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Chapter 2.2.1 : Gold-Based Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples

Authors: Thilo Eickhorst1, Hannes Schmidt2
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Affiliations: 1: Soil Microbial Ecology, University of Bremen, Germany; 2: Soil Microbial Ecology, University of Bremen, Germany
Content Type: Reference
Publication Year: 2016

Category: Environmental Microbiology

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Abstract:

The advent of new sophisticated spectroscopic and tomographic techniques arise interest in the study of environmental conditions within microbial habitats on a submicroscopic level. These methods are based on electromagnetic radiation and result in either elemental characterization or structure visualization. Both aspects are relevant for the investigation of microbe-habitat interactions why a correlative detection of microbial cells would be useful. Fluorescence in situ hybridization is an ideal technique to identify and localize microorganisms but requires a cell-detection via fluorescence microscopy which has a limited optical and elemental resolution. Therefore the utilization of nanogold as marker for in situ hybridization approaches is of great potential. Gold labels can be visualized with one of the aforementioned techniques on resolutions beyond light microscopy and allow the identification and localization of single microbial cells in their habitat in situ. The basic principal and potential of this method is described in this chapter giving an overview on the development steps of gold-targeted cell detection as well. Selected results exemplarily show applications in environmental microbiology both via fluorescence microscopy and electron microscopy including elemental mapping.

Citation: Eickhorst T, Schmidt H. 2016. Gold-Based Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples, p 2.2.1-1-2.2.1-10. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.2.1
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Gold-Based In Situ Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples
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Citation: Eickhorst T, Schmidt H. 2016. Gold-Based Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples, p 2.2.1-1-2.2.1-10. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.2.1
/content/10.1128/9781555818821.ch2.2.1.ch2.2.1fig01
FIGURE 1

Workflow diagram of the gold-FISH method. Modified from ( ); HRP: horseradish peroxidase, Au: 1.4 nm nanogold particle, AF488: Alexa Fluor 488. Reprinted from ( ) with permission from the publisher. doi: 10.1128/9781555818821.ch2.2.1.f1

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Image of FIGURE 1
FIGURE 1

Workflow diagram of the gold-FISH method. Modified from ( ); HRP: horseradish peroxidase, Au: 1.4 nm nanogold particle, AF488: Alexa Fluor 488. Reprinted from ( ) with permission from the publisher. doi: 10.1128/9781555818821.ch2.2.1.f1

Citation: Eickhorst T, Schmidt H. 2016. Gold-Based Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples, p 2.2.1-1-2.2.1-10. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.2.1
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FIGURE 2

Gold-FISH detected bacteria on polycarbonate filters. (a) hybridized with mono-gold-FISH, probe GAM42a-biotin and AF 488 fluoro-nanogold–streptavidin conjugate. (b) hybridized with gold-FISH, probe GAM42a-HRP, biotinylated tyramide, and AF 488 fluoro-nanogold–streptavidin conjugate. (c) Mixed culture of and strain AK199 hybridized with gold-FISH, probe ROS537-HRP, biotinylated tyramide, and AF 488 fluoro-nanogold–streptavidin conjugate. Numbers indicate corresponding images of (1) fluorescence microscopy, (2) SEM, and (3) BSE. (d) DAPI counterstain of image (c1). (E) Mixed culture of and strain AK199 hybridized with probe NONEUB. Scale bars: fluorescent micrographs 20 µm, SEM/BSE 2 µm. SEM and BSE images reprinted from ( ) with permission from the publisher. doi: 10.1128/9781555818821.ch2.2.1.f2

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Image of FIGURE 2
FIGURE 2

Gold-FISH detected bacteria on polycarbonate filters. (a) hybridized with mono-gold-FISH, probe GAM42a-biotin and AF 488 fluoro-nanogold–streptavidin conjugate. (b) hybridized with gold-FISH, probe GAM42a-HRP, biotinylated tyramide, and AF 488 fluoro-nanogold–streptavidin conjugate. (c) Mixed culture of and strain AK199 hybridized with gold-FISH, probe ROS537-HRP, biotinylated tyramide, and AF 488 fluoro-nanogold–streptavidin conjugate. Numbers indicate corresponding images of (1) fluorescence microscopy, (2) SEM, and (3) BSE. (d) DAPI counterstain of image (c1). (E) Mixed culture of and strain AK199 hybridized with probe NONEUB. Scale bars: fluorescent micrographs 20 µm, SEM/BSE 2 µm. SEM and BSE images reprinted from ( ) with permission from the publisher. doi: 10.1128/9781555818821.ch2.2.1.f2

Citation: Eickhorst T, Schmidt H. 2016. Gold-Based Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples, p 2.2.1-1-2.2.1-10. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.2.1
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FIGURE 3

Application of gold-FISH to environmental samples. (a) Bacteria in marine sediment, probe EUBI-III, scale bar 20 µm. (b) on the root surface of L. (wetland rice), probe: ALF968; (b1) fluorescence microscopy, scale bar 20 µm; (b2) corresponding SEM-image, scale bar 10 µm; (b3) SEM image at higher magnification (red frame in image b2), scale bar 1 µm. (c) associated with marine diatomes, probe: CF319a, (c1) SEM image, scale bar 5 µm; (c2) BSE-image of red frame in image (c1), scale bar 5 µm; (c3) merged image of SEM (reddish color) and BSE (bluish color) of red frame in (c2), scale bar 1 µm. Parts a and b1–b3 reprinted from ( ) with permission from the publisher. doi: 10.1128/9781555818821.ch2.2.1.f3

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Image of FIGURE 3
FIGURE 3

Application of gold-FISH to environmental samples. (a) Bacteria in marine sediment, probe EUBI-III, scale bar 20 µm. (b) on the root surface of L. (wetland rice), probe: ALF968; (b1) fluorescence microscopy, scale bar 20 µm; (b2) corresponding SEM-image, scale bar 10 µm; (b3) SEM image at higher magnification (red frame in image b2), scale bar 1 µm. (c) associated with marine diatomes, probe: CF319a, (c1) SEM image, scale bar 5 µm; (c2) BSE-image of red frame in image (c1), scale bar 5 µm; (c3) merged image of SEM (reddish color) and BSE (bluish color) of red frame in (c2), scale bar 1 µm. Parts a and b1–b3 reprinted from ( ) with permission from the publisher. doi: 10.1128/9781555818821.ch2.2.1.f3

Citation: Eickhorst T, Schmidt H. 2016. Gold-Based Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples, p 2.2.1-1-2.2.1-10. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.2.1
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FIGURE 4

Digital image analysis, elemental mapping, and artifacts of gold-FISH applications. (a) associated with marine diatoms, probe: CF319a, scale bar 5 µm; (a1) SEM image, (a2) result of digital image analysis, purple areas indicate detected bacterial cells and yellow areas indicate non-specific gold deposition; (b) SEM-EDS analysis of a gold-FISH detected cell on a diatom, scale bar 1 µm; (c) elemental mapping of silicon (Si-KA, c1), gold (Au-LA, c2), and merged data including SEM image (c3), scale bar 5 µm; (d) SEM and BSE image of nonspecific gold deposition in soil samples hybridized with gold-FISH, scale bar 5 µm; (e) nonspecific gold deposition on polycarbonate filters in the vicinity of gold-FISH detected cells, scale bar 5 µm. Parts d1 and d2 reprinted from ( , supplementary data) with permission from the publisher. doi: 10.1128/9781555818821.ch2.2.1.f4

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Image of FIGURE 4
FIGURE 4

Digital image analysis, elemental mapping, and artifacts of gold-FISH applications. (a) associated with marine diatoms, probe: CF319a, scale bar 5 µm; (a1) SEM image, (a2) result of digital image analysis, purple areas indicate detected bacterial cells and yellow areas indicate non-specific gold deposition; (b) SEM-EDS analysis of a gold-FISH detected cell on a diatom, scale bar 1 µm; (c) elemental mapping of silicon (Si-KA, c1), gold (Au-LA, c2), and merged data including SEM image (c3), scale bar 5 µm; (d) SEM and BSE image of nonspecific gold deposition in soil samples hybridized with gold-FISH, scale bar 5 µm; (e) nonspecific gold deposition on polycarbonate filters in the vicinity of gold-FISH detected cells, scale bar 5 µm. Parts d1 and d2 reprinted from ( , supplementary data) with permission from the publisher. doi: 10.1128/9781555818821.ch2.2.1.f4

Citation: Eickhorst T, Schmidt H. 2016. Gold-Based Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples, p 2.2.1-1-2.2.1-10. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.2.1
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Tables

Gold-Based In Situ Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples
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Citation: Eickhorst T, Schmidt H. 2016. Gold-Based Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples, p 2.2.1-1-2.2.1-10. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.2.1
/content/10.1128/9781555818821.ch2.2.1.ch2.2.1tab01
TABLE 1

Selection of commercially available nanogold particles and conjugates

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TABLE 1

Selection of commercially available nanogold particles and conjugates

Citation: Eickhorst T, Schmidt H. 2016. Gold-Based Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples, p 2.2.1-1-2.2.1-10. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.2.1
/content/10.1128/9781555818821.ch2.2.1.ch2.2.1tab02
TABLE 2

Original approaches for gold-based detection of single cells

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TABLE 2

Original approaches for gold-based detection of single cells

Citation: Eickhorst T, Schmidt H. 2016. Gold-Based Hybridization for Phylogenetic Single-Cell Detection of Prokaryotes in Environmental Samples, p 2.2.1-1-2.2.1-10. In Yates M, Nakatsu C, Miller R, Pillai S (ed), Manual of Environmental Microbiology, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818821.ch2.2.1

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