Solid-phase Cytometry Research Articles

Co-transport of Microplastics and a surrogate for Human Enteric Viruses in a saturated column packed with Quartz Sand

Groundwater can be contaminated with infective human enteric viruses from various sources, such as wastewater treatment plant discharge, landfills, septic tanks, agricultural practices, and artificial groundwater recharge. Anthropogenic pollutants, such as microplastics, may exhibit an affinity to transport biocolloids (bacteria, viruses) further and reduce their degradation rates in the natural environment. Human enteric viruses (poliovirus, hepatitis A, rotavirus, and adenovirus) can adsorb to the abiotic surface of microplastics and are simultaneously present in wastewater discharge. These newly formed clumps of pathogens and microplastics could penetrate deeper into soils as vectors for preferential flow and threaten groundwater systems, triggering a higher risk for drinking water and possibly followed by a disease outbreak. The mechanisms behind the adsorption of human enteric viruses on microplastic surfaces and their potential role in prolonging virus survival and promoting environmental transport remain unclear. This study aims to explore the possibility of co-transport of microplastics and human enteric viruses in saturated porous media, using PRD1 bacteriophage as a surrogate. PRD1 bacteriophages have been widely used as surrogates of rotavirus because they share many fundamental properties and features. Column experiments were performed using quartz sand (soil grain size: 0.60 - 1.30 mm) as a porous media in a 30 cm long and 7 cm diameter column. The column experiments were conducted by maintaining Darcy velocity of 2.65 m/day. Three different influent solution scenarios were considered in the experiments: PRD1 mixed with microplastics, PRD1 alone, and microplastics alone. The enumeration of PRD1 in the effluent solution was performed using quantitative polymerase chain reaction (qPCR) as well as the culture method, in order to differentiate between infective and inactive virus transport. Microplastics were quantified using Solid-Phase Cytometry (SPC). Results were analyzed by calculating the collision and sticking efficiencies of the microplastics and PRD1 using the classical colloid filtration theory and Hydrus 1D modeling tool. There was no evidence of interference or inhibition of microplastics on the performance of qPCR and DNA extraction in the methodological setup. Additionally, the efficacy of qPCR and DNA extraction methods did not yield significantly different results across any of the influent solution conditions. Preliminary results suggest that the presence of microplastics enhanced the transport of PRD1, which led to reduced attachment of PRD1 in the porous media. The concentration of infective phages showed a delayed sharp increase, indicating that there may be a sorption mechanism that delays their breakthrough. It is possible that a portion of the active phages possess a higher sticking efficiency and that population heterogeneity contributes to this phenomenon. A comprehensive understanding of the processes that govern virus transport with globally distributed microplastics is crucial for protecting public health.

A rapid sterility method using solid phase cytometry for cell based preparations and culture media and buffers.

In this report, we introduce a rapid sterility testing method for cell-based preparations and its in-process control media/buffers. The selected Rapid Sterility Test (RST) in this work is based on the ScanRDI® system, which detects fluorescently labeled microorganisms with solid phase cytometry. ScanRDI® has been chosen due to its sensitivity for detecting viable microorganisms down to one microbial cell with a shorter time to detection compared to Compendial Sterility Test (CST) method. The RST was validated for a CAR-T cell-therapy product with 4 days of Time to Detection (TTD) and evaluated for in-process control of media/buffers with real time detection method successfully according to USP <1223>, Ph. Eur. 5.1.6, Ph. Eur. 2.6.27 and PDA Technical Report No. 33. The validation parameters included limit of detection and equivalence in routine operations, specificity, robustness, ruggedness and repeatability. For the validation, a combination of pharmacopoeial ATCC strains as well as in-house isolates were used. In addition, the evaluation study of this RST for in-process control of media/buffers was assessed by performing the limit of detection and equivalence with four representative microorganisms. Where applicable, results were statistically evaluated to demonstrate equivalence and no significant difference of the rapid method as compared to the CST method. All acceptance criteria of the validation/evaluation have been met and the solid phase cytometry technology was successfully validated as an alternative sterility test for cell-based preparations and its in-process control of media/buffer.

Predictive Potential of Flow Cytometry Crossmatching in Deceased Donor Kidney Transplant Recipients Subjected to Peritransplant Desensitization.

Recipient sensitization is a major risk factor of antibody-mediated rejection (ABMR) and inferior graft survival. The predictive effect of solid-phase human leukocyte antigen antibody testing and flow cytometry crossmatch (FCXM) in the era of peritransplant desensitization remains poorly understood. This observational retrospective single-center study with 108 donor-specific antibody (DSA)-positive deceased donor kidney allograft recipients who had undergone peritransplant desensitization aimed to analyze variables affecting graft outcome. ABMR rates were highest among patients with positive pretransplant FCXM vs. FCXM-negative (76 vs. 18.7%, p < 0.001) and with donor-specific antibody mean fluorescence intensity (DSA MFI) > 5,000 vs. <5,000 (54.5 vs. 28%, p = 0.01) despite desensitization. In univariable Cox regression, FCXM positivity, retransplantation, recipient gender, immunodominant DSA MFI, DSA number, and peak panel reactive antibodies were found to be associated with ABMR occurrence. In multivariable Cox regression adjusted for desensitization treatment (AUC = 0.810), only FCXM positivity (HR = 4.6, p = 0.001) and DSA number (HR = 1.47, p = 0.039) remained significant. In conclusion, our data suggest that pretransplant FCXM and DSA number, but not DSA MFI, are independent predictors of ABMR in patients who received peritransplant desensitization.

Recent Progress on the Electrochemical Biosensing of Escherichia coli O157:H7: Material and Methods Overview.

Escherichia coli O157:H7 (E. coli O157:H7) is a pathogenic strain of Escherichia coli which has issued as a public health threat because of fatal contamination of food and water. Therefore, accurate detection of pathogenic E. coli is important in environmental and food quality monitoring. In spite of their advantages and high acceptance, culture-based methods, enzyme-linked immunosorbent assays (ELISAs), polymerase chain reaction (PCR), flow cytometry, ATP bioluminescence, and solid-phase cytometry have various drawbacks, including being time-consuming, requiring trained technicians and/or specific equipment, and producing biological waste. Therefore, there is necessity for affordable, rapid, and simple approaches. Electrochemical biosensors have shown great promise for rapid food- and water-borne pathogen detection. Over the last decade, various attempts have been made to develop techniques for the rapid quantification of E. coli O157:H7. This review covers the importance of E. coli O157:H7 and recent progress (from 2015 to 2020) in the development of the sensitivity and selectivity of electrochemical sensors developed for E. coli O157:H7 using different nanomaterials, labels, and electrochemical transducers.

DETECTION ESCHERICHA COLI BACTERIA: A REVIEW OF IMAGE PROCESSING METHODS

There have been numerous chemical studies to detect Eschericha Coli Bacteria or better known as E.coli bacteria. However, studies using image processing to detect E.coli bacteria have been restricted. There is an urgent need to incorporate image processing into systematic reviews for the identification of E.coli in water or food samples. The E.coli detection process takes between 24 hours to 48 hours as the bacteria needs to go through the growth phase as a result of a mixture of water samples and chemical reagents and then placed in an incubator at certain temperatures and humidity. This article review several approaches for E.coli bacteria detection from standard technique, portable hybrid imaging system, hybrid smartphone and microfluidic biosensor, smartphone and paper microfluidics and goes to fibre optics. The colour change after several hours is an indicator for detecting the presence of E.coli bacteria in the water sample. Although standard techniques such as Gold Nanoparticles, Solid phase Cytometry, Viable Plate Count and Polymerase Chain Reaction (PCR) for detection the presence of E.coli, the use of this technique involving the transfer of water samples to the laboratory, trained staff, longer sample monitoring process, expensive costs and complex procedures. E.coli detection techniques are also going through a revolutionary phase as technology advances by using a hybrid technique that combines standard technique and image processing. E.coli growth monitoring and detection process is now easier as it can be viewed using a portable device, smartphone or via a website in real-time.

Spatiotemporal Dynamics of Total Viable Vibrio spp. in a NW Mediterranean Coastal Area.

A cellular approach combining Direct Viable Counting and Fluorescent In Situ Hybridization using a one-step multiple-probe technique and Solid Phase Cytometry (DVC-FISH-SPC) was developed to monitor total viable vibrios and cover the detection of a large diversity of vibrios. FISH combined three probes in the same assay and targeted sequences located at different positions on the 16S rRNA of Vibrio and Aliivibrio members. We performed a 10-month in situ study to investigate the weekly dynamics of viable vibrios relative to culturable counts at two northwestern Mediterranean coastal sites, and identified the key physicochemical factors for their occurrence in water using a multivariate analysis. Total viable and culturable cell counts showed the same temporal pattern during the warmer season, whereas the ratios between both methods were inverted during the colder seasons (<15°C), indicating that some of the vibrio community had entered into a viable but non-culturable (VBNC) state. We confirmed that Seawater Surface Temperature explained 51–62% of the total variance in culturable counts, and also showed that the occurrence of viable vibrios is controlled by two variables, pheopigment (15%) and phosphate (12%) concentrations, suggesting that other unidentified factors play a role in maintaining viability.

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Sterility tests described in official compendia are carried out by membrane filtration or by direct inoculation into suitable culture media. About 14 days are needed to provide results and release products for sale, so speed is of the essence in rapid microbiological methods. Solid phase cytometry is a fast innovative method for testing the sterility of injectable medications. It is based on the detection of viable cells by using reagent viability markers which permeate the cell membrane, and are cleaved by non-specific sterases to form fluorochrome, which is detected by a Chem Scan RDI®. This study set out to evaluate this technology when applied to the sterility test in a 0.9% sodium chloride injection solution, using Chem Scan RDI® equipment. Microorganisms recommended by the official compendia Clostridium sporogenes NCTC12935 (ATCC 11437), Pseudomonas aeruginosa NCTC12924 (ATCC 9027), Staphylococcus aureus NCTC10788 (ATCC 6538), Bacillus subtilis NCTC10400(ATCC 6633), Aspergillus brasiliensis NCPF2275 (ATCC16404) and Candida albicans NCPF3179(ATCC 10231), and two “in house” microorganisms, Micrococcus luteus and Staphylococcus epidermidis, obtained from monitoring the pre-sterilization bioburden, were evaluated in order to validate the proposed method. When the solid phase cytometry method was compared to the traditional membrane filtration sterility test for all the microorganisms tested, it was found to be significantly faster in that it reduced analysis time from 14 days to approximately 3 h. Key words: Solid phase cytometry, sterility test by membrane filtration, validation.

Optimization of solid phase and flow cytometry crossmatch assays through the use of DTT treated patient sera

Aim To determine the effects of DTT treatment of patient sera on C1 interference in mixed bead, single antigen, C1q and flow cytometric assays. Methods For all experiments, patient sera were treated with 0.005M DTT. C1qrs (C1) interference was confirmed for 10 sera samples by dilution studies. We compared single antigen bead (SAB) results for these sera with and without DTT treatment. 4 Of these same sera known to contain complement binding antibodies were used to evaluate the effects of DTT treatment in the C1q assay. The assay for C1q was followed as per manufacturer’s instructions with the exception of the heat inactivation step. We compared our results to IgG subclass testing. 26 T cell and 24 B cell flow crossmatch assays (XM) were run at RT, 4 °C, and with DTT treatment. Results C1 interference was eliminated from all solid phase assays. Repeat testing of the 4 sera using PE-conjugated monoclonal antibodies to the 4 IgG Subclasses confirmed that IgG1 antibodies were present and were binding to the same specificities that had been blocked in the SAB assay due to C1 interference. A modified C1q procedure in which DTT treatment could be done without interfering with the C1q reagent was developed. Results of the modified procedure correlated very well with results of both the IgG subclass testing and the SAB DTT validation. All XMs that showed decreased event counts/C1 interference at RT (30 of 50) showed higher event counts in 4 °C and DTT XM methods, demonstrating that C1 interference was eliminated in 100% of the affected XMs. 100% specificity was seen in both the 4 °C and DTT methods, however, both the RT and 4 °C XMs had one false negative. Lower MFI values were seen in the RT and 4 °C XMs when compared with DTT treated XMs. On average, positive T cell XM values increased by 920 MFI at 4 °C and 1793 MFI with DTT; positive B cell XM values increased by 4894 MFI at 4 °C and 5036 MFI with DTT. All XMs run using DTT achieved expected positive or negative results based on the presence/absence of DSA. Conclusion We have eliminated false negative reactivity in solid phase PRA and flow cytometry XM by incorporating DTT treatment into all assays.

Enumerating Microorganism Surrogates for Groundwater Transport Studies Using Solid-Phase Cytometry

Investigations on the pollution of groundwater with pathogenic microorganisms, e.g. tracer studies for groundwater transport, are constrained by their potential health risk. Thus, microspheres are often used in groundwater transport studies as non-hazardous surrogates for pathogenic microorganisms. Even though pathogenic microorganisms occur at low concentrations in groundwater, current detection methods of microspheres (spectrofluorimetry, flow cytometry and epifluorescence microscopy) have rather high detection limits and are unable to detect rare events. Solid-phase cytometry (SPC) offers the unique capability of reliably quantifying extremely low concentrations of fluorescently labelled microorganisms or microspheres in natural waters, including groundwater. Until now, microspheres have been used in combination with SPC only for instrument calibration purposes and not for environmental applications. In this study, we explored the limits of the SPC methodology for its applicability to groundwater transport studies. The SPC approach proved to be a highly sensitive and reliable enumeration system for microorganism surrogates down to a minimum size of 0.5 μm, in up to 500 ml of groundwater, and 0.75 μm, in up to 1 ml of turbid surface water. Hence, SPC is proposed to be a useful method for enumerating microspheres for groundwater transport studies in the laboratory, as well as in the field when non-toxic, natural products are used.Electronic supplementary materialThe online version of this article (doi:10.1007/s11270-013-1827-3) contains supplementary material, which is available to authorized users.

Whole cell hybridisation for monitoring harmful marine microalgae

Fluorescence in situ hybridisation (FISH) is a powerful molecular biological tool to detect and enumerate harmful microorganism in the marine environment. Different FISH methods are available, and especially in combination with automated counting techniques, the potential for a routine monitoring of harmful marine microalgae is attainable. Various oligonucleotide probes are developed for detecting harmful microalgae. However, FISH-based methods are not yet regularly included in monitoring programmes tracking the presence of harmful marine microalgae. A limitation factor of the FISH technique is the currently available number of suited fluorochromes attached to the FISH probes to detect various harmful species in one environmental sample at a time. However, coupled automated techniques, like flow cytometry or solid-phase cytometry, can facilitate the analysis of numerous field samples and help to overcome this drawback. A great benefit of FISH in contrast to other molecular biological detection methods for harmful marine microalgae is the direct visualisation of the hybridised target cells, which are not permitted in cell free formats, like DNA depending analysis methods. Therefore, an additional validation of the FISH-generated results is simultaneously given.

Rapid quantification of viable fungi in hospital environments: analysis of air and surface samples using solid-phase cytometry

Environmental surveillance is important in high-risk areas of hospitals to prevent fungal infections in immunosuppressed patients. Conventional culture methods for enumerating environmental fungi are time-consuming. In this field study, a solid-phase cytometry technique (SPC) and a more conventional culture-based method to quantify fungal contamination of hospital air and surface samples were compared. For the air sampling, a liquid cyclone air sampler was used with a flow rate of 300L/min for 10min in each of four hospital locations. Surface swabbing was done in two locations, with two different swab types. Samples from all areas were processed by SPC and by culture on malt extract agar. The mean airborne concentrations of viable fungi determined by SPC were about 1.5-fold higher than the mean concentrations obtained with the culture-based method. These differences for air samples were significant in three hospital environments. No significant difference was observed for surface samples between the two swab types and between the two analytical methods. One of the prominent advantages of SPC was its rapidity in comparison with the culture-based method (5h versus 5 days). In this study, we showed that SPC allows for rapid monitoring of viable fungi in hospital environments. SPC can thus be used to provide an early warning and a rapid implementation of corrective measures. Viable fungi detection may be an important tool to assess infectious risk in wards with immunosuppressed patients.

A new combination of microbial indicators for monitoring composting bioaerosols

Bioaerosols emitted from composting plants are a cause of concern because of their potential impact on occupational health and neighboring residential areas. The aim of this study was to identify microbial indicators that are most useful for monitoring bioaerosol emittance and dispersal by industrial composting plants. Seven microbial indicators were measured in air collected outdoors in natural environments and at eleven composting plants. The indicators were: cultivable bacteria and fungi, total bacteria (epifluorescent microscopy), viable bacteria (solid-phase cytometry) and quantification by qPCR of three microbial indicators which had been previously shown as strongly associated with composting. For each indicator, the increase in concentrations due to the turning of composting piles as compared to the background concentration obtained in natural environments and upwind of composting plants was determined. Based on these results, the most effective combination of three indicators was selected for monitoring composting bioaerosol emissions: viable bacteria as one general indicator of bioaerosol emission and two bacterial phylotypes specific to composting bioaerosol: NA07, affiliated to Saccharopolyspora sp. and NC38, affiliated to the Thermoactinomycetaceae. This set of indicator was then quantified on-site and at increasing distances downwind during the turning of compost windrows in thermophilic phase. Composting activity was considered to affect bioaerosol emission when the concentrations of the three indicators were higher than their respective background levels. For all the composting sites studied, an impact was measureable up to distances of 100 m. Further away, the impact was not systematically observed as it depended on meteorological conditions (wind speed) and on levels of bioaerosol emissions.

Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

A new protocol for rapid, specific, and sensitive cell-based quantification of Vibrio cholerae/Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6 V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific for V. cholerae and V. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantify V. cholerae/V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 × 10(1) cells ml(-1) in the large saline lake Neusiedler See to 5.6 × 10(4) cells ml(-1) in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantify V. cholerae/V. mimicus in ecological studies as well as for risk assessment and monitoring programs.

Rapid assessment of microbiological purity of dialysis water: the promise of solid-phase cytometry assessment and the epifluorescence microscopy method

assessment of glomerular density) and lower glomerular density score. Interestingly, glomeruli with segmental sclerosis, included in the glomerular density metric, were also more common in the group with lower glomerular density (2 versus 0.2%.) The infrequent and highly variable presence of segmental sclerosis likely underlies the lack of statistically significant difference in sclerosis between groups with higher versus lower glomerular density. Exclusion of patients with decreased renal function at time of biopsy likely explains why this lesion was so rare. Not surprisingly, interstitial fibrosis >10% was linked to lower glomerular density than interstitial fibrosis < 10%, with similar distinction when the interstitial fibrosis threshold was set at 20%. Yet, these variables of interstitial fibrosis/tubular atrophy did not predict outcome in univariate or multivariate analysis nor did global sclerosis per se .T he robust predictive value of the simple metric of glomerular density thus may have complex underlying pathophysiological mechanisms that may not be easy to unravel. Nonetheless, in this group of patients who were indistinguishable at the onset, this information, in conjunction with a response of proteinuria to therapeutic intervention, significantly contributed to identification of patients with poor prognosis. Regardless of the causality of the decrease in number of glomeruli relative to cortical area, this simple technique may offer additional guidance to identify patients in whom we should target our most aggressive interventions.

Relationship between the culturability of stressed Listeria monocytogenes cells in non-selective and selective culture media and the cellular esterase activity measured by solid phase cytometry

Solid phase cytometry in conjunction with fluorescent probe was applied to rapidly quantify cellular esterase activity of Listeria monocytogenes cells. Viability of cells stressed by several treatments (starvation, NaCl, lactic acid and peracetic acid) was assessed simultaneously by their esterase activity estimated by fluorescence intensity and by their ability to multiply in liquid and solid, non-selective and selective, culture media. It was determined that cell physiological state has a significant impact on the cellular fluorescence intensity which was very dependent on the stress suffered by cells. No general relationship was observed between the bacterial populations observed by cytometry and the populations able to grow on culture media. The link between the cell culturability in non-selective and selective media and the esterase activity was always dependent on the stress suffered. Nevertheless, it was also established that solid phase cytometry is an efficient, sensitive and accurate tool to characterize the ability of non-selective and selective enrichment broths to allow the repair of stressed L. monocytogenes cells by examining the increase in the fraction of the most esterase active cells during the course of resuscitation.

Applicability of solid-phase cytometry and epifluorescence microscopy for rapid assessment of the microbiological quality of dialysis water

Currently, the gold standard to assess the microbiological quality of dialysis water is the determination of heterotrophic plate counts (HPC). The long waiting time of the HPC method and the fact that most bacteria are not culturable on agar plates provokes the search for rapid alternative methods for monitoring the microbiological quality of dialysis water. We tested the applicability of total viable counts (TVC) and total direct counts (TDC), determined via solid-phase cytometry and epifluorescence microscopy (EFM), in comparison to the standard HPC determination method in 113 samples from 13 dialysis water treatment units (59 drinking water and 54 dialysis water samples). Additionally, for a set of dialysis water samples (n = 22) endotoxin concentrations were also determined. TVC showed high correlation with HPC and results were of comparable magnitude for most investigated dialysis water samples [median: 3 cells/colony forming units (CFU) 100 mL(-1)]. However, in one dialysis water sample, HPC values (5800 CFU 100 mL(-1)) were >35-fold lower than TVC values (2.05 × 10(5) cells 100 mL(-1)) indicating severe limits of the HPC method to assess the microbiological quality of dialysis water. For drinking water, TVC (median: 4.8 × 10(4) cells 100 mL(-1)) was on average one order of magnitude higher than HPC (median: 2.5 × 10(3) cells 100 mL(-1)). TDCs (median dialysis water: 1.1 × 10(4) cells 100 mL(-1) and median drinking water: 4.9 × 10(6) cells 100 mL(-1)) were always several orders of magnitude higher than HPC or TVC. We propose that the TVC/solid-phase cytometry approach is a reliable and rapid alternative to the culture-dependent approach for assessment of the microbiological quality of dialysis water, especially when fast results are needed. TDC determined via EFM lacks sensitivity and reliability for assessing microbial concentrations in low-cell dialysis water samples since the limits of detection and quantification are high.

Total and Viable Legionella pneumophila Cells in Hot and Natural Waters as Measured by Immunofluorescence-Based Assays and Solid-Phase Cytometry

A new method was developed for the rapid and sensitive detection of viable Legionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viable L. pneumophila cells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter(-1), and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viable L. pneumophila cells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 10(3) viable but nonculturable L. pneumophila cells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viable L. pneumophila in both man-made water systems and environmental water samples.

Intensive exploitation of a karst aquifer leads to Cryptosporidium water supply contamination

Groundwater from karst aquifers is an important source of drinking water worldwide. Outbreaks of cryptosporidiosis linked to surface water and treated public water are regularly reported. Cryptosporidium oocysts are resistant to conventional drinking water disinfectants and are a major concern for the water industry. Here, we examined conditions associated with oocyst transport along a karstic hydrosystem, and the impact of intensive exploitation on Cryptosporidium oocyst contamination of the water supply. We studied a well-characterized karstic hydrosystem composed of a sinkhole, a spring and a wellbore. Thirty-six surface water and groundwater samples were analyzed for suspended particulate matter, turbidity, electrical conductivity, and Cryptosporidium and Giardia (oo)cyst concentrations. (Oo)cysts were identified and counted by means of solid-phase cytometry (ChemScan RDI ®), a highly sensitive method. Cryptosporidium oocysts were detected in 78% of both surface water and groundwater samples, while Giardia cysts were found in respectively 22% and 8% of surface water and groundwater samples. Mean Cryptosporidium oocyst concentrations were 29, 13 and 4/100 L at the sinkhole, spring and wellbore, respectively. Cryptosporidium oocysts were transported from the sinkhole to the spring and the wellbore, with respective release rates of 45% and 14%, suggesting that oocysts are subject to storage and remobilization in karst conduits. Principal components analysis showed that Cryptosporidium oocyst concentrations depended on variations in hydrological forcing factors. All water samples collected during intensive exploitation contained oocysts. Control of Cryptosporidium oocyst contamination during intensive exploitation is therefore necessary to ensure drinking water quality.

Sensitive counting of viable Enterobacteriaceae in seawaters and relationship with fecal indicators

A fluorescent in situ hybridization based assay was used to enumerate viable Enterobacteriaceae members in seawaters by solid phase cytometry. The method was specific, highly sensitive (1cell/100ml) and allowed the quantification of VNC Enterobacteriaceae cells during an osmotic stress. Investigations on contaminated coastal seawater revealed a strong correlation between Enterobacteriaceae counts and standard fecal indicators.

Rapid detection of Escherichia coli in waters using fluorescent in situ hybridization, direct viable counting and solid phase cytometry

We developed an improved Fluorescent In Situ Hybridization FISH-based method to detect viable Escherichia coli cells by solid phase cytometry (SPC), and results were compared to those obtained by the standard culture method. The method includes a direct viable count (DVC) assay, multi-probes labelled and unlabelled (helpers) to detect specifically viable E. coli cells and to enhance SPC cell counts. We demonstrate that helpers increase the fluorescence intensity of hybridized E. coli cells as detected by SPC and assess the high specificity of the DVC-FISH procedure on a large panel of cultured strains. Application to seawater, freshwater and wastewater samples showed a good correlation between SPC cells counts and standard plate counts. The high specificity of the procedure was demonstrated as well as its accuracy for detecting and counting viable E. coli cells in environmental samples. The developed approach may be used to monitor faecal contamination sources and to investigate the occurrence of viable E. coli in natural environments.