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Chapter 39 : Cell Identification by Fluorescence In Situ Hybridization

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Abstract:

This chapter presents an updated collection of protocols for the identification of individual microbial cells by fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes. FISH of whole cells starts with a fixation of the sample containing the target cell types. Fixation stabilizes macromolecules and cytoskeletal structures, thus preventing lysis of the cells during hybridization, and at the same time permeabilizes the cell walls for fluorescently labeled oligonucleotide probes. The fixed cells are transferred onto gelatin-coated slides and incubated in a buffer containing the specific probe at a temperature near but below the melting point of the probe-rRNA hybrid. The subsequent washing step will remove unbound probe and leave only those probe-rRNA pairs intact that have no mismatches in the hybrid. Consequently, only target cells that contain the full signature sequence on their rRNA will be stained. Finally, hybridized cells can be enumerated by epifluorescence microscopy or by flow cytometry.

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39

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Microbial Ecology
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Sodium Dodecyl Sulfate
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16s rRNA Sequencing
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Anaerobic Ammonium Oxidation
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Figures

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FIGURE 1

Example of an rRNA alignment showing the probe sequence, the target sequence, and possible false identities.

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39
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Image of FIGURE 2
FIGURE 2

Theoretical dissociation profiles of a nucleic acid probe from a perfectly matched (bold line) and an imperfectly matched immobilized target nucleic acid (dashed line). The probeconferred signal (axis), which is directly proportional to the sensitivity, is shown over temperature and formamide concentration (axis), which represents the hybridization stringency. Note that the temperature of dissociation of the perfect hybrid is higher than that of the imperfect hybrid. The primary goal of the probe design is to maximize the difference between the temperature of dissociation of a probe from target and nontarget nucleic acid. The bar above the dissociation profiles indicates hybridization stringencies with high and low specific discrimination of target and nontarget nucleic acid, respectively.

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39
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References

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Tables

Generic image for table
TABLE 1

Dye labels frequently used for oligonucleotide probes and their characteristics

Fluorescein and derivatives are pH sensitive and exhibit maximum fluorescence at pH ≥9.

ε, molar extinction coefficient.

Data compiled from Amersham Biosciences, San Francisco, CA.

Data compiled from reference 50 and Invitrogen, Carlsbad, CA.

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39
Generic image for table
TABLE 2

Standard hybridization buffer

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39
Generic image for table
TABLE 3

Formamide concentration of some frequently applied probes

For horseradish-labeled probes, use 20% more formamide in hybridization buffer. See text for details.

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39
Generic image for table
TABLE 4

Standard washing buffer

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39
Generic image for table
TABLE 5

Corresponding NaCl concentration in 50 ml of washing buffer

Calculated using the formula from Lathe ( ). Note that the addition of EDTA contributes to the Na_ concentration. Therefore the required volume of 5 M NaCl solution in the washing buffer is reduced by 100 μl.

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39
Generic image for table
TABLE 6

Hybridization buffer for CARD-FISH

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39
Generic image for table
TABLE 7

Washing buffer for CARD-FISH

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39
Generic image for table
TABLE 8

Common failure symptoms during FISH, their possible causes, and suggestions for improvements

Citation: Fuchs B, Pernthaler J, Amann R. 2007. Cell Identification by Fluorescence In Situ Hybridization, p 886-896. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch39

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