Total Coliforms

Background

Coliforms (coliform bacteria) are rod-shaped gram-negative bacteria that are commonly used as an indicator of the sanitary quality of water. Coliforms are ubiquitous in the environment but are prevalent in the intestine and faecal material of warm-blooded animals. Though not particularly harmful the presence of coliforms in water samples is indicative of contamination by more harmful microorganisms. The established methodology for quantification of coliforms involves a laborious laboratory procedure. Samples (usually 100 ml) are passed through a filter and the filter is then placed in a sterile plate with a selective growth medium and incubated for 24 hours at 44.5 °C. This elevated temperature heat shocks non-faecal coliform bacteria and suppresses their growth. Faecal coliform colonies develop a blue colour due to a reaction with an aniline dye in the growth medium. As each cell present in the sample develops into a separate colony highly contaminated sources require dilution to ensure the plate that can be accurately counted. Historically the term faecal coliforms has been used to describe this group, however, the more accurate term "thermotolerant coliform is becoming the preferred nomenclature.

Why is it Important?

Coliforms are widely monitored for both environmental, legislative and public health reasons:

• the test is used under the Water Framework Directive (WFD) to assess bathing water quality (see Table 2);
• it is a parameter monitored under the Shellfish Waters Directive;
• used to confirm potable water is safe for human consumption (e.g. wells, boreholes and reservoirs);
• the coliform test can be used to identify sources of organic pollution in river networks and monitor CSOs.

Challenges to monitoring coliforms

There are numerous issues associated with the laboratory procedure for quantification of coliform counts:

• lead times until results are available (> 24h) that can lead to unacceptable public health risks;
• the test is time consuming and expensive;
• it fails to recreate natural processes;
• results are not always simple to interpret as a low value can be due to high organic content that is not readily degraded or that degradation is inhibited by toxins;
• it is prone to contamination and requires a 'clean' laboratory;
• a significant degree of uncertainty is associated with the test measurement variability in certified laboratories as high as 20%; and that is just the measurement in the laboratory and does not involve all of the potential issues of the sampling and transportation process
• degree of dilution required is often unknown before analysis

It is clear that a move from traditional laboratory testing to in-situ (real-time) monitoring would help to alleviate most of the problems outlined above. It would particularly improve spatial temporal resolution of monitoring that would be directly beneficial to basin managers, water companies and legislators alike.

Proteus - the real-time solution for coliform monitoring

Figure 1. Image of the Proteus for coliform analysis.

The Proteus is a new product providing users with a robust, repeatable, low maintenance sensor platform for measuring coliforms in real-time. The Proteus is underpinned by comprehensive research exploring the use of in-situ fluorescence as a technique for real-time coliform measurement. The Proteus is a multi-parameter instrument that can incorporate a range of optical sensors. The configuration for microbial monitoring includes a tryptophan-like fluorescence (TLF) sensor, turbidity sensor (see Fig. 2) and thermistor which provides users a real time measurement of total coliforms, negating the need for the standard laboratory analysis. Using a robust correction algorithm the tryptophan signal is corrected, in real time, for known interferrants. The result is a repeatable and highly accurate measurement that can provide instantaneous coliform measurements.

Figure 2. Optical configuration of the Proteus for coliform measurement with a schematic of the measurement set up (left) and the tryptophan like fluorescence (TLF) measurement region highlighted in optical space (right).

Figure 3. Example of the relationship between the Proteus and laboratory measurements for a source to sea study in an agricultural catchment.

The science...

Fluorescence spectroscopy is a selective and sensitive optical technique enabling in-situ, real-time measurement of dissolved organic matter. Molecules absorb light of a specific wavelength and orbiting electrons are excited to a higher energy state .The electrons then emit light of a specific wavelength to return to the base state.

The dissolved organic matter pool can be mapped in optical space based on its fluorescent properties (see Fig. 2). The TLF peak (red) represents a mixture of free amino acids, peptides and proteins but importantly also those bound into the cells of microbes. Numerous published studies have correlated TLF with faecal coliform counts and our site specific or generic calibrations are able to provide users with highly accurate and repeatable coliform measurements (Fig. 3).

Applications

• River pollution monitoring
• Pollution source tracing surveys
• Bathing water monitoring
• Borehole / well monitoring
• Reservoir monitoring
• Combined Sewage Overflow (CSO) event detection
• Monitoring clean water systems for coliform ingress