitations, including single cell flow cytometry (Zengler et al. 2002; Müller and Nebe-von-Caron 2010), microfluidic culture coupled with FISH and flow cytometry (Liu et al. 2011), and the use of complex media and simulated environmental niches (Stevenson et al. 2004; Delavat et al. 2012), at present molecular methods appear more capable at detecting rare and specialized taxa than conventional cultivation-based approaches. Thus the use of culturedependent assays in environmental assessment is limited and will not be discussed in detail in this review; interested readers are referred to Stewart Rev Environ Sci Biotechnol (2012) for a review of recent advances in cultivation techniques targeting 'unculturable' microorganisms. "
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