26
distinctly stained cells are identified through flow cytometry or epifluorescence
microscopy (Pernthaler et al. 2002). The suitable application of fluorophore-labelled
rRNA-targeted probes as phylogenetic strains for cultivation-independent identification of microbes was first reported by DeLong et al. (1989) Since then, this technique has become a reliable and rapid method for identification of microorganisms
in the environment (Sekar et al. 2003).
FISH is one of the commonly applied tools for localizing, identifying, and isolating desired microbial taxa in environmental microbiology. Single-cell techniques
are promising for studying microbial community composition, and the efficiency
can be further improved through FISH technology (Amann and Fuchs 2008). FISH
identified the complex Nitrospira community which persisted in wastewater treatment plants, suggesting the stable coexistence of a Nitrospira strain that shared
highly similar ecological niches with other microbial species (Gruber-Dorninger
et al. 2015). Guangyin et al. (2016) analysed the electro-methanosynthesis process
in a twin-chambered microbial electrolytic cell equipped with a graphite-covered
hybrid biocathode through FISH. Microbial community structure of PAHscontaminated river sediments was explored by using temperature gradient gel electrophoresis (TGGE) and a catalysed reported deposition (CARD-FISH) (Zoppini
et al. 2016).
Fluorophore signal intensity is the most limiting factor in studying soil microbial
communities using FISH technology. To overcome poor fluorescence problems in
FISH, a recent advance is introduction of a single oligonucleotide combinatorial
probe labelling called multi-labelled FISH (MiL-FISH). In MiL-FISH, the singleoligonucleotide probe combinatorial labelling is used to improve the signal intensity and image quality of individual microbial cells in environmental samples
(Schimak et al. 2016).
3.3 Denaturing Gradient Gel Electrophoresis (DGGE)
Denaturing gradient gel electrophoresis (DGGE) is another popular 16S rRNAbased microbial fingerprinting technique that has been actively applied in exploring
the microbial communities of natural ecosystems (Drigo et al. 2009; Janczyk et al.
2010). Amplification of the desired 16S rDNA fragment, mostly the V3 region and
different amplicons, is followed by its further subjection to denaturing polyacrylamide gel electrophoresis (DGGE). Different amplicons have different electrophoretic mobility inside the gel, which leads to separation of the amplicons and
generation of polymorphic bands in the gel. The community members can be identified by hybridization with taxon-specific probes. The differentiating bands could
also be characterized by cloning and sequencing (Muyzer 1999; Jousset et al. 2010).
DGGE has various advantages over other fingerprinting techniques as it offers rapid
and intensive phylogenetic characterization and generates the structural profile of
microbial communities. Alternatively, slight modification in DGGE such as associating it with other fingerprinting techniques could increase the resolution of the
3 Methods for Exploring Soil Microbial Diversity
distinctly stained cells are identified through flow cytometry or epifluorescence
microscopy (Pernthaler et al. 2002). The suitable application of fluorophore-labelled
rRNA-targeted probes as phylogenetic strains for cultivation-independent identification of microbes was first reported by DeLong et al. (1989) Since then, this technique has become a reliable and rapid method for identification of microorganisms
in the environment (Sekar et al. 2003).
FISH is one of the commonly applied tools for localizing, identifying, and isolating desired microbial taxa in environmental microbiology. Single-cell techniques
are promising for studying microbial community composition, and the efficiency
can be further improved through FISH technology (Amann and Fuchs 2008). FISH
identified the complex Nitrospira community which persisted in wastewater treatment plants, suggesting the stable coexistence of a Nitrospira strain that shared
highly similar ecological niches with other microbial species (Gruber-Dorninger
et al. 2015). Guangyin et al. (2016) analysed the electro-methanosynthesis process
in a twin-chambered microbial electrolytic cell equipped with a graphite-covered
hybrid biocathode through FISH. Microbial community structure of PAHscontaminated river sediments was explored by using temperature gradient gel electrophoresis (TGGE) and a catalysed reported deposition (CARD-FISH) (Zoppini
et al. 2016).
Fluorophore signal intensity is the most limiting factor in studying soil microbial
communities using FISH technology. To overcome poor fluorescence problems in
FISH, a recent advance is introduction of a single oligonucleotide combinatorial
probe labelling called multi-labelled FISH (MiL-FISH). In MiL-FISH, the singleoligonucleotide probe combinatorial labelling is used to improve the signal intensity and image quality of individual microbial cells in environmental samples
(Schimak et al. 2016).
3.3 Denaturing Gradient Gel Electrophoresis (DGGE)
Denaturing gradient gel electrophoresis (DGGE) is another popular 16S rRNAbased microbial fingerprinting technique that has been actively applied in exploring
the microbial communities of natural ecosystems (Drigo et al. 2009; Janczyk et al.
2010). Amplification of the desired 16S rDNA fragment, mostly the V3 region and
different amplicons, is followed by its further subjection to denaturing polyacrylamide gel electrophoresis (DGGE). Different amplicons have different electrophoretic mobility inside the gel, which leads to separation of the amplicons and
generation of polymorphic bands in the gel. The community members can be identified by hybridization with taxon-specific probes. The differentiating bands could
also be characterized by cloning and sequencing (Muyzer 1999; Jousset et al. 2010).
DGGE has various advantages over other fingerprinting techniques as it offers rapid
and intensive phylogenetic characterization and generates the structural profile of
microbial communities. Alternatively, slight modification in DGGE such as associating it with other fingerprinting techniques could increase the resolution of the
3 Methods for Exploring Soil Microbial Diversity