High throughput, real-time detection of Naegleria lovaniensis in natural river water using LED-illuminated Fountain Flow Cytometry.
OBJECTIVE
To test Fountain Flow Cytometry (FFC) for the rapid and sensitive detection of Naegleria lovaniensis amoebae (an analogue for Naegleria fowleri) in natural river waters.


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.


CONCLUSIONS
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.
Johnson
P E
PE
Department of Physics and Astronomy, University of Wyoming, Laramie, WY 82070, USA. pjohnson@uwyo.edu
Deromedi
A J
AJ
Lebaron
P
P
Catala
P
P
Havens
C
C
Pougnard
C
C
D000818:Animals; D003470:Culture Media; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis; D005602:France; D009253:Naegleria / Q000302:isolation & purification*; D010799:Phycoerythrin / Q000032:analysis; D015203:Reproducibility of Results; D045483:Rivers / Q000469:parasitology*; D014481:United States; D014876:Water Pollution / Q000032:analysis
D003470:Culture Media; D005456:Fluorescent Dyes; D010799:Phycoerythrin