Flow cytometry quantifies microbes in drinking water in minutes

Swiss researchers have discovered that flow cytometry can now quantify microbial cells in drinking water—and do so in minutes rather than days. Stemming from work at aquatic research institute Eawag (Dübendorf, Switzerland) and extensive tests both in Switzerland and abroad, the optical techinque has been incorporated into the Swiss Food Compendium (SLMB) by the Federal Office of Public Health (FOPH; Bern, Switzerland).

For over 100 years, the conventional method used to assess drinking water quality 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 E.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° to 45°C (mesophilic). According to the global standard, the number of colonies formed should not exceed 300/mL.

But the cultivation-based method is both time-consuming (in the case of the HPC, results are available after 3–10 days) and incomplete, as it counts only a fraction of the living cells present in samples. This is because the method only detects those bacteria that can grow and form colonies under the specified conditions—generally 0.01–1% of the total. So the limit of 300 colony-forming units per milliliter (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. Cultivation of E. coli and Enterococci does, however, normally yield reliable results.

In flow cytometry, cells in a sample are 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 laser light. The resultant scatter and fluorescence signals are picked up by detectors, and analytical software is used to classify each individual particle (cell). The technique 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 harbors 100 to 10,000 times more living cells than the conventional plate count method would suggest.

In December 2012, the FOPH incorporated the flow cytometry method into the Swiss Food Compendium as a recommended test method. Instead of the HPC, which is no longer considered relevant for food hygiene purposes, flow cytometry can now be used to determine total cell count in a water sample within minutes. Also, the ratio of larger to smaller cells can be determined (for example, cells with a high or low nucleic acid content), which can help to pinpoint water treatment facility problems.

Switzerland is the first country worldwide to have adopted flow cytometry for quantifying microbial cells in water, but other countries such as The Netherlands, says Eawag drinking water specialist Stefan Koetzsch, will follow soon. He adds that flow cytometry is ideally suited for monitoring an entire water 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 flow cytometry method, which would permit "online" monitoring of bacterial cell counts.