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Aims: To test Fountain FlowTM Cytometry (FFC) for the rapid and sensitive detection of Naegleria lovaniensis amoebae (an analogue for Naegleria fowleri) in natural river waters.
Methods and Results: Samples were incubated with one of two fluorescent labels to facilitate detection: ChemChrome V6, a viability indicator, and an R-phycoerytherin (RPE) immunolabel to detect N. lovaniensis specifically. The resulting aqueous sample was passed as a stream in front of a light-emitting diode, which excited the fluorescent labels. The fluorescence was detected with a digital camera as the sample flowed toward the imager. Detections of N. lovaniensis were made in inoculated samples of natural water from eight rivers in France and the United States. FFC enumeration yielded results that are consistent with other counting methods: solid-phase cytometry, flow cytometry, and hemocytometry, down to concentrations of 0·06 amoebae ml−1, using a flow rate of 15 ml min−1.
Conclusions: This study supports the efficacy of using FFC for the detection of viable protozoa in natural waters and indicates that use of RPE illuminated at 530 nm and detected at 585 nm provides a satisfactory means of attenuating background.
Significance and Impact of the Study: Because of the severe global public health issues with drinking water and sanitation, there is an urgent need to develop a technique for the real-time detection of viable pathogens in environmental samples at low concentrations. FFC addresses this need.
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Pathogenic micro-organisms are known to cause widespread waterborne disease worldwide, accounting for nearly 6000 deaths per day, mostly in children (WHO/UNICEF 2002). Contamination in drinking and bathing water by pathogenic micro-organisms is one of the greatest causes of preventable disease (WHO/OEDC 2003). The ability to routinely screen raw and processed waters for pathogenic micro-organisms, including protozoa, is essential to protect public health. Water monitoring usually includes: bulk water filtration, concentration, incubation, and culturing. Alternative detection methods, such as polymerase chain reaction (PCR) and conventional flow cytometry, suffer from interference from background particulates and require expensive laboratory equipment and highly trained personnel for analyses of micro-organisms in bulk water particulate concentrates (Sluter et al. 1997). Methods based on culturing often require days from sample acquisition to result, which often means that drinking water is consumed prior to results of contaminant analyses. For environmental water analyses, it is often necessary to collect samples in the field, necessitating shipment of water samples to laboratories. These factors preclude routine monitoring of pathogenic micro-organisms. In addition, for those pathogen-positive samples requiring confirmatory analyses, the technology should be sample-nondestructive (Ford 1999).
In the previous work (Johnson et al. 2002, 2006; Johnson 2004, 2006), we described a novel approach for the detection of bacteria in aqueous samples using a methodology which we call Fountain Flow™ Cytometry (FFC). FFC is an imaging system for the detection and enumeration of micro-organisms with fluorescent labels in aqueous samples. This system is a precursor to a rapid, field-portable, low-cost screening technology that will allow rapid identification and quantification of contamination, and provide early warning screening of water supplies. The FFC in Johnson et al. (2006) was used to detect Escherichia coli in buffer and natural water at moderately low concentrations (down to c. 200 bacteria ml−1) and low flow rates (2·1 ml h−1). This FFC used an argon-ion laser for illumination and a commercial CCD (charge-coupled device) camera to image fluorescent bacteria for detection and enumeration.
In this paper, we test an inexpensive light-emitting diode (LED)-illuminated FFC to detect viable amoebae, Naegleria lovaniensis, at concentrations of 0·06–3·0 amoebae ml−1 and a flow rate of 15 ml min−1 (Fig. 1). Naegleria lovaniensis is used as an analogue for the highly pathogenic amoeba Naegleria fowleri. In addition, we address the problem of detection of amoebae in natural river water with high levels of background autofluorescence from organic and nonorganic particulates, by selecting dyes and filters which avoid bandpasses corresponding to high emissivity from natural pigments, such as chlorophyll a and b.
Figure 1. Schematic diagram of a light-emitting diode (LED)-illuminated epifluorescent Fountain Flow Cytometer. A sample of fluorescently tagged cells flows through the flow cell toward the CMOS camera and fore-optics. The cells are illuminated in the focal plane by an LED. When the cell(s) pass through the CMOS camera focal plane they are imaged by the camera and lens assembly through the transparent flow cell window, and a filter that isolates the wavelength of fluorescence emission. The fluid in which the cells are suspended then passes by the window and out the flow cell drain tube.
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For this study, amoebae were stained with ChemChrome V6 (CV6) (Chemunex, Paris, France), a viability dye, and an R-phycoerytherin (RPE) immunolabel specific to N. lovaniensis, prior to inoculation into buffer or natural river water. One motivation for these tests was to determine the effectiveness of using two dyes emitting at two clearly separated wavelengths; one to specifically detect N. lovaniensis and the second to determine its viability as well as to confirm the detection. Another requirement was to avoid false-positive detections in the immunolabel bandpass which would confuse amoeba-sized organic detritus with N. lovaniensis. These experiments were nonsimultaneous two-colour measurements, a precursor to eventual simultaneous measurements of N. lovaniensis in natural river water.
Four sets of experiments were made in this study. Data were taken on two nearly identical FFC systems, one in the United States and another in France. These experiments were performed to: (i) determine the sensitivity of the FFC system and to optimize the excitation/emission filters; (ii) to determine the occurrence of false-positive events; (iii) to validate FFC counts of amoebae in buffer by comparison between FFC and hemocytometry; and (iv) to validate FFC counts of amoebae in natural river water by comparison between FFC and solid-phase cytometry and flow cytometry.