Flow cytometry (FCM) can now be officially used for the
quantification of microbial cells in drinking water. The new
analytical method – developed at Eawag and extensively tested both
in Switzerland and abroad – has been incorporated into the Swiss
Food Compendium (SLMB) by the Federal Office of Public Health
(FOPH). FCM provides much more realistic results than the
conventional method, in which bacterial colonies are grown on agar
plates. The results demonstrate that even good-quality drinking
water harbours 100 to 10,000 times more living cells than the
conventional plate count method would suggest.
For over 100 years, the method used to assess the microbiological
safety of drinking water has remained essentially unchanged:
bacteria present in water are allowed to grow on solid nutrient
media (incubated at a warm temperature), and the colonies formed are
then counted. The intestinal bacteria Escherichia coli and
enterococci serve as indicators of faecal contamination. At the same
time, the heterotrophic plate count (HPC) is determined as a measure
of general microbiological quality. This method quantifies all the
microorganisms present which can reproduce at temperatures of around
20–45°C (mesophilic). According to the global standard, the number
of colonies formed should not exceed 300 per millilitre.
Cell counts significantly underestimated
The cultivation-based method has two major drawbacks: it is
time-consuming – results are only available after 3–10 days in the
case of the HPC – and only a fraction of the living cells actually
present in samples are counted. This is because the method only
detects those bacteria which can grow and form colonies under the
specified conditions – generally 0.01–1% of the total. Thus, the
limit of 300 colony-forming units per millilitre (CFU/mL) also
specified in the Swiss Ordinance on Food Hygiene (HyV) is based on a
significant underestimate of the actual number of microorganisms
present. The cultivation of E. coli and enterococci does, however,
normally yield reliable results. (Total cell counts for different
types of water are shown in Fig. 1.)
Total cell count and fingerprint
In December 2012, the FOPH incorporated method no. 333 “Determining
the total cell count and ratios of high and low nucleic acid content
cells in freshwater using flow cytometry” into the Swiss Food
Compendium as a recommended test method. Instead of the HPC, which
is no longer considered relevant for food hygiene purposes, FCM (see
Box) can now be used to determine the total cell count in a water
sample within a matter of minutes. Unlike the AMC, this count
provides a realistic indication of the microbial content of water
and – at least indirectly – allows conclusions to be drawn about
contamination. In addition, with the same method, the ratio of
larger to smaller cells can also be determined (i.e. cells with a
high or low nucleic acid content). This is seen by experts as the
“fingerprint” of drinking water: sudden changes in this value may
indicate, for example, damage or misconnections in the water
network, or faults at water treatment facilities.
New standard method
Switzerland is the first country worldwide to have adopted this
advanced method for the quantification of microbial cells in water.
Eawag drinking water specialist Stefan Koetzsch believes that other
countries, such as the Netherlands, will follow soon. Given the much
higher total cell counts, should the federal authorities now specify
new limits? “No,” says Koetzsch, “that would not be appropriate; nor
would it really be possible since the microbiological composition of
water will depend on its particular origin, and high cell counts do
not in themselves provide conclusive evidence of possible pathogens”
(see Fig. 1). However, Koetzsch and his colleagues are convinced
that FCM will become established as a new standard in the monitoring
of drinking water. The method is ideally suited for monitoring an
entire supply system (from drinking water abstraction through
treatment and distribution to consumers), optimizing processes and
identifying problems. Efforts are already underway to develop an
automated version of the method, which would permit “online”
monitoring of bacterial cell counts.
How does flow cytometry work?
Flow cytometry was developed for applications in the
field of medicine, where it has been used since the 1980s,
e.g. for counting (relatively large) blood cells. When this
method is employed for drinking water analysis, the
(generally small) cells contained in a sample are first
stained with fluorescent dyes, which bind to DNA. The cells
are then passed in single file through a glass capillary,
where they are exposed to a beam of light from a laser. The
resultant scatter and fluorescence signals are picked up by
detectors, and analytical software is used to classify each
individual particle (cell).
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SOURCE: Eawag
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