Thermal and chemical disinfection of water and biofilms: only a temporary effect in regard to the autochthonous bacteria.

Thermal and chemical disinfection of technical water systems not only aim at minimizing the level of undesired microorganisms, but also at preventing excessive biofouling, clogging and interference with diverse technical processes. Typically, treatment has to be repeated in certain time intervals, as the duration of the effect is limited. The transient effect of disinfection was demonstrated in this study applying different treatments to water and biofilms including heat, chlorination, a combination of hydrogen peroxide and peracetic acid and monochloramine. Despite the diverse treatments, the reduction in live bacteria was followed by regrowth in all cases, underlining the universal validity of this phenomenon. The study shows that autochthonous bacteria can reach the concentrations given prior to treatment. The reason is seen in the nutrient concentration that has not changed and that forms the basis for regrowth. Nutrients are released by disinfection from lysed cells or are still fixed in dead biomass that is subsequently scavenged by necrotrophic growth. Treatment cycles therefore only provide a transient reduction of water microbiology if nutrients are not removed. When aiming at greater sustainability of the effect, biocidal treatment has to be equally concerned about nutrient removal by subsequent cleaning procedures as about killing efficiency.

Microbiological tap water profile of a medium-sized building and effect of water stagnation.

Whereas microbiological quality of drinking water in water distribution systems is routinely monitored for reasons of legal compliance, microbial numbers in tap water are grossly understudied. Motivated by gross differences in water from private households, we applied in this study flow cytometry as a rapid analytical method to quantify microbial concentrations in water sampled at diverse taps in a medium size research building receiving chlorinated water. Taps differed considerably in frequency of usage and were located in laboratories, bathrooms, and a coffee kitchen. Substantial differences were observed between taps with concentrations (per mL) in the range from 6.29 x 10(3) to 7.74 x 10(5) for total cells and from 1.66 x 10(3) to 4.31 x 10(5) for intact cells. The percentage of intact cells varied between 7% and 96%. Water from taps with very infrequent use showed the highest bacterial numbers and the highest proportions of intact cells. Stagnation tended to increase microbial numbers in water from those taps which were otherwise frequently used. Microbial numbers in other taps that were rarely opened were not affected by stagnation as their water is probably mostly stagnant. For cold water taps, microbial numbers and the percentage of intact cells tended to decline with flushing with the greatest decline for taps used least frequently whereas microbial concentrations in water from hot water taps tended to be somewhat more stable. We conclude that microbiological water quality is mainly determined by building-specific parameters. Tap water profiling can provide valuable insight into plumbing system hygiene and maintenance.

Use of propidium monoazide and increased amplicon length reduce false-positive signals in quantitative PCR for bioburden analysis.

Rapid microbiological methods (RMMs) as an alternative to conventional cultivation-based bioburden analysis are receiving increasing attention although no single technology is currently able to satisfy the needs of the health care industry. Among the RMMs, quantitative PCR (qPCR) seems particularly suited. Its implementation is, however, hampered by false-positive signals originating from free DNA in PCR reagents or from dead cells in the samples to be analysed. In this study, we assessed the capability of propidium monoazide (PMA) to inactivate exogenous DNA in PCR reagents and thus to minimise its impact in bioburden analysis. PMA is a membrane-impermeant dye that intercalates into DNA and covalently binds to it upon photoactivation leading to strong inhibition of PCR amplification. PMA is currently used mainly for treatment of microbiological samples to exclude signals from membrane-compromised cells, but is also very useful for suppression of exogenous DNA signals. In addition to testing the effect of different PMA concentrations on non-template controls and target DNA, we demonstrate the effect of amplicon length on the exclusion of background amplification. Targeting a 1,108-bp 16S rRNA gene fragment using universal bacterial primers and PCR reagents treated with 5 µM PMA resulted in complete suppression of signals from exogenous DNA within 50 cycles of amplification, while a limit of detection of 10 copies of Escherichia coli genomic DNA per PCR reaction was achieved. A combined PMA treatment of sample and PCR reagents furthermore improved the selective detection of live cells making this method appear a highly attractive RMM.

Exploring the use of flow cytometry for understanding the efficacy of disinfection in chlorine contact tanks.

A pilot scale chlorine contact tank (CCT) with flexible baffling was installed at an operational water treatment plant (WTP), taking a direct feed from the outlet of the rapid gravity filters (RGF). For the first time, disinfection efficacy was established by direct microbial monitoring in a continuous reactor using flow cytometry (FCM). Disinfection variables of dose, time, and hydraulic efficiency (short circuiting and dispersion) were explored following characterisation of the reactor's residence time distributions (RTD) by tracer testing. FCM enabled distinction to be made between changes in disinfection reactor design where standard culture-based methods could not. The product of chlorine concentration (C) and residence time (t) correlated well with inactivation of microbes, organisms, with the highest cell reductions (N/N0) reaching <0.025 at Ctx¯ of 20 mg.min/L and above. The influence of reactor geometry on disinfection was best shown from the Ct10. This identified that the initial level of microbial inactivation was higher in unbaffled reactors for low Ct10 values, although the highest levels of inactivation of 0.015 could only be achieved in the baffled reactors, because these conditions enabled the highest Ct10 values to be achieved. Increased levels of disinfection were closely associated with increased formation of the trihalomethane disinfection by-products. The results highlight the importance of well-designed and operated CCT. The improved resolution afforded by FCM provides a tool that can dynamically quantify disinfection processes, enabling options for much better process control.

Multiparameter viability assay for stress profiling applied to the food pathogen Listeria monocytogenes F2365.

A novel generic approach for stress profiling was applied to Listeria monocytogenes strain F2365. This food-borne pathogen was exposed to gradients of five different stresses of increasing intensity, typically ranging from moderate to lethal conditions. The stress factors included heat, acidic pH, a detergent disinfectant, an oxidant, and hyperosmotic conditions. In addition to CFU counts and lag time, five different molecular viability parameters were measured by fluorescence-based assays, including membrane integrity, membrane potential, esterase activity, redox activity, and intracellular pH stability. The last was measured by our recently invented real-time viability assay. Exposure to all stresses resulted in clear dose-response relationships for all viability parameters with the exception of hyperosmotic conditions. A statistical analysis showed strong correlations for (i) the growth parameters plate counts and lag times, (ii) the enzyme-associated functions redox and esterase activity, and (iii) the membrane-associated pH stability and membrane integrity. Results indicated a pronounced difference in the susceptibilities of the measured parameters depending on the stress factor applied. However, at relatively high stress intensities, all of the viability parameters became affected independent of the stress factor. Applications of the approach presented here include studies on the mechanism of action of unknown compounds with biocidal activity and a comparative analysis of the severities of the impact of stress conditions of interest. It appears that a meaningful evaluation of the impact of mild stress conditions can be obtained only through measurement of multiple viability parameters.

Real-time detection of viable microorganisms by intracellular phototautomerism.

BACKGROUND: To date, the detection of live microorganisms present in the environment or involved in infections is carried out by enumeration of colony forming units on agar plates, which is time consuming, laborious and limited to readily cultivable microorganisms. Although cultivation-independent methods are available, they involve multiple incubation steps and do mostly not discriminate between dead or live microorganisms. We present a novel generic method that is able to specifically monitor living microorganisms in a real-time manner. RESULTS: The developed method includes exposure of cells to a weak acid probe at low pH. The neutral probe rapidly permeates the membrane and enters the cytosol. In dead cells no signal is obtained, as the cytosolic pH reflects that of the acidic extracellular environment. In live cells with a neutral internal pH, the probe dissociates into a fluorescent phototautomeric anion. After reaching peak fluorescence, the population of live cells decays. This decay can be followed real-time as cell death coincides with intracellular acidification and return of the probe to its uncharged non-fluorescent state. The rise and decay of the fluorescence signal depends on the probe structure and appears discriminative for bacteria, fungi, and spores. We identified 13 unique probes, which can be applied in the real-time viability method described here. Under the experimental conditions used in a microplate reader, the reported method shows a detection limit of 10(6) bacteria ml(-1), while the frequently used LIVE/DEAD BacLight Syto9 and propidium iodide stains show detection down to 10(6) and 10(7) bacteria ml(-1), respectively. CONCLUSIONS: We present a novel fluorescence-based method for viability assessment, which is applicable to all bacteria and eukaryotic cell types tested so far. The RTV method will have a significant impact in many areas of applied microbiology including research on biocidal activity, improvement of preservation strategies and membrane permeation and stability. The assay allows for high-throughput applications and has great potential for rapid monitoring of microbial content in air, liquids or on surfaces.

Discrimination between live and dead cellsin bacterial communities from environmental water samples analyzed by 454 pyrosequencing.

SUMMARY: The preferential detection of cells with intact membranes by sample treatment with propidium monoazide (PMA) in combination with PCR amplification is gaining in popularity. This study evaluates the effect of PMA on 454 pyrosequencing profiles of environmental water samples from a canal in Amsterdam and seawater (with sediment) left untreated or exposed to elevated temperatures (50, 60, or 85 °C) for 10 min. Community analysis was based on the extraction of genomic DNA followed by PCR amplification of 16S rRNA genes using universal bacterial primers. Whereas the highest temperature in combination with PMA treatment completely suppressed PCR amplification, PCR products from the other samples were subjected to massively parallel tag sequencing. PMA treatment did not substantially affect the sequence profiles of non-heated samples, but heat exposure resulted in a clear difference in the relative proportions of certain groups. This difference was significantly more pronounced in heated seawater than in heated canal water. The effect of the chosen experimental conditions on the membrane integrity of cells was supported by BacLight LIVE/DEAD staining in combination with flow cytometry, which confirmed an increase in the uptake of propidium iodide in samples exposed to high temperatures.

Microbiological examination of water and aerosols from four industrial evaporative cooling systems in regard to risk of Legionella emissions and methodological suggestions for surveillance.

The hygienic risk associated with evaporative cooling systems in Germany is currently only assessed by determining concentrations of Legionella spp. in the corresponding cooling waters. Relevant for the health risk is however the load of Legionella in emitted aerosols. In this work aerosol emissions from four industrial cooling systems (A - D) were analyzed. A microbiological air bioburden factor (MABF) is suggested to be useful to assess the overall microbiological load of emitted air and to judge the efficiency of droplet separation and overall microbiological retention. Whereas the MABF by itself only serves as a technical quality assurance (QA) parameter, the hygienic relevance has to be seen in combination with the assessment of Legionella either contained in the aerosol or in the cooling water. Plate counting of colonies was an appropriate method to quantify Legionella spp. in aerosols given the short time of flight at the chosen sampling locations and resulting low risk of desiccation. qPCR data on the other hand proved more reproducible than the culture approach to quantify Legionella spp. concentrations in cooling water-. The application of qPCR also allowed to assess the relative proportion of Legionella pneumophila within the total pool of Legionella which adds epidemiological relevance to risk assessment. A traffic light system was proposed to guide interpretation of qPCR data. The four industrial systems greatly differed in all measured parameters leading to different associated risks.

Lysis Performance of Bacteriophages with Different Plaque Sizes and Comparison of Lysis Kinetics After Simultaneous and Sequential Phage Addition.

Background: Although bacteriophages see a revival for specifically removing undesired bacteria, there is still much uncertainty about how to achieve the most rapid and long-lasting clearance. Materials and Methods: This study investigated the lysis kinetics of three distinct environmental coliphages, reproducibly forming different plaque sizes (big, medium, and small). Lysis performance by individual phages was compared with the one obtained after simultaneous or sequential addition of all three phages. Kinetics was monitored by density absorbance or by flow cytometry, with the latter having the advantage of providing higher sensitivity. Results: Plaque size happened to correlate with lysis kinetics in liquid suspensions, with phages producing big (phage B), medium (phage M), and small (phage S) plaques showing maximal bacterial clearance under the chosen conditions within ∼6, 12, and 18 h, respectively. Use of a phage cocktail (all three phages added simultaneously) resulted in slower initial lysis compared with the fastest lysing phage with the greatest plaque size alone, but it showed longer efficacy in suppression. When adding phages sequentially, overall lysis kinetics could be influenced by administering phages at different time points. The lowest bacterial concentration after 36 h was obtained when administering phages in the sequence S, M, and B although this combination initially took the longest to achieve bacterial clearance. Conclusions: Results support that timing and order of phage addition can modulate strength and duration of bacterial suppression and, thus, influence the overall success of phage treatment.

Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology.

The use of DNA-based molecular detection tools for bacterial diagnostics is hampered by the inability to distinguish signals originating from live and dead cells. The detection of live cells is typically most relevant in molecular diagnostics. DNA-intercalating dyes like ethidium monoazide and propidium monoazide (PMA) offer a possibility to selectively remove cells with compromised cell membranes from the analysis. Once these dyes enter a cell, they bind to DNA and can be covalently crosslinked to it by light exposure. PCR amplification of such modified DNA is strongly inhibited. In this study we evaluated the suitability of propidium monoazide treatment to exclude isopropanol-killed cells from detection in defined mixtures using diagnostic microarray technology. The organisms comprised Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Serratia marcescens, and Escherichia coli O157:H7. PCR products obtained from amplification of chaperonin 60 genes (cpn60; coding for GroEL) were hybridized to a custom-designed microarray containing strain-specific cpn60-based 35-mer oligonucleotide probes. Results were compared with data from quantitative PCR, which confirmed that PMA could successfully inhibit amplification of DNA from killed cells in the mixtures. Although microarray data based on analysis of end-point PCR amplicons is not quantitative, results showed a significant signal reduction when targeting killed cells and consistently agreed with qPCR results. Treatment of samples with PMA in combination with diagnostic microarray detection can therefore be considered beneficial when analyzing mixtures of intact and membrane-compromised cells. Minimization of detection signals deriving from dead cells will render data more relevant in studies including pathogen risk assessment.

Optimization of viability qPCR for selective detection of membrane-intact Legionella pneumophila.

Although a number of viability qPCR assays have been reported to selectively detect signals from membrane-intact Legionella pneumophila, the efficient suppression of amplification of DNA from dead membrane-compromised bacteria remains an ongoing challenge. This research aimed at establishing a new oligonucleotide combination that allows for a better exclusion of dead Legionella pneumophila on basis of the mip gene. Propidium monoazide (PMA) was chosen as viability dye. An oligonucleotide combination for the amplification of a 633 bp sequence was established with 100% specificity for different Legionella pneumophila strains compared with 17 other Legionella species tested. Apart from increasing amplicon length, the study aimed at optimizing dye incubation time and temperature. An incubation temperature of 45 °C for 10 min was found optimal. Dye treatment of heat-killed bacteria in the presence of EDTA improved signal suppression, whereas deoxycholate also affected signals from live intact bacteria. Suppression of signals from heat-treated bacteria was found to be approx. twice as efficient compared to a commercial kit, although the detection sensitivity is superior when targeting short amplicons. With a limit of detection of 10 genome copies per PCR well and a 6-log signal reduction of bacteria killed at 80 °C, the assay appears useful for applications where pathogen numbers are not limiting and where the priority is on the distinction between intact and damaged Legionella pneumophila for the evaluation of hygienic risk and of disinfection efficiency.

Effect of turbidity on water disinfection by chlorination with the emphasis on humic acids and chalk.

Chlorine is globally the most widely used chemical for water disinfection. Whereas disinfection efficiency is well known to depend on water pH and temperature, the effect of turbidity is less well studied. Although turbidity is measured online in most drinking water works and most countries where regulations exist have set limits of <1 NTU for water leaving the works, the composition of turbidity is typically unknown. Given the heterogeneous nature of substances contributing to turbidity, the aim of this work was to study the effect of selected compounds on chlorination efficacy. The effect of humic acids and chalk on the inactivation of the indicator bacteria Escherichia coli and Enterococcus faecalis was assessed at neutral pH at different turbidity levels using both plate counting and flow cytometry in combination with membrane integrity staining. For humic acids, a turbidity of 1 NTU (corresponding to 2 mg L-1) was identified as a critical threshold, which when exceeded was found to have a negative impact on chlorine disinfection. Chalk, on the other hand, had no measurable impact up to 5 NTU. The observation applied to both bacterial species with identical conclusions from the two diagnostic methods. Results corroborate that different turbidity causing substances affect chlorination efficiency to very different extents with chlorine demand by organic material probably being the most important determinant. In the case of turbidities >1 NTU, turbidity measurement benefits from the consideration of the organic content as mere NTU values do not allow predicting an impact on chlorination efficiency.

Comparing flow cytometry with culture-based methods for microbial monitoring and as a diagnostic tool for assessing drinking water treatment processes.

Flow cytometry (FCM) and the ability to measure both total and intact cell populations through DNA staining methodologies has rapidly gained attention and consideration across the water sector in the past decade. In this study, water quality monitoring was undertaken over three years across 213 drinking water treatment works (WTW) in the Scottish Water region (Total n = 39,340). Samples subject to routine regulatory microbial analysis using culture-based methods were also analysed using FCM. In addition to final treated water, the bacterial content in raw water was measured over a one-year period. Three WTW were studied in further detail using on-site inter-stage sampling and analysis with FCM. It was demonstrated that there was no clear link between FCM data and the coliform samples taken for regulatory monitoring. The disinfectant Ct value (Ct = mg·min/L) was the driving factor in determining final water cell viability and the proportion of intact cells (intact/total cells) and the frequency of coliform detections in the water leaving the WTW. However, the free chlorine residual, without consideration of treatment time, was shown to have little impact on coliform detections or cell counts. Amongst the three treatment trains monitored in detail, the membrane filtration WTW showed the greatest log removal and robustness in terms of final water intact cell counts. Flow cytometry was shown to provide insights into the bacteriological quality of water that adds significant value over and above that provided by traditional bacterial monitoring.

The absence or presence of a lytic coliphage affects the response of Escherichia coli to heat, chlorine, or UV exposure.

Disinfection aims at maximal inactivation of target organisms and the sustainable suppression of their regrowth. Whereas many disinfection efforts achieve efficient inactivation when the effect is measured directly after treatment, there are questions about the sustainability of this effect. One aspect is that the treated bacteria might recover and regain the ability to grow. In an environmental context, another question is how amenable surviving bacteria are to predation by omnipresent bacteriophages. Provisional data suggested that bacteria when subjected to sublethal heat stress might develop a phage-resistant phenotype. The result made us wonder about the susceptibility to phage-mediated lysis for bacteria exposed to a gradient of chlorine and UV-LED disinfection strengths. Whereas bacteria exposed to low sublethal chlorine doses still underwent phage-mediated lysis, the critical chlorine Ct of 0.5 mg min/L eliminated this susceptibility and induced phage resistance in the cells that survived treatment. In the case of UV, even the smallest tested dose of 2.8 mJ/cm2 abolished phage lysis leading to direct regrowth. Results suggest that bacteria surviving disinfection might have higher environmental survival chances directly after treatment compared to non-treated cells. A reason could possibly lie in their compromised metabolism that is essential for phage replication.

Turbidity composition and the relationship with microbial attachment and UV inactivation efficacy.

Turbidity in water can be caused by a range of different turbidity causing materials (TCM). Here the characteristics and attachment of bacteria to TCMs was assessed and the resultant impact on UV disinfection determined. TCMs represent potential vehicles for bacterial penetration of water treatment barriers, contamination of potable supplies and impact on subsequent human health. The TCMs under investigation were representative of those that may be present in surface and ground waters, both from the source and formed in the treatment process. The TCMs were chalk, Fe (III) hydroxide precipitate, kaolin clay, manganese dioxide and humic acids, at different turbidity levels representative of source waters (0, 0.1, 0.2, 0.4, 1, 2, and 5 NTU). Escherichia coli and Enterococcus faecalis attachment followed the order of Fe(III)>chalk, with little to no attachment seen for MnO2, humic acids and clay. The attachment was postulated to be due to chalk and Fe(III) particles having a more neutral surface charge resulting in elevated aggregation with bacteria compared to other TCMs. The humic acids and Fe(III) were the TCMs which influenced inactivation of E. coli and E. faecalis due to decreasing UV transmittance (UVT) with increasing TCM concentration. The presence of the Fe(III) TCM at 0.2 NTU resulted in the poorest E. coli inactivation, with 2.5 log10 reduction at UV dose of 10mJcm-2 (kd of -0.23cm2mJ-1) compared to a 3.9 log10 reduction in the absence of TCMs. E. faecalis had a greater resistance to UV irradiation than E. coli for all TCMs. Effective disinfection of drinking water is a priority for ensuring high public health standards. Uniform regulations for turbidity levels for waters pre-disinfection by UV light set by regulators may not always be appropriate and efficacy is dependent on the type, as well as the amount, of turbidity present in the water.

Flow cytometry-based evaluation of the bacterial removal efficiency of a blackwater reuse treatment plant and the microbiological changes in the associated non-potable distribution network.

The study evaluated the changes in bacterial numbers across a full-scale membrane bioreactor (MBR) blackwater reuse system. Flow cytometry was used to quantify total and intact bacterial concentrations across the treatment train and during distribution of the recycled water. Membrane passage reduced bacterial numbers by up to 5-log units resulting in coliform-free permeate. A 2-log increase in bacterial cell concentration was subsequently observed after the granular activated carbon unit followed by a reduction in intact cells after chlorination, which corresponds to an overall intact bacteria removal of 3.4-log units. In the distribution network, the proportion of intact cells greatly depended on the free chlorine residual, with decreasing residual enabling regrowth. An initial target of 0.5 mg L-1 free chlorine ensured sufficient suppression of intact cells for up to 14 days (setting the time intervals for system flushes at times of low water usage). Bacterial regrowth was only observed when the free chlorine concentration was below 0.34 mg L-1. Such loss of residual chlorine mainly applied to distant points in the distribution network from the blackwater reuse treatment plant (BRTP). Flushing these network points for 5 min did not substantially reduce cell numbers. At points closer to the BRTP, on the other hand, flushing reduced cell numbers by up to 1.5-log units concomitant with a decreasing proportion of intact cells. Intact cell concentrations did not correlate with DOC, total nitrogen, or soluble reactive phosphate, but it was shown that dead biomass could be efficiently converted into new biomass within seven days.

Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR.

One of the major drawbacks of DNA-based microbial diagnostics is its inability to discriminate between live and dead bacteria. Due to the persistence of DNA in the environment after cells have lost their viability, DNA-based assays cannot assess pathogenic risk since signals can originate from both live and dead cells. Presented here is a potential application of the novel chemical propidium monoazide (PMA), which results in the selective suppression of DNA detection from dead cells. PMA can only penetrate dead cells with permeabilized cell membranes. Upon intercalation into the DNA, covalent crosslinkage of PMA to DNA is achieved through light exposure. This modification prevents the DNA from being amplified by PCR. The method, in combination with quantitative PCR as a diagnostic tool, successfully monitored the disinfection efficacy of hypochlorite, benzalkonium and heat on several model pathogens. Threshold cycle numbers increased with increasing disinfection strength after PMA treatment of samples compared to non-PMA treated samples. With some disinfectant-specific differences, monitoring viability loss with membrane integrity as an indicator seemed to be more conservative than monitoring viability loss with plate counts. Loss of viability after short UV-exposure could not be monitored with PMA as UV light affects viability by inducing DNA damage without directly affecting membrane permeability.

The feasibility of improved live-dead distinction in qPCR-based microbial source tracking.

PCR-based microbial source tracking (MST) has become a useful tool to identify dominant sources of fecal pollution in water. The method has previously been successfully combined with viability PCR (using propidium monoazide) allowing the preferential detection of membrane-intact bacteria. This study aimed at further improving the selectivity for intact cells when targeting host-specific markers in Bacteroidales bacteria. One approach was to increase amplicon sizes that had been shown to be useful for other applications of viability PCR. For this purpose, two different amplicon sizes were compared when targeting either the genus of Bacteroidales or subgroups thereof specifically associated with human and ruminant fecal material. When applied to different environmental samples, the proposed proportion of intact cells could drop by up to 38% (for sewage treatment effluent from 64 to 26%) when targeting longer sequences. Furthermore co-incubation of the viability dye with dimethylsulfoxide (DMSO) was found to be beneficial, although this observation is currently still empirical. When examining signal decay of artificially contaminated unfiltered river water over six weeks, the PMA treatment effect was observed from the beginning, but the ratio of intact and damaged cells remained constant over time with signals disappearing at the same rate independent of PMA treatment. In this instance the contribution of other factors to overall signal decay seemed more important than loss of membrane integrity.

When are bacteria dead? A step towards interpreting flow cytometry profiles after chlorine disinfection and membrane integrity staining.

Flow cytometry is increasingly employed by drinking water providers. Its use with appropriate fluorescent stains allows the distinction between intact and membrane-damaged bacteria, which makes it ideally suited for assessment of disinfection efficiency. In contrast to plate counting, the technology allows the visualization of the gradual loss of membrane integrity. Although this sensitivity per se is very positive, it creates the problem of how this detailed viability information compares with binary plate counts where a colony is either formed or not. Guidelines are therefore needed to facilitate interpretation of flow cytometry results and to determine a degree of membrane damage where bacteria can be considered 'dead'. In this study we subjected Escherichia coli and environmental microorganisms in real water to increasing chlorine concentrations. Resulting flow cytometric patterns after membrane integrity staining were compared with culturability and in part with redox activity. For laboratory-grown bacteria, culturability was lost at lower disinfectant concentrations than membrane integrity making the latter a conservative viability parameter. No recovery from chlorine was observed for four days. For real water, loss of membrane integrity had to be much more substantial to completely suppress colony formation, probably due to the heterogenic composition of the natural microbial community with different members having different susceptibilities to the disinfectant.

Schrödinger's microbes: Tools for distinguishing the living from the dead in microbial ecosystems.

While often obvious for macroscopic organisms, determining whether a microbe is dead or alive is fraught with complications. Fields such as microbial ecology, environmental health, and medical microbiology each determine how best to assess which members of the microbial community are alive, according to their respective scientific and/or regulatory needs. Many of these fields have gone from studying communities on a bulk level to the fine-scale resolution of microbial populations within consortia. For example, advances in nucleic acid sequencing technologies and downstream bioinformatic analyses have allowed for high-resolution insight into microbial community composition and metabolic potential, yet we know very little about whether such community DNA sequences represent viable microorganisms. In this review, we describe a number of techniques, from microscopy- to molecular-based, that have been used to test for viability (live/dead determination) and/or activity in various contexts, including newer techniques that are compatible with or complementary to downstream nucleic acid sequencing. We describe the compatibility of these viability assessments with high-throughput quantification techniques, including flow cytometry and quantitative PCR (qPCR). Although bacterial viability-linked community characterizations are now feasible in many environments and thus are the focus of this critical review, further methods development is needed for complex environmental samples and to more fully capture the diversity of microbes (e.g., eukaryotic microbes and viruses) and metabolic states (e.g., spores) of microbes in natural environments.