MICROBIAL MONITORING DURING BIOAUGMENTATION WITH DEHALOCOCCOIDES - Bioaugmentation for Groundwater Remediation - page 183

Figure 6.7. Determination of target cell concentrations using fluorescent in situ hybridization
(FISH) (courtesy of Natuschka Lee, Technische Universit ¨ tM¨ nchen, Germany).
cells can be screened using fluorescent microscopy for genes of interest (Figure 6.7 ). This target
specificity is the principal difference between FISH and general fluorescent staining using
DNA intercalators. Although more labor intensive than molecular techniques such as qPCR,
FISH permits investigation of specific target genes or activities in their native environment
without the need for DNA extraction or amplification. FISH originally was developed as a tool
for clinical diagnostics in medical research but it has been routinely used in environmental
microbiology since the 1990s. The following section provides a brief description of FISH in the
context of environmental microbiology and bioremediation.
6.6.2 Description and General Methodology
FISH analysis uses fluorescently-labeled molecular probes in conjunction with a series
of techniques permitting transmittance of the probes into cells. Direct examination of the
FISH-labeled cells using fluorescent microscopy permits the determination of target cell
concentrations. Much like the qPCR probes described earlier, FISH probes are short (approxi-
mately 20-30 nucleotides), complementary pieces of DNA that contain a fluorophore (fluores-
cent molecule) at one end. They differ from qPCR fluorescent probes in two key aspects:
(1) FISH probes lack the quenching molecules at the terminal end, and thus will continually
fluoresce, and (2) FISH probes used in environmental microbiology generally bind with RNA,
not DNA. The use of RNA (versus DNA) as a target binding-site stems from the higher
RNA concentrations present within a cell. These higher concentrations result in greater signal
intensity as a larger number of probes are able to bind with target sites. The ability to hybridize a
sample with multiple probes (each of which with a specific fluorescence) targeting different
sequences, also adds to the power of this technique.
FISH probes generally fall into two categories: group-specific and functional probes.
Group-specific probes bind to rRNA with specificities ranging from domain- (e.g., bacterial
and archaeal) to strain-level (e.g., Dhc strain BAV1 and Dhc strain VS). These probes are useful
in delineating the types of microbes present, such as determining the Dhc concentration in a
groundwater sample. Functional probes bind to RNA coding for enzymes involved in specific
metabolic processes, such as sulfate-reduction or ammonia-oxidation. The use of functional