This chapter describes the application of fluorescent molecular probes used with immunofluorescence microscopy (IFM) and fluorescence in situ hybridization (FISH) techniques for studies of microbial autecology, with an emphasis on soil and root-associated microbes.  Proper filter sets must be used to match the specific optical requirements for excitation and emission of fluorescent light by different fluorochromes.  Fluorescein isothiocyanate (FITC), and to a lesser extent tetramethyl rhodamine isothiocyanate (TRITC), are common fluorochromes for single labeling experiments of IFM.  There are two general approaches in immunofluorescence staining: direct and indirect.  Both approaches involve the production, in an immunologically competent animal (e.g., rabbit), of a primary specific antibody against the antigen of interest.  The exciting innovations in image analysis technology featured in Center for Microbial Ecology Image Analysis System (CMEIAS) v. 3.0 software will undoubtedly enhance the ecological analysis of in situ bacterial colonization using immunofluorescence and other discriminating microscopy techniques operating at single cell resolution.  A large online probeBase database provides an overview of more than 700 published oligonucleotide probes and their characteristics for prokaryotic rRNAs suitable for FISH.  The potential ability of FISH-MAR techniques to target the ecological niche for physiological groups of microorganisms in environmental samples may help to close the gap to the general enzymatic measurements, which are also very much increased in sensitivity.  CMEIAS can extract an abundance of quantitative information on microbial community structure from the multiprobe FISH image. 

In situ detection of R. leguminosarum bv.  trifolii on the root surface of its host, white clover (Trifolium repens), using indirect IFM with a monoclonal antibody specific for its lipopolysaccharide O-antigen. ( A) Conventional epifluorescence.  Immunofluorescent cells have aggregated at the tip of a root hair, where the host lectin, trifoliin A, accumulates. ( B) Confocal laser scanning epifluorescence.  Immunofluorescent cells have developed a microcolony on the rhizoplane. 

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FIGURE 2

In situ detection of R. leguminosarum bv.  trifolii on the root surface of its host, white clover (Trifolium repens), using indirect IFM with a monoclonal antibody specific for its lipopolysaccharide O-antigen. ( A) Conventional epifluorescence.  Immunofluorescent cells have aggregated at the tip of a root hair, where the host lectin, trifoliin A, accumulates. ( B) Confocal laser scanning epifluorescence.  Immunofluorescent cells have developed a microcolony on the rhizoplane. 

Use of optical sectioning with the laser scanning confocal microscope to detect R. leguminosarum bv.  trifolii cells within the overlap of a markedly curled root hair of white clover.  This step precedes primary host infection in the Rhizobium-legume symbiosis.  Bacteria are stained by immunofluorescence using a monoclonal antibody against their somatic O-antigen.  Bar, 10 µm. 

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FIGURE 3

Use of optical sectioning with the laser scanning confocal microscope to detect R. leguminosarum bv.  trifolii cells within the overlap of a markedly curled root hair of white clover.  This step precedes primary host infection in the Rhizobium-legume symbiosis.  Bacteria are stained by immunofluorescence using a monoclonal antibody against their somatic O-antigen.  Bar, 10 µm. 

Frequency (left) and 2-D scatterplots (right) of the CMEIAS plotless spatial distribution analyses of the first and second nearest neighbor distances (NND) separating each individual bacterium shown in Fig.  4. 

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FIGURE 5

Frequency (left) and 2-D scatterplots (right) of the CMEIAS plotless spatial distribution analyses of the first and second nearest neighbor distances (NND) separating each individual bacterium shown in Fig.  4. 

Geostatistical analysis of the bacterial colonization of the root surface shown in Fig.  4. ( Left) Semivariogram autocorrelation plot and best-fit spherical model of the local spatial density; (right) 2-D kriging map derived from the autocorrelation model depicting the local density of bacteria interpolated over the entire spatial domain. 

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FIGURE 6

Geostatistical analysis of the bacterial colonization of the root surface shown in Fig.  4. ( Left) Semivariogram autocorrelation plot and best-fit spherical model of the local spatial density; (right) 2-D kriging map derived from the autocorrelation model depicting the local density of bacteria interpolated over the entire spatial domain. 

(Top) 2-D projection of a 3-D confocal image stack showing microcolonies of two different ribotypes of nitrite-oxidizing bacteria, genus Nitrospira, in a biofilm from a wastewater treatment plant.  The image was rendered by the program DAIME and provided courtesy of Frank Maixner and Holger Daims (University of Vienna, Vienna, Austria). ( Bottom) Formamide concentration series for the determination of optimal hybridization conditions for a newly designed 16S rRNA-targeted oligonucleotide probe using the program DAIME.  Data courtesy of Sebastian Lücker and Holger Daims (University of Vienna, Vienna, Austria). 

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FIGURE 7

(Top) 2-D projection of a 3-D confocal image stack showing microcolonies of two different ribotypes of nitrite-oxidizing bacteria, genus Nitrospira, in a biofilm from a wastewater treatment plant.  The image was rendered by the program DAIME and provided courtesy of Frank Maixner and Holger Daims (University of Vienna, Vienna, Austria). ( Bottom) Formamide concentration series for the determination of optimal hybridization conditions for a newly designed 16S rRNA-targeted oligonucleotide probe using the program DAIME.  Data courtesy of Sebastian Lücker and Holger Daims (University of Vienna, Vienna, Austria). 

(Left) Bacteria with ectomycorrhizal hyphae.  The ectomycorrhizal fungus Fagirhiza pallida was retrieved from roots of beech trees in the Kranzberg forest near Freising, Germany.  For FISH analysis, the following probes were applied: EUB338Mix Cy3, Bet42a Fluos, and Gam42a Cy5.  Due to concomitant binding of probes, beta-proteobacteria are stained yellow (Beta42a Fluos plus EUB338Mix Cy3) and other Bacteria (non-beta- or -gamma proteobacteria) are labeled red (EUB338Mix Cy3).  Another unidentified organism that hybridized with none of the three fluorescent probes is included in the image because of its blue autofluorescence. ( Right) A. brasilense on wheat roots.  Wheat seedlings were inoculated with A. brasilense Sp245, and their colonization was visualized by FISH analysis.  The following oligonucleotide probes were applied: EUB-338Mix Cy3 and Abras-1420-Cy5.  Due to concomitant binding of both probes, the bacterial cells display magenta fluorescence.  The computer-rendered orthogonal view displays the third dimension in the 3-D-z-stack of xy-optisections. 

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FIGURE 8

(Left) Bacteria with ectomycorrhizal hyphae.  The ectomycorrhizal fungus Fagirhiza pallida was retrieved from roots of beech trees in the Kranzberg forest near Freising, Germany.  For FISH analysis, the following probes were applied: EUB338Mix Cy3, Bet42a Fluos, and Gam42a Cy5.  Due to concomitant binding of probes, beta-proteobacteria are stained yellow (Beta42a Fluos plus EUB338Mix Cy3) and other Bacteria (non-beta- or -gamma proteobacteria) are labeled red (EUB338Mix Cy3).  Another unidentified organism that hybridized with none of the three fluorescent probes is included in the image because of its blue autofluorescence. ( Right) A. brasilense on wheat roots.  Wheat seedlings were inoculated with A. brasilense Sp245, and their colonization was visualized by FISH analysis.  The following oligonucleotide probes were applied: EUB-338Mix Cy3 and Abras-1420-Cy5.  Due to concomitant binding of both probes, the bacterial cells display magenta fluorescence.  The computer-rendered orthogonal view displays the third dimension in the 3-D-z-stack of xy-optisections. 

Distribution of microbial abundance in the community represented by the FISH image of Fig.  8, left panel.  CMEIAS was used to segment the microbes in the image according to their foreground color ranges and then measure the cumulative cell bio-volume for each microbial ribotype. 

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FIGURE 9

Distribution of microbial abundance in the community represented by the FISH image of Fig.  8, left panel.  CMEIAS was used to segment the microbes in the image according to their foreground color ranges and then measure the cumulative cell bio-volume for each microbial ribotype. 

27.  Cottrell,M. T.,, and D.L. Kirchmann.2003.Contribution of major bacterial groups to bacterial biomass production (thymidine and leucine incorporation) in the Delaware estuary. Limnol.  Oceanogr.48:168– 178. 

28.  Daims,H.,, A.Bruhl,, R.Amann,, K.H. Schleifer, and, M.Wagner.1999.The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst.  Appl.  Microbiol.22:434– 444. 

41.  Dazzo,F. B.,, and J.Wopereis.2000.Unraveling the infection process in the Rhizobium-legume symbiosis by microscopy, p. 295–347.InE.Triplett (ed.),  Prokaryotic Nitrogen Fixation: a Model System for the Analysis of a Biological Process. Horizon Scientific Press, Norwich,United Kingdom.