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Keywords:

  • cell detection;
  • environmental monitoring;
  • flow cytometry;
  • Naegleria lovaniensis;
  • protozoa detection;
  • water monitoring

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

image

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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

FFC measurements of river water without amoebae

FFC measurements of uninoculated river water were made on 2 July 2005, in order to make dye and filter selections (Table 1). Eleven-millilitre samples were measured with a flow rate of 3·3 ml min−1. Five hundred frames were taken on each sample with an exposure time of 400 ms. There was no significant ‘dead’ time between exposures. The Seine, Loire, Garonne, Rhone, and Vienne were sampled in January 2005. The Tech River water samples were taken in May 2005. The Laramie River was sampled on 30 June 2005. All samples were refrigerated prior to use. Water samples that showed high turbidity or a high rate of false-positive detections were filtered with a 20-μm paper filter (Whatman, Springfield Mill, UK) before being measured with the FFC. The July 2005 Laramie River sample was not taken directly from the Laramie River but from a stagnant marsh on the river bank. This sample was visibly green, even after filtration, and the water exhibited a high fluorescent background. This location was chosen to sample water with an unusually high content of chlorophyll-bearing particulate matter.

Table 1.   Enumeration of filtered and unfiltered river water, showing false detections from null (uninoculated) samples. Excitation/emission pairs were chosen to explore the use of RPE (excitation either at 470 or 530 nm and emission at 585 nm), RPE-CY5 (530-nm excitation and 675-nm emission), and FluoProbes 647 (excitation at 625 nm and emission at 670 nm). Water was filtered when its turbidity was high and/or preliminary measurements showed unacceptably high count rates. July measurements were made with a 4-mm diameter flow orifice
SampleRiverWater filteredExcitation filter wavelength/bandpassEmission filter/ bandpassLEDTotal false detections in 11 ml
  1. Abbreviations: RPE, R-phycoerytherin; LED, light-emitting diode.

a0702LaramieFiltered450-nm LED/500-nm short pass585 nm/40 nmLuxeon III Royal Blue81
d0702LaramieFiltered450-nm LED/500-nm short pass585 nm/40 nmLuxeon III Royal Blue58
e0702LaramieFiltered625-nm LED/650-nm short pass670 nm/40 nmLuxeon III Red147
f0702LaramieFiltered530 nm/30 nm585 nm/40 nmLuxeon V Green0
g0702SeineFiltered530 nm/30 nm585 nm/40 nmLuxeon V Green0
h0702LoireUnfiltered530 nm/30 nm585 nm/40 nmLuxeon V Green1
i0702GaronneUnfiltered530 nm/30 nm585 nm/40 nmLuxeon V Green0
j0702RhoneUnfiltered530 nm/30 nm585 nm/40 nmLuxeon V Green0
k0702SeineUnfiltered530 nm/30 nm585 nm/40 nmLuxeon V Green0
l0702TechFiltered530 nm/30 nm585 nm/40 nmLuxeon V Green1
m0702VienneFiltered530 nm/30 nm585 nm/40 nmLuxeon V Green1
h0707LaramieFiltered530 nm/30 nm675 nm/50 nmLuxeon V Green42

All FFC detections in this study were made using our Biocount™ software (Johnson 2006; Johnson et al. 2006). The uninoculated river water samples measured in July 2005 (Table 1) were analysed with an unusually low threshold for detection, 10 ADU (analogue-to-digital units or the digitized units of fluorescent intensity measured by the FFC). This is normally the detection threshold that is used for bacteria. Our detections for amoebae were based on a 100 ADU threshold for RPE and a 3000 ADU threshold for CV6, consistent with the histograms shown in Fig. 2. Thus, false-positive rates for all the samples are quite conservative.

image

Figure 2.  Histogram of detections of labelled N. lovaniensis. Panel A: ChemChrome V6 (CV6)-labelled Naegleria lovaniensis in 50 ml of phosphate-buffered saline (PBS)/2 at a concentration of 2·0 amoebae ml−1. Intensities were computed by summing the individual pixel intensities in a 100 × 100 box (in the 2 × 2 binned image). Panel B: R-phycoerytherin (RPE)-labelled N. lovaniensis in 50 ml of PBS/2 at a concentration of 2·0 amoebae ml−1. Intensities were computed by summing the individual pixel intensities in a 100 × 50 box (in the 2 × 2 binned image). (A smaller box was used than for CV6 because the streak lengths of the lower intensity RPE-stained amoebae were shorter.)

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Amoebae samples

For our experiments, N. lovaniensis was obtained from two sources: Pr. Pierre Pernin (Faculté de Pharmacie, Lyon) and American Type Culture Collection (ATCC #30569). Naegleria lovaniensis samples were maintained and cultured in 3 ml of SCGYEM liquid medium (De Jonckheere 1977) for 3 days in the dark at 37°C prior to use. Tubes were tilted 10–15° during incubation. All measurements were made on the trophozoite form of N. lovaniensis.

Stain selection

For this project, we chose to illustrate the efficacy of detecting viable amoebae at relatively low concentrations using the CV6 viability dye (Chemunex, Paris) and a N. lovaniensis antibody (Indicia Diagnostic, Oullins, France) labelled with RPE. CV6 is converted to the fluorescent fluorescein molecule when activated by esterase activity. Fluorescein fluorescence is sensitive to pH, with its peak in emission and absorption occurring between a pH of 8·0 and 9·0 (Invitrogen Corporation 2005).

Cell staining with CV6 in buffer

The staining protocol used for CV6 is that described in Parthuisot et al. (2000). Cells were prepared by centrifuging a sample (1–10 ml) of a 3-day culture of N. lovaniensis at 500–1000 g for 10 min. The resulting pellet was washed and resuspended twice in the same volume of PBS/2 buffer [a 50% concentration of phosphate-buffered saline (PBS)] as the original sample. Then, 10 μl of CV6, in the concentration provided by the manufacturer, was added to 1 ml of this sample. The resulting mixture was incubated at room temperature for 30 min in the dark for measurements made prior to February 2006. In February 2006, mixtures were further diluted by 10× before adding CV6 and then incubated at 37°C for 30 min in the dark to optimize the fluorescent signal. Small portions, 10 μl, were then removed for hemocytometer enumeration or flow cytometer enumeration. Afterwards, N. lovaniensis were inoculated into PBS/2 or natural water samples from the Laramie and Tech Rivers. PBS/2 had a pH of 7·59, Laramie River water a pH of 7·62, and Tech River water a pH of 7·72. Laramie River water was filtered through a 20-μm paper filter (Whatman) to remove detritus. Tech River water was filtered using either a 5-μm polycarbonate filter (Nuclepore TrackEtch Membrane, Whatman) or a 50-μm mesh filter (Buisine, Clermont de l’Oise, France) to remove large particles. Water was filtered before inoculation with amoebae.

Cell staining with RPE in buffer

RPE-immunofluorescent staining was performed on formaldehyde-fixed samples (2% final concentration). The antibody specific for N. lovaniensis was conjugated with biotin and revealed by streptavidin conjugated with RPE. A staining mix was prepared with two parts of biotinylated primary antibody anti-N. lovaniensis and five parts of RPE-conjugated streptavidin (Dako, Glostrup, Denmark) in the dark for 30 min at room temperature. The staining mix was diluted 1/14 with amoebae suspended in PBS/2 with 0·1% Tween 20. The final antibody concentration was 40 μg ml−1. Cells were then incubated with the antibody for 30 min at room temperature in the dark. Finally, amoebae were inoculated into PBS/2 or natural water samples from the Laramie and Tech Rivers.

Hemocytometer enumeration of amoebae in buffer

For comparison of FFC-measured cell concentrations in buffer with an independent method, we initially chose hemocytometer counting. Both techniques count fluorescent cells and should therefore yield consistent results. We made three sets of comparisons between FFC and hemocytometry measurements using CV6- and RPE-labelled N. lovaniensis, using PBS/2 as the solute. Three-day liquid cultures of the N. lovaniensis were used. Ten microlitres of amoebae suspended in PBS/2 were loaded into a hemocytometer cell mounted on the stage of an Olympus BH-2 epifluorescent microscope and counted with a total magnification of 100×. Further serial dilutions of the sample were then enumerated with the FFC.

At least 150 hemocytometer counts per slide were made in order to produce a high degree of accuracy in the resulting enumeration. The number of grids counted varied according to the concentration, between 5 and 40 grids, in order to obtain a total of 150–200 counts (cell detections). Hemocytometer counts where made before and after an FFC run, repeating to within 10%. Similarly, for each sample enumerated with the FFC, the sample was counted in multiples of 500 frames (each frame consisting of a 400-ms exposure) with each set of 500 frames representing a 50-ml sample.

In hemocytometer measurements of RPE-labelled amoebae, both amoebae and brightly fluorescing smaller particles were seen by microscope. The latter appear to be RPE dye particles. Only the amoebae were counted. A stock solution of N. lovaniensis with a concentration of 17 900 ml−1, as measured by a FACSCalibur flow cytometer (Becton Dickinson), was used to prepare dilutions and the hemocytometer measurements were used to confirm that concentration within 5%. A population of relatively weak intensity detections appears in the FFC measurements and appears to coincide with this population of small fluorescing particles. Our choice of FFC detection threshold, 100 ADU, appears to separate amoebae detections from small particle detections.

Solid-phase cytometer enumeration of amoebae in Tech River samples

ChemScan solid-phase cytometer counts of fixed RPE-labelled N. lovaniensis were used for comparison with FFC measurements of fixed RPE-labelled N. lovaniensis, both inoculated into Tech River water at concentrations of 0·06–3·0 amoebae ml−1. After labelling, samples were filtered through a 2-µm polycarbonate 25-mm membrane (Nuclepore, Whatman). The membrane was transferred onto the sample holder of the ChemScan system. The system is able to differentiate between labelled micro-organisms and autofluorescent particles present in the sample based on the optical and electronic characteristics of the generated signals (Walner et al. 1997; Pougnard et al. 2002). Illumination was provided by a water-cooled argon laser emitting at 488 nm. The fluorescence emission was collected in the orange channel (540–570 nm). In this study, the holder was previously overlaid with a support pad (black membrane; Chemunex) soaked with 100 µl of PBS. ChemScan detections were confirmed by manual microscope examination in order to eliminate false-positive events.

Flow cytometer enumeration of amoebae in Tech River samples

Flow cytometer counts of CV6-labelled N. lovaniensis inoculated into Tech River water were performed with a FACSCalibur flow cytometer equipped with an air-cooled laser providing 15 mW at 488 nm. Cell discrimination was based on green fluorescence collected in the FL1 channel (530 ± 15 nm). Cells were enumerated during a fixed time (2–5 minutes for each sample) at a given flow rate, calibrated at the beginning and at the end of each analysis session. Because the low concentrations involved in our FFC samples (0·08 to 2 ml−1) were under the detection limit for flow cytometry, flow cytometry measurements were made of two independent stock solutions which were diluted from c. 100 000 amoebae ml−1 to the final desired concentration in the 500-ml samples used for FFC measurements.

FFC enumeration of Tech River samples

FFC samples were placed in an open glass cylindrical container with a magnetic stir bar and introduced into the FFC with a peristaltic pump. The magnetic stirrer prevented sedimentation of amoebae in the sample during the sampling process. In general, we found that measurements of CV6-labelled amoebae showed a decrease in intensity of c. 30 min to 1 h after CV6 incubation, so that measurements had to be made quickly after sample preparation. Subsequent FFC measurements were made with additional serial dilutions. For each 50-ml sample enumerated with the FFC, the sample was counted in multiples of 100 frames (each frame consisting of a 400-ms exposure) with each set of 100 frames representing a 10-ml subsample. A mean and standard deviation was produced from the ensemble of the 100-frame subsamples representing a single sample.

Peristaltic pump rates for the two peristaltic pumps used in our experiments (Rainin Rabbit, Oakland, CA; Reglo, Ismatech, Glattbrugg, Switzerland) were continuously calibrated during the sampling period by weighing the FFC effluent on a digital scale. Variations in pump rate between such calibrations were within approximately 5%. Although the nominal pump rate throughout this study is 15 ml min−1, all data were adjusted to the measured pump rate.

All FFC data taken in 2005 were taken with the filters listed in Table 1. All 2006 FFC data were taken with SEMRock filters (Rochester, NY, USA) designated for RPE (CY3-4040B filter set, with an excitation filter at 531 nm, 40-nm bandwidth and an emission filter at 593 nm and 40-nm bandwidth) and FITC (FITC-3540B filter set with an excitation filter at 482 nm, 35-nm bandwidth and an emission filter at 536 nm, and a 40-nm bandwidth).

FFC measurements of the rate of false-positive detections

FFC measurements of uninoculated samples of Laramie River water (referred to as Laramie River* in Table 2) were made on 23 and 26 January 2006 for water collected on 19 January 2006 and refrigerated prior to use. This sample was collected from the flowing river and filtered through a 20-μm paper filter (Whatman). The samples were shaken immediately before being introduced into the FFC. In addition, a magnetic stirrer was used to prevent sedimentation of particulates in the sample prior to introduction into the FFC.

Table 2.   Measurements of the rate of false-positive detections. Measurements 2 to 5 and 7 to 8 were made to determine the rate of false-positive detections in Laramie River* (flowing) water, Tech River water, and buffer using the sample thresholds used for Naegleria lovaniensis detection shown in Figs 3, 4, and 6, i.e. 100 ADU for the RPE filter set and 3000 ADU for the CV6 data set. In addition, measurements 1 and 6 were used to detect amoebae using the ‘wrong’ filter set, i.e. using the RPE LED and filter set (and detection threshold) to detect CV6-labelled amoebae and the CV6 LED and filter set (and detection threshold) to detect RPE-labelled amoebae
MeasurementDate Solute/filterAmoebae concentrationLED and filter setNo. of 50-ml samplesFalse detections
  1. Abbreviations: RPE, R-phycoerytherin; LED, light-emitting diode; CV6, ChemChrome V6; ADU, analogue-to-digital units; PBS, phosphate-buffered saline.

120 Jan 2006PBS/22·1 ml−1 w CV6RPE248, 36
226 Jan 2006PBS/2NoneRPE30, 0, 0
323 Jan 2006Distilled waterNoneRPE10
423 Jan 2006Laramie R.*/20 μmNoneRPE325, 23, 23
526 Jan 2006Laramie R.*/20 μmNoneCV630, 0, 0
626 Jan 2006Laramie R.*/20 μm1·5 ml−1 w RPECV630, 0, 0
713 Feb 2006Tech R./50 μmNoneRPE539, 27, 29, 30, 34
814 Feb 2006Tech R./5 μmNoneRPE42, 6, 1, 3
915 Feb 2006Tech R./50 μmNoneCV650, 1, 0, 0, 1

Validation of FFC counts of amoebae in Tech River water with ChemScan and flow cytometry

Measurements of inoculated and uninoculated samples of filtered Tech River samples were made on 14–16 February 2006 to validate FFC enumeration. To validate FFC counting of RPE-labelled amoebae, 1-l samples were prepared by inoculation with RPE-labelled amoebae at concentrations estimated to be approximately 0·04, 0·2, 1·0, and 2·0 amoebae ml−1. Subsequently five 50-ml samples were measured by FFC at each concentration and five 50-ml samples were measured by ChemScan. The ChemScan counts only the relatively circular amoebae and not the irregular red fluorescent particles (Pougnard et al. 2002). Similarly, the threshold for FFC counting RPE-labelled N. lovaniensis is set to ignore the same red fluorescent particles.

To validate FFC counting of CV6-labelled amoebae, it was necessary to use flow cytometry results for comparison, as filtration of unfixed cells for the ChemScan mechanically destroys the cell membrane. An initial stock solution of CV6-labelled amoebae was measured to allow preparation of 500-ml samples at concentrations estimated to be 0·04, 0·2, 1·0, and 2·0 amoebae ml−1. Two to five 50-ml subsamples of each 500-ml sample were measured by FFC at each concentration. Finally, intermediate dilutions used to prepare each 500-ml sample were then measured by FACSCalibur flow cytometry and the concentrations determined for comparison with FFC.

FFC counts of amoebae labelled in Tech River water

Measurements made to this point were made of buffer and Tech River water inoculated with N. lovaniensis stained with fluorescent labels in buffer. Measurements of inoculated Tech River samples stained with fluorescent labels in Tech River water were made on 22 September and 6 October 2006. To validate FFC counting of RPE-labelled amoebae, 1-l samples were prepared by dilution of a stock solution of RPE-labelled amoebae at a known concentration determined by BD FACsCalibur flow cytometry. The resulting samples had predicted concentrations of 0·0, 0·5, 1·0, and 2·0 amoebae ml−1. Subsequently five 30-ml samples were measured by FFC at each concentration.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

FFC measurements of river water to determine the rate of false-positive detections from fluorescent detritus

Table 1 summarizes the results of a series of experiments on natural river water, filtered and unfiltered, used to determine the best fluorochrome and filters to use in order to avoid emission from chlorophyll a and b in organic particles. The lowest number of counts is found in samples illuminated with a green LED through a 530-nm (30-nm bandpass) filter and imaged through a 585-nm (40-nm bandpass) filter. The excitation/illumination bandpasses correspond to a broad minimum in the absorption and emission spectra of chlorophyll a and b which extends from 500 to 600 nm. In addition, this filter set corresponds to the filter set appropriate for RPE, when illuminated in the green. Three other bandpasses were examined. One corresponds to RPE, when illuminated in the blue (470-nm excitation, 585-nm emission). The second corresponds to FluoProbes 647 (625-nm excitation, 670-nm emission; Interchim, Montluçon, France). The third corresponds to RPE-CY5 (530-nm excitation, 675-nm emission). The only LED/filter set that produced a low rate of false detections was the green-illuminated RPE filter set. This produced ≤1 false detections per 11 ml of water in both filtered and unfiltered water samples from the eight rivers sampled. In addition, the sample with clearly the highest load of organic material, the Laramie River, when filtered, produced no false detections. It should be noted that false detections can also be produced from mineral grains suspended in water samples. This is hard to predict without prior observations or prior knowledge of the mineral properties of the river bed over which the sampled water flows. However, it should be noted that when higher detection thresholds are used, such as the ones that we found appropriate for the detection of RPE- and CV6-labelled N. lovaniensis, the Laramie River showed no false-positive detections in 50-ml samples through the CV6 LED/filter set combination, but did exhibit false positives in the RPE LED/filter set combination. We discuss this next.

Measurements of CV6- and RPE-labelled amoebae in buffer

Comparisons of FFC and hemocytometer counts of CV6- and RPE-labelled amoebae in PBS/2 are shown in Fig. 3. The data show a linear relationship within the 1–σ error bars (calculated empirically from the five subsamples comprising each sample). Three to five 50-ml samples were used at each concentration. In addition, we measured uninoculated samples with the FFC, all of which show zero counts. Best-fit lines were computed for the data shown in Fig. 3. The slopes of these two lines, 0·95 ± 0·16 (for CV6) and 0·99 ± 0·20 (for RPE), correspond to the detection efficiencies using these fluorochromes, which should ideally be 1·0. (Errors in the slopes are calculated for 95% confidence limits.) The differences between 1·0 and the measured detection efficiencies are not significant. The R2 of the fits are 0·94 and 0.93 for CV6 and RPE, respectively.

image

Figure 3.  Comparison between Fountain Flow Cytometer (FFC) and hemocytometer measurements of Naegleria lovaniensis in phosphate-buffered saline (PBS)/2. Each data point represents a 50-ml sample measured at a flow rate of 15 ml min−1. Concentrations of amoebae in inoculated samples range from 0·2 to 2·0 amoebae ml−1. A best-fit line is drawn through the data. Panel A: Comparison of Fountain Flow Cytometer and hemocytometer measurements of ChemChrome V6 (CV6)-labelled N. lovaniensis samples enumerated on 20 January 2006. Panel B: Comparison of FFC and hemocytometer measurements of R-phycoerytherin (RPE)-labelled N. lovaniensis samples enumerated on 21 January 2006.

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In order to test the hypothesis that FFC enumeration errors were consistent with Poisson (counting) statistics, a reduced chi-squared test was performed for each sample in Fig. 3, representing the number of cells counted in n subsamples. The reduced χ2 is given by χ2 = σ2/m(n–1), where σ is the standard deviation measured from the n subsamples comprising a single sample and m is the mean number of cells counted by FFC in each subsample. The maximum value of reduced χ2 for the data points shown in Fig. 3 was 0·62. This corresponds to a probability >64% (for each point) that the measured variance can be attributed to counting statistics.

FFC measurements of the rate of false-positive detections

The samples used in this study were chosen among those which may contain high fluorescence background and/or nonspecific fluorescent particles. Although the number of false-positive events was generally highly acceptable, it was high in a few samples. Table 2 shows nine sets of measurements made to determine the rate of false-positive detections in uninoculated samples of distilled water, PBS/2, Laramie River water, and Tech River water. Measurements 2 to 5 and 7 to 8 were made to determine the rate of false-positive detections in Laramie River water, Tech River water, and buffer using the sample thresholds used for N. lovaniensis detection shown in Fig. 3, i.e. 100 ADU for the RPE filter set and 3000 ADU for the CV6 data set. In addition, measurements 1 and 6 were used to detect amoebae using the wrong filter set, i.e. using the RPE LED and filter set (and detection threshold) to detect CV6-labelled amoebae and the CV6 LED and filter set (and detection threshold) to detect RPE-labelled amoebae.

Validation of FFC using ChemScan enumeration

Figure 4 shows a plot of FFC counts vs ChemScan counts for 50-ml samples of inoculated Tech River samples (amoebae incubated with stain in buffer), filtered with a 5-μm polycarbonate filter (Whatman), spanning the range of 0·06–3·0 amoebae ml−1, at a sampling rate of approximately 15 ml min−1. The best-fit line to the data has a slope of 0·83 ± 0·05 (95% confidence limit) and an intercept of 4·4. The R2 of the fit is 0·98. The background of uninoculated (null) samples was approximately three false counts per 50 ml. The slope of the fit indicates a higher count rate with solid-phase cytometry than with FFC (by about 15%), while the FFC measurements agree well with hemocytometry (Fig. 3). A microscope photograph of an RPE-labelled amoeba and a red-fluorescent particle is shown in Fig. 5.

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Figure 4.  Comparison of Fountain Flow Cytometer and ChemScan measurements of R-phycoerytherin (RPE)-labelled Naegleria lovaniensis samples in 5-μm-filtered Tech River water enumerated on 14 February 2006. Each data point represents a 50-ml sample measured at a flow rate of 15 ml min−1. There are five replicate measurements at each concentration, although some data points are plotted on top of each other. A best-fit line is drawn through the data. Concentrations of amoebae in inoculated samples range from 0·06 to 3·0 amoebae ml−1.

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image

Figure 5.  An epifluorescence microscope image of a red-fluorescent particle, above, and an R-phycoerytherin (RPE)-labelled Naegleria lovaniensis, below, made with a 530–550-nm excitation filter and a 590-nm emission filter. The length of the horizontal line in the lower left represents 20 μm.

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Validation of FFC using flow cytometry

Figure 6 shows a plot of FFC counts vs FACSCalibur counts for 50-ml samples of inoculated Tech River water (amoebae incubated with stain in buffer), filtered with a 50-μm filter (Buisine), spanning the range of 0·08–2·0 amoebae ml−1, at a sampling rate of approximately 15 ml min−1. The best-fit line to the data has a slope of 0·81 ± 0·10 (95% confidence limit) and an intercept of –1·0. The R2 of the fit is 0·97. The slope of the fit indicates a lower count rate with FFC than with flow cytometry.

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Figure 6.  Comparison of Fountain Flow Cytometry and BD FACSCalibur Flow Cytometry measurements of ChemChrome V6 (CV6)-labelled Naegleria lovaniensis samples in 50-μm filtered Tech River water, enumerated on 16 February 2006. Each data point represents a 50-ml sample measured at a flow rate of 15 ml min−1. A best-fit line is drawn through the data. Concentrations of amoebae in inoculated samples range from 0·08 to 2·0 amoebae ml−1. Panel A shows the full set of data; Panel B is an expanded view of data at lower concentrations.

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In order to test the hypothesis that FFC enumeration errors were consistent with Poisson (counting) statistics in the flow cytometry and ChemScan comparison data shown in Figs 4 and 6, a reduced chi-squared test was performed for each sample. The maximum value of reduced χ2 for the data points shown in Figs 4 and 6 was 0·63. This corresponds to a probability >63% (for each point) that the measured variance can be attributed to counting statistics.

Figure 7 shows a plot of FFC counts vs FACSCalibur counts for 30-ml samples of inoculated Tech River water (incubated with stain in Tech River water). First, the N. lovaniensis were stained with label (either CV6 or RPE) in Tech River water filtered with a 50-μm filter (Buisine) and then diluted with 50-μm filtered Tech River water to achieve sample concentrations of 0·0, 0·5, 1·0, and 2·0 amoebae ml−1. In the case of CV6, N. lovaniensis were incubated with the label in Tech River water filtered with a 0·2-μm filter and then diluted with 50-μm filtered Tech River water to yield sample concentrations of 0·5, 1·0, and 2·0 amoebae ml−1. The best-fit line to the RPE data has a slope of 1·03 ± 0·14 (95% confidence limit) and an intercept of 2·0. The R2 of the fit is 0·96. The best-fit line to the CV6 data has a slope of 0·88 ± 0·28 (95% confidence limit) and an intercept of –0·2. The R2 of the fit is 0·91. The slope of these fits is consistent with a 100% counting efficiency. The maximum value of reduced χ2 for the data points shown in Fig. 7 was 0·90. This corresponds to a probability >46% (for each point) that the measured variance can be attributed to counting statistics.

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Figure 7.  Comparison of Fountain Flow Cytometry (FFC) and BD FACSCalibur Flow Cytometry measurements of ChemChrome V6 (CV6)- and R-phycoerytherin (RPE)-labelled Naegleria lovaniensis samples in 50-μm filtered Tech River water. Each data point represents a 30-ml sample measured at a flow rate of 15 ml min−1. Concentrations of amoebae in inoculated samples range from 0·0 to 2·0 amoebae ml−1. A best-fit line is drawn through the data. Panel A: Comparison of FFC and flow cytometry of RPE-labelled N. lovaniensis samples enumerated on 22 September 2006. Panel B: Comparison of FFC and flow cytometry measurements of CV6-labelled N. lovaniensis samples enumerated on 6 October 2006.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Rate of false-positive detections

One of the primary questions addressed in this study is whether or not the selection of dyes and filters used can produce an acceptable rate of FFC false detections of N. lovaniensis in natural river water. Data shown in Table 2 were taken to address this question. In measurement 1, false-positive detections were made at RPE bandpasses when analysing a sample inoculated with CV6-stained amoebae. This is of concern when trying to discriminate between viable N. lovaniensis and other viable protozoa, both of which could have considerable emission from CV6 leaking through the RPE detection bandpass, even when illuminated in the green. However, one must keep in mind that in this work we have set the threshold for CV6 detections 30 times higher than the threshold for RPE detections (3000 ADU vs 100 ADU), owing to the fact that CV6 emission was found to be c. 30 times brighter than RPE emission from N. lovaniensis in these experiments. Lowering the intensity of the CV6 label, e.g. lowering the concentration of the staining solution, could produce similar fluorescent intensities from CV6 and RPE, allowing the use of similar detection thresholds for CV6 and RPE, and would result in much lower false-positive detections in the RPE bandpass. Indeed, if the intensity of false-positive detections in measurement 1 were reduced by only a factor of three, none would have been counted.

In measurement 4, significant counts, 23–25 false-positive detections per 50 ml, were made for Laramie River samples only when using RPE illumination/detection bandpasses. This appears to be because of the detection of nonchlorophyll-bearing particles. Measurements 7 to 9 were made to determine the rate of false-positive detections in Tech River water using the same thresholds as before (100 and 3000 ADU). These latter measurements were used to determine the level of background counts in the validation measurements discussed next. Tech River water was initially filtered with a 50-μm mesh filter (Buisine) to remove detritus. However, as with measurement 4 of the Laramie River, measurements at RPE wavelengths, chosen to avoid chlorophyll emission, showed an unexpectedly high rate of false counts, 27–39 per 50-ml sample. We were forced to filter samples with a 5-μm polycarbonate filter (Whatman) to lower the false count rate to an acceptable level for the RPE validation experiments. In practice, any filter much smaller than c. 50 μm will not pass N. lovaniensis. However, measurements of the false detection rate at CV6 wavelengths, which do not avoid chlorophyll emission, showed an acceptable rate of 0–1 per 50-ml sample. This is because of the high threshold that we can use for CV6-labelled amoebae.

Measurements of N. lovaniensis stained with RPE in Tech River water, filtered with a 50-μm filter, showed no significant increase in false count rate or decrease in counting efficiency from samples stained in buffer and diluted in Tech River water. However, incubation of N. lovaniensis with CV6 in 50-μm filtered Tech River water showed an unacceptable increase in the rate of false positives, over staining in buffer, presumably because of the presence of organic matter. Therefore, we chose to filter Tech River water with a 0·2-μm filter to CV6-label N. lovaniensis, although 50-μm Tech water was used as a diluent after staining. Using CV6 for 50-μm filtered natural water samples is only practical where a second fluorochrome, e.g. RPE, is used to confirm the detection.

For both the Laramie and Tech Rivers, it is possible that the false detections at RPE wavelengths are from small mineral particles or organic particles that are fluorescing in the RPE bandpass from a fluorescent source other than chlorophyll. In other words, false detections could be confused mainly with nonviable amoebae. Use of both the CV6 and RPE probes and bandpasses simultaneously to discriminate viable N. lovaniensis should lower the false detection rate even further.

Comparison of FFC with hemocytometry, solid-phase cytometry, and flow cytometry

Although FFC enumeration of N. lovaniensis in PBS/2 shows agreement with hemocytometry, within counting statistics, there is less agreement with solid-phase cytometry and conventional flow cytometry. FFC undercounts amoebae by 17% compared with solid-phase cytometry and 19% compared with flow cytometry. It is possible that flow cytometric measurements overcount amoebae or that FFC undercounts amoebae as a result of the detection threshold used on each system. One should note that the FFC results compared with hemocytometry were taken by one system and the FFC results compared with flow cytometry and solid-phase cytometry were taken with a second, nearly identical system.

Summary

FFC enumeration of N. lovaniensis yields results for CV6- and RPE-labelled amoebae that are consistent with other counting methods, including ChemScan, flow cytometry, and hemocytometry enumeration, down to concentrations of 0·06 amoebae ml−1, using a flow rate of 15 ml min−1. The results of this study indicate that use of RPE illuminated at 530 nm and detected at 585 nm provides a satisfactory means of attenuating background from natural waters, particularly waters contaminated with chlorophyll-bearing particles. The rate of false positives for inoculated Tech River water (amoebae incubated with stain in buffer), was 0–1 per 50-ml sample for 50-μm filtered water when measured with CV6 bandpasses and a detection threshold appropriate for CV6-labelled N. lovaniensis. The simultaneous measurement of emission at CV6 and RPE bandpasses should further reduce the rate of false detections.

Future work

We have designed and are currently beginning fabrication of a two-colour version of the FFC, allowing simultaneous CV6- and RPE-measurements. This will allow simultaneous detection of a specific micro-organism and its viability, as well as eliminating false counts which appear only at a single bandpass. This new technology may be of great interest in the fields of sanitary microbiology and microbial ecology. It may be used for the rapid detection and enumeration of viable pathogens present at low concentrations in natural waters, but it should also apply to any protozoa when specific fluorescent probes are available.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors acknowledge funding from Electricité de France, the US Geological Survey, the Wyoming Water Development Commission, and the NSF STTR Grant Program, under Grant No. DMI-9810567, in support of this project. In addition, P.E. Johnson is grateful for a sabbatical fellowship from the Université Pierre et Marie Curie (Paris 6).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • De Jonckheere, J. (1977) Use of an axenic medium for differentiation between pathogenic and non-pathogenic Naegleria fowleri isolates. Appl Environ Microbiol 33, 751757.
  • Ford, T.E. (1999) Microbiological safety of drinking water: United States and global perspectives. Environ Health Perspect 107, 191206.
  • Invitrogen Corporation (2005) The Handbook – A Guide to Fluorescent Probes and Labeling Technologies. [Online.] Available at http://probes.invitrogen.com/handbook.
  • Johnson, P.E. (2004) Apparatus and methods for high throughput analysis of samples in a translucent flowing liquid. U.S. Patent No. 6,765,656.
  • Johnson, P.E. (2006) Method and system for counting particles in a laminar flow with an imaging device. U.S. Patent Application No. 11/328,033.
  • Johnson, P.E., Votaw, A.S. and Deromedi, A.J. (2002) Biodetection with flow cytometry: better, faster, cheaper. In Biodetection Technologies, Vol. 1. pp. 7183. Massachusetts: Brookline.
  • Johnson, P.E., Deromedi, A.J., Lebaron, P., Catala, P. and Cash, J. (2006) Rapid detection and enumeration of Escherichia coli in aqueous samples using Fountain FlowTM Cytometry. Cytometry Part A 69A, 12121221.
  • Parthuisot, N., Catala, P., Lemarchand, K., Baudart, J. and Lebaron, P. (2000) Evaluation of ChemChrome V6 for bacterial viability assessment in waters. J Appl Microbiol 89, 370380.
  • Pougnard, C., Catala, P., Drocourt, J.L., Legastelois, S., Pernin, P., Pringuez, E. and Lebaron, P. (2002) Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry. Appl Environ Microbiol 68, 31023107.
  • Sluter, S.D., Tzipori, S. and Widmer, G. (1997) Parameters affecting polymerase chain reaction detection of waterborne Cryptosporidium parvum oocysts. Appl Microbiol Biotechnol 48, 325330.
  • Walner, G., Tillman, D., Haberer, K., Cornet, P. and Drocourt, J.L. (1997) The ChemScan system: a new method for rapid microbiological testing of water. Eur J Parenter Sci 2, 123126.
  • WHO/OEDC (2003) Assessing the Microbial Safety of Drinking Water. Geneva: World Health Organization.
  • WHO/UNICEF (2002) Global Water Supply and Sanitation Assessment 2000 Report. Geneva: World Health Organization.