5(6)-Carboxyfluorescein diacetate as an indicator of Caenorhabditis elegans viability for the development of an in vitro anthelmintic drug assay.

A new in vitro assay for anthelmintic activity using Caenorhabditis elegans is based on the ability of 5(6)-carboxyfluorescein diacetate (CFDA) to indicate the worm's viability. It is shown for the first time that the treatment of a suspension of worms with a solution of 5(6)-carboxyfluorescein diacetate (4.2%) for 30min transiently induces fluorescence in dead worms only, allowing a fast and efficient determination of the proportion of dead worms by fluorescence microscopy. The proposed test has been validated using mixtures of populations of living and killed C. elegans and proved to be selective, linear in the range 0-100%, accurate and precise. The suitability of the assay to detect anthelmintic activity was then evaluated by studying the toxicity against C. elegans of a series of known anthelmintic compounds (mebendazole, levamisole, niclosamide, pyrantel, piperazine, and thiabendazole) with various modes of action. The worms were exposed to each drug at two concentrations, 50 and 100microg/ml for piperazine, niclosamide, pyrantel and 5 and 10microg/ml for the others. We observed that, in the tested range of doses, piperazine and niclosamide were only moderately toxic, yielding 13.1 and 17.5% of dead worms; due to their mode of action and/or specificity, the low toxicity of these compounds was as expected. The marked activities of all the other compound fully agree with those described in the literature and obtained by other more laborious techniques. These validation data indicate that the proposed in vitro anthelmintic assay using 5(6)-carboxyfluorescein diacetate allows for sensitive measurement of worm viability.