Biomedical Engineering Reference
In-Depth Information
depends not only on the stringencies described above, but also on the location of targeted
sequences within the RNA strand. In fact, some probes theoretically hybridize based on
sequence comparisons with target RNA, but in reality, poor signal intensities result from
such physical problems as steric hindrance; the probe simply cannot reach the targeted region
within the folded RNA. Additionally, hybridization of multiple different probes within a single
cell can be problematic. Thus, FISH allows determination of cell identity (e.g., a
Dhc
) using
group-specific probes, or metabolic activity (e.g., sulfate-reduction) using functional probes,
but rarely both at the same time.
Non-specific and mismatched binding present problems when using FISH to analyze
environmental samples. Non-specific binding results from probes adsorbing or hybridizing
with background material such as humics or organic debris. Since non-specific binding gener-
ally appears visually different from fluorescently-labeled cells, careful visual observation by
personnel skilled in fluorescent microscopy can usually distinguish and account for this.
Discriminating between cells labeled as a result of mismatched binding is much more difficult.
Mismatched binding occurs when probes hybridize with less than perfectly complementary
RNA sequences. While software has been developed to predict the overall quality of a probe
design and to estimate the possibility of mismatches, the enormous genetic variability within
microbes almost guarantees some mismatched hybridizations will occur in environmental
samples. Careful selection of thoroughly tested probes currently remains the best solution
for overcoming this interference. In addition, mineral matrices often autofluoresce under
various wavelengths of light, and may add to the background 'noise' of a given sample, leading
to under- or over-estimates during quantification.
6.6.4 Conjunctive Technologies
A number of technologies used in conjunction with FISH have arisen over the past decade.
These technologies attempt to link the identity of organisms (as determined by FISH) with
their physiological activity. One such method utilizes radiolabeled compounds (e.g.,
14
C-lactate,
32
P-phosphate, etc.) that become incorporated into DNA by microbes capable of consuming or
incorporating those substrates. The cells are screened using microautoradiography to determine
the metabolically active cells and FISH to determine identity. This process, termed MAR-FISH,
possesses its own limitations, but generally combines the strengths of two technologies in
examining microbial populations.
Another technology rapidly gaining acceptance uses FISH to fluorescently label target
cells, followed by rapid sorting and cell counting using flow cytometry. Flow cytometry can
screen thousands of microbial cells per minute, permitting rapid quantification and/or identifi-
cation of microbes from environmental samples in a substantially more accurate manner than
manual enumeration. Cost is the principal limitation of this technology at this time, preventing
its routine use in environmental sampling.
6.6.5 Conclusion
FISH provides a generally unbiased method of analyzing microbial populations in their
natural conditions. Although not explicitly mentioned within this text, numerous variations on
FISH exist, with most emphasizing methods for improving the signal intensity and/or quality.
These methods include the use of longer polynucleotide probes that target 10-100
longer
portions of RNA and fluorescent signal enhancements using catalyzed reporters. As these
technologies develop, they will undoubtedly affect the ability of remediation practitioners to
properly assess the need and/or effect of bioaugmentation at contaminated sites.
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