Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring

Highlights

• Routine drinking water monitoring still relies on heterotrophic plate counts (HPC).

• Flow cytometry (FCM) is proposed as a better method for process monitoring.

• No good correlation was found between FCM and HPC data (n = 3,675).

• Good correlations were found between FCM and ATP data (n = 1,441).

• FCM advantages are: relevance, speed, accuracy, costs and automation potential.

Abstract

Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically < 1% of all bacteria). FCM measurements are reproducible with relative standard deviations below 3% and can be available within 15 min of samples arriving in the laboratory. High throughput sample processing and complete automation are feasible and FCM analysis is arguably less expensive than HPC when measuring more than 15 water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water.

Introduction

Drinking water treatment and distribution systems are designed and operated to safeguard the hygienic quality and ensure the aesthetic quality of the water from source to tap. With this in mind, monitoring is a non-negotiable and legislated requirement worldwide. There is a recognised and accepted need to monitor, characterise and understand the general microbiological performance/response of individual treatment steps, especially under changing environmental and operational conditions (Reasoner, 1990, Lautenschlager et al., 2013, Pinto et al., 2012). There is, furthermore, the need to monitor the general microbiological behaviour of treated water during distribution, particularly to detect potential contamination or deterioration due to biologically unstable water or distribution systems (Prest et al., 2016a, Prest et al., 2016b, Pinto et al., 2014). From a water utility perspective, microbiological methods used for such general water quality monitoring would ideally meet the criteria of being relevant, simple, reliable, rapid and cost-effective.

Heterotrophic plate counts (HPC) is the descriptive term for a group of similar methods used routinely by water utilities for general microbiological monitoring of drinking water. The method enumerates a variety of heterotrophic bacteria that are cultivable on semi-solid nutrient-rich media under defined incubation conditions (Allen et al., 2004, Rice et al., 2012, Gensberger et al., 2015). The basic HPC method was proposed well over a century ago (Koch, 1881) and was for a considerable time regarded as indicative of the hygienic quality of drinking water (Sartory, 2004). However, during the 1980's and 1990's it was decisively concluded that HPC measurements have no hygienic relevance (WHO, 2003a, WHO, 2003b, Sartory, 2004). Increasingly, HPC was regarded as a process variable to monitor a range of events and/or processes relevant to the general microbiological quality of drinking water in treatment and distribution systems (Reasoner, 1990, WHO, 2003a, WHO, 2003b, Sartory, 2004). For most of the previous century, HPC was regarded as the best available technology for drinking water process monitoring, and HPC data contributed towards considerable advances in our understanding of drinking water microbiology (Chowdhury, 2012).

In the last two decades, a number of powerful quantitative and molecular methods have emerged for water analysis (e.g., adenosine tri-phosphate (ATP) analysis, flow cytometry (FCM), 16S rRNA gene amplification and sequencing). Application of these new techniques showed that bacterial communities in drinking water were vastly more abundant and complex than what was previously understood from research based on cultivation-dependent methods (Berry et al., 2006, Hoefel et al., 2003). Current evidence suggests that the drinking water microbiome consists of as many as 9,000 distinct taxa, with total numbers ranging between 1,000–500,000 bacteria mL⁻¹ (Proctor and Hammes, 2015, Bautista-de los Santos et al., 2016).

FCM is one exciting “new” method capable of rapidly and accurately counting and characterising practically all bacteria in drinking water. FCM has already been used for microbiological characterisation and quantification in natural aquatic habitats for several decades (Legendre and Yentsch, 1989, Trousellier et al., 1993), but was only recently introduced as a method for drinking water analysis (Hoefel et al., 2003, Hoefel et al., 2005a, Hoefel et al., 2005b, Hammes et al., 2008). All early drinking water FCM studies confirmed the growing awareness of the considerable numerical divide between the total bacteria and the fraction of cultivable bacteria in drinking water (Hoefel et al., 2003, Hammes et al., 2008). Multiple drinking water studies comparing FCM and HPC data argued that FCM is more meaningful for use as a process variable, and questioned the future relevance of HPC measurements (Hoefel et al., 2005a, Hammes et al., 2008, Ho et al., 2012, Liu et al., 2013b, Gillespie et al., 2014).

Here we evaluate the last 15 years of FCM developments and applications in the field of drinking water analysis, and we argue that routine HPC analysis no longer qualifies as the best available technology for the above-stated criteria of relevance, simplicity, reliability, speed and cost-effectiveness. The purpose of this critical review is to initiate a constructive discussion on whether FCM can and should replace HPC as the primary process variable in routine microbiological water quality monitoring. We approached this by briefly assessing the history, advantages and disadvantages of HPC as a process variable, followed by a consideration of several alternative methods that may be suitable as alternatives. We then argue the case for FCM as the method of choice, covering both the advantages and disadvantages of the methodology. We also compare FCM to HPC and ATP with extensive data sets collected over the last decade and outline how FCM could be applied as a monitoring method in the future.