Review of Coliforms - National Health and Medical Research Council

Fluorescence In-situ Hybridisation (FISH)
MICROBIAL INDICATORS OF DRINKING WATER QUALITY
Solid-state cytometry is a methodology that is rapidly taking off in water microbiology,
utilising either chromogenic substrates (see Appendix A) or the molecular labelling method
called fluorescence in situ hybridisation (FISH).
FISH detection makes use of gene probes with a fluorescent marker, typically targeting
the 16S ribosomal RNA (16S rRNA) (Amann et al., 1995). Concentrated and fixed cells are
permeabilised and mixed with the probe. Incubation temperature and addition of chemicals
can influence the stringency of the match between the gene probe and the target sequence.
Since the signal of a single fluorescent molecule within a cell does not allow detection, target
sequences with multiple copies in a cell have to be selected (eg. there are 10 2 -10 4 copies
of 16S rRNA in active cells). A number of FISH methods for the detection of coliforms and
enterococci have been developed (Fuchs et al., 1998; Meier et al., 1997; Patel et al., 1998).
A number of studies indicate that FISH detection-based methods may better report the
presence of infective pathogens and viable, but not necessarily culturable indicator bacteria.
As a further extension of the FISH approach, peptide nucleic acid probes targeted against
the 16S rRNA molecule were designed and used to detect E. coli from water (Prescott et al.,
1998). The probe was labelled with biotin, which was subsequently detected with streptavidin
horseradish-peroxidase and the tyramide signal amplification system. E. coli cells were
concentrated by membrane filtration prior to hybridisation and the labelled cells detected
by a commercial laser-scanning solid-state cytometer (eg. ChemScan TM ) within 3h. Detection
and enumeration is also possible by the use of a flow cytometer (Fuchs et al., 1998). These
cytometers, however, are expensive, and high sample throughput is necessary to justify
their purchase.
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