use alone does not provide any perception of the metabolic function of microorganisms. However, it may be combined with different techniques such as microautoradiography to describe the practical properties of microorganisms in their
natural surroundings (Wagner et al. 2006). Two kinds of FISH probes based on
conserved or precise areas of 16S rRNA genes may be advanced: domain- or
organization-specific probes and stress-unique probes. Domain- or institutionspecific probes discriminate or indicate participants of larger phylogenetic assemblages, even as stress-particular probes quantify or investigate the abundance of a
specific species or presence inside a microbial network (Dubey et al. 2006). Richardson et al. (2002) blended institution-unique FISH and T-RFLP in the characterization of microbial groups involved in TCE biodegradation. From the FISH
analysis, the authors found that a wide variety of organisms consisting of
Cytophaga, Flavobacterium, and Bacteroides had greater abundance than the TCE
degrader Dehalococcoides ethenogenes in the microbial consortium. However, the
lack of useful gene evaluation meant that the relative abundance of these organisms
and their ecological importance for TCE biodegradation could not be established.
FISH has been used to analyze microbial community composition in PAH-infected
soils, in particular detecting the 16S and 23S rDNA genes (Van Herwijnen et al.
2006; Hesham et al. 2012; Chang et al. 2014). However, unique care must be taken
using FISH on soil or sediment samples.
FISH techniques are frequently used with different genetic fingerprinting strategies including DGGE (Onda et al. 2002; Collins et al. 2006) and T-RFLP (Richardson et al. 2002; Kotsyurbenko et al. 2004; Jardillier et al. 2005; Collins et al. 2006)
for the enumeration and characterization of microbial populations from contaminated resources. The downside of FISH is that a confined variety of probes may be
utilized in a single hybridization experiment and the fluorescence history thus can be
complex in some samples (Dubey et al. 2006; Sanz and Kochling 2007). An earlier
examination of the sample and the maximum microorganisms that may be detected
is important (i.e., rRNA sequence) for the selection of specific probes. A predominant obstacle of the same FISH technique is its restricted sensitivity because
bacterial cells with decreased ribosome content that frequently appear in oligotrophic environments, such as maximum soil habitats, are not satisfactorily stained for
microscopic analyses. Modifications of FISH, such as the catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) and fluorescence in situ
hybridization micro-autoradiography (FISH-MAR) are being used as alternatives
to circumvent these issues and measure the potential for PAH degradation in soils
(Teira et al. 2007; Lekunberri et al. 2010).
In CARD-FISH, signal intensities of hybridized cells are multiplied by enzymatic
signal amplification using horseradish peroxidase (HRP)-categorized oligonucleotide probes in combination with tyramide signal amplification (TSA). TSA is based
on the patented catalyzed reporter deposition (CARD) method using derivatized
tyramide. In the presence of small quantities of hydrogen peroxide, immobilized
HRP converts the categorized substrate (tyramide) into a short-lived, extremely
reactive intermediate. The activated substrate molecules then very swiftly react
with, and covalently bind to, electron-rich areas of adjacent proteins. This binding
18 Molecular Technologies for Assessment of Bioremediation and. . .
455
natural surroundings (Wagner et al. 2006). Two kinds of FISH probes based on
conserved or precise areas of 16S rRNA genes may be advanced: domain- or
organization-specific probes and stress-unique probes. Domain- or institutionspecific probes discriminate or indicate participants of larger phylogenetic assemblages, even as stress-particular probes quantify or investigate the abundance of a
specific species or presence inside a microbial network (Dubey et al. 2006). Richardson et al. (2002) blended institution-unique FISH and T-RFLP in the characterization of microbial groups involved in TCE biodegradation. From the FISH
analysis, the authors found that a wide variety of organisms consisting of
Cytophaga, Flavobacterium, and Bacteroides had greater abundance than the TCE
degrader Dehalococcoides ethenogenes in the microbial consortium. However, the
lack of useful gene evaluation meant that the relative abundance of these organisms
and their ecological importance for TCE biodegradation could not be established.
FISH has been used to analyze microbial community composition in PAH-infected
soils, in particular detecting the 16S and 23S rDNA genes (Van Herwijnen et al.
2006; Hesham et al. 2012; Chang et al. 2014). However, unique care must be taken
using FISH on soil or sediment samples.
FISH techniques are frequently used with different genetic fingerprinting strategies including DGGE (Onda et al. 2002; Collins et al. 2006) and T-RFLP (Richardson et al. 2002; Kotsyurbenko et al. 2004; Jardillier et al. 2005; Collins et al. 2006)
for the enumeration and characterization of microbial populations from contaminated resources. The downside of FISH is that a confined variety of probes may be
utilized in a single hybridization experiment and the fluorescence history thus can be
complex in some samples (Dubey et al. 2006; Sanz and Kochling 2007). An earlier
examination of the sample and the maximum microorganisms that may be detected
is important (i.e., rRNA sequence) for the selection of specific probes. A predominant obstacle of the same FISH technique is its restricted sensitivity because
bacterial cells with decreased ribosome content that frequently appear in oligotrophic environments, such as maximum soil habitats, are not satisfactorily stained for
microscopic analyses. Modifications of FISH, such as the catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) and fluorescence in situ
hybridization micro-autoradiography (FISH-MAR) are being used as alternatives
to circumvent these issues and measure the potential for PAH degradation in soils
(Teira et al. 2007; Lekunberri et al. 2010).
In CARD-FISH, signal intensities of hybridized cells are multiplied by enzymatic
signal amplification using horseradish peroxidase (HRP)-categorized oligonucleotide probes in combination with tyramide signal amplification (TSA). TSA is based
on the patented catalyzed reporter deposition (CARD) method using derivatized
tyramide. In the presence of small quantities of hydrogen peroxide, immobilized
HRP converts the categorized substrate (tyramide) into a short-lived, extremely
reactive intermediate. The activated substrate molecules then very swiftly react
with, and covalently bind to, electron-rich areas of adjacent proteins. This binding
18 Molecular Technologies for Assessment of Bioremediation and. . .
455