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 fecal 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 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". 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.
Box: 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).
Before the flow cytometry method could be applied for water analysis
in day-to-day practice, it had to undergo rigorous testing. Financial
support for this standardization and validation was provided by the
Commission for Technology and Innovation (CTI) and the Swiss Gas and
Water Industry Association (SVGW). A total of 24 partners from academia,
administration and public/private laboratories participated in this
process.
Story Source:
The above story is reprinted from materials provided by EAWAG: Swiss Federal Institute of Aquatic Science and Technology.
Courtesy: ScienceDaily
No comments:
Post a Comment