How Flow Cytometry Helps Water Utilities Better Understand the Microbial Fingerprint

Providing clean, safe water is the single-biggest priority for water utilities. To continue doing so, these municipal / local water treatment facilities must perform regular and reliable monitoring. This includes testing that quickly and accurately ensures there has not been contamination of any kind. Unfortunately, the most widely used microbial testing methods today only capture a fraction of the microbial population or resort to unprecise proxy parameters to assess the microbial load in a sample. As a result, these common methods present only a very limited picture of what is going on in the water. For municipalities needing to deliver safe water all times, it’s important to understand what some of those limitations are, and what better options are available.

Heterotrophic place count (HPC), the commonly used method to assess total microbial load in water is based on cell cultivation and has several limitations:

  • It’s a time-consuming process that takes three to seven days to complete. How does a utility company react during this window of uncertainty? Wait and take no action until they have results? Or take the wrong action that may lead to a waste of time, energy, and/or additional biocides (such as disinfectants) left behind in water unnecessarily? Neither option is ideal. 
  • It’s extremely insensitive. Only a small fraction of the viable bacteria present in water, usually less than 1%, grow on the cultivation media (agar). Most of the cells present in the sample are not cultivable on HPC nutrient media and, therefore, are not quantified by HPC. This results in a somewhat hygienic indication of contamination but a far cry from a robust process parameter. 
  • It does not allow for the identification of the specific types of bacteria present in the water, which can be important for determining the potential health risks associated with the contamination. Water utilities therefore perform additional tests to screen for hygiene issues (e.g., testing for total coliforms). These tests however suffer from similar limitations as HPC because they are also based on cell cultivation.

A faster alternative is to quantify free cellular Adenosine Triphosphate (ATP) in a water sample. ATP can be found in all living cells, including algae, protozoa, bacteria, and fungi. When cellular ATP is present, it indicates that there is active microbial contamination in water. ATP testing, although cultivation-independent, also suffers from similar limitations as described above:

  • It is very insensitive in practice, because the amount of cellular ATP measured is influenced by many factors and is just a proxy parameter which is used to estimate bioburden. ATP is therefore not suitable for detecting minor microbiological changes, for example in the early phase of exponential bacterial outgrowth or for accurately assessing the effects of water treatment.
  • And again, ATP is an unspecific testing method that cannot zoom in to detect and quantify specific organisms present in a sample.

A more direct and accurate testing alternative to HPC and ATP is the counting of single bacterial cells using a high-speed optical method called Flow Cytometry (FCM). FCM can provide highly accurate assessments of the Intact Cell Count (ICC), also known as Total Viable Count, and the Total Cell Count (TCC), also known as Total Bacteria Count, in water.

ICC is a measure of the number of viable (i.e., living) bacteria present in a water sample. The ICC of a water sample can provide accurate, timely and valuable information about the microbial state of the water. Changes in ICC help to understand and quantify potential risks, recognize build-up of bacterial populations, detect maintenance need of equipment, and generally assess the microbial state of water in a much more precise and direct way as done before.

Typical external factors that affect the ICC at fresh water sources, in treatment, or in distribution are rain events, flooding, agricultural runoff, infiltration of surface water into the ground water supply, release of biofilm, or effects of microbially influenced corrosion (see our other blog post). Biofilms are thin layers of microorganisms that can form on the surfaces of pipes and other infrastructure, and they can harbor large numbers of bacteria.

Understanding the dynamics of ICC over time in a water distribution system can help water utility operators identify potential sources of contamination and take targeted steps to address them. For example, if the ICC of a water sample is significantly higher than expected in normal conditions, it could indicate the need for additional treatment or failure in water management processes or equipment. In such a case, rqmicro’s flow cytometry kits allow for subsequent testing for specific risks, like fecal contamination (E.coli) or presence of Legionella. 

What’s more? Fingerprinting!

Using flow cytometry, the bacteria in water can be differentiated into two groups, bacteria of low nucleic acid content (LNA) and of high nucleic acid content (HNA). LNA and HNA refer to the amount of DNA or RNA present in their cells. Low nucleic acid (LNA) bacteria have a lower amount of DNA or RNA compared to high nucleic acid (HNA) bacteria. The difference in nucleic acid content can be due to differences in the size of the genome or the relative amount of DNA or RNA present in the cell.

LNA bacteria are often found in environments where there is a lack of nutrients, such as in soil or water. They tend to have smaller genomes and may have a reduced number of genes. HNA bacteria, on the other hand, are found in a variety of environments and tend to have larger genomes with a greater number of genes.

The nucleic acid content of a bacterial species can have an impact on its growth and survival. For example, LNA bacteria may have a slower growth rate and may be more resistant to stress conditions, such as extreme temperatures or pH, compared to HNA bacteria. However, HNA bacteria may have a greater capacity for adaptation and evolution due to their larger genome size. The LNA/HNA ratio, also known as the microbial fingerprint, analyzed over time, can provide useful insights into the microbiological state of the water and allows to detect changes in the microbial population with even higher sensitivity than the cell number allows. 

How to quantify bacteria in water using Flow Cytometry?

So, if measuring Intact Cell Count (ICC) or Total Cell Count (TCC) is preferable, how does one test for it? Using a technology called Flow Cytometry, or FCM

FCM is an optical technology for electronic cell counting that allows multiparametric analysis of the properties of thousands of cells per second. Cells which have been labelled with a fluorescent dye pass in front of a laser in a confined stream of fluid. The laser excites electrons in the fluorescent dye that then return in fractions of a second to their previous state by emitting light. The analog light signal gets transformed into digital data that is further processed into the desired format of the user.

One of the major advantages of FCM is its ability to analyze both small and large numbers of cells quickly and accurately, especially when compared to cell cultivation. It is a highly sensitive method that can provide substantially more insight into water microbiology than the competing methods. FCM started off in the 1970s and has since become a routine tool in in-vitro diagnostics and life sciences research and pharma. Widespread adoption of FCM outside of these applications has been hindered by:

  • The complexity of the method
  • The maintenance requirements of the equipment
  • The technical/scientific requirements towards the operator

rqmicro.COUNT has been developed to resolve these barriers and finally brings FCM to main street.


With rqmicro.COUNT, water utilities can easily and efficiently detect and quantify bacteria in water on-site at key treatment or critical control points using flow cytometry. Among the benefits: 

  • Accurate: High measuring accuracy and sensitivity on single-cell level
  • Rapid: First results are available after 30 minutes from taking the sample
  • Versatile: Test kits available for unspecific cell counting (ICC and TCC) and specific pathogens (E. coli, Legionella) on the same platform
  • Connected: Synchronizes with a secure cloud platform which enables online data analysis, automated alarms and sharing of reports
  • Easy to use: Ready-to-use reagent tubes and single-use cartridges make flow cytometry as convenient as never before

Contact us for more information today.

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Recognize the Bacterial Fingerprint of Drinking Water with Portable Flow Cytometry