EVALUATION OF FLUOROCHROMES AND EXCITATION SOURCES FOR IMMUNOFLUORESCENCE IN WATER SAMPLES

Fluorescent labelling methods for detecting microorganisms in water ha ve Limited sensitivity partly due to the natural autofluorescence from environmental particles, The aim of this study was to examine the aut ofluorescence of water samples to determine the optimal excitation sou rce and fluorescent labels for minimising background autofluorescence and therefore enhancing sensitive detection of Cryptosporinium oocysts , Particles concentrated from water were examined using fluorimetry at a wide range of excitation wavelengths to determine their autofluores cent properties, Two major peaks were identified emitting at 390 to 51 0 nm and at 640 to 700 nm, Flow cytometry was used to define the optic al properties of oocysts immunofluorescently labelled with a range of fluorochromes. Concentrated water samples were analysed using flow cyt ometry and the number of particles with fluorescence and Light scatter properties similar to the fluorescently labelled oocysts recorded, Fl uorescein isothiocyanate exited at 488 nm was the most suitable label for oocysts in untreated water with less than 70 particles having opti cal properties similar to labelled oocysts, detected in 10 litre conce ntrates. The fluorochromes CY3, phycoerythrin (PE), and tetramethylrho damine B thioisocyanate (TRITC) excited at 542 nm were the most suitab le labels for oocysts in drinking water with less than 40 particles ha ving optical properties similar to labelled oocysts, detected in 100 l itre concentrates. (C) 1997 Wiley-Liss, Inc.