A method for assessing the migratory response of Naegleria fowleri utilizing [3H]uridine-labeled amoebae

A new procedure is described to assay the migratory response of Naegleria fowleri (ATCC 30894) amoebae to potential chemoattractants. The method utilizes a blind-well Boyden chemotaxis chamber, two micropore filters of different construction, and amoebae-labeled with [3H]uridine. The technique was standardized by determining the influence of incubation time, filter construction, filter pore size and geometry, amoebae to filter pore ratio, and chemoattractant concentration. Radiolabeled amoebae were placed in Boyden chambers that contained the combination of an upper polycarbonate filter with distinct pores with a diameter of 8 microns and a lower filter of nitrocellulose with a 150-micron depth to separate the wells. A ratio of two amoebae to one filter pore and a 2-h incubation period were chosen to obtain optimal migration conditions. Nerve cell extract was used as the chemoattractant. The migratory responses of both highly pathogenic and weakly pathogenic strains of N. fowleri to nerve cell extract were compared using either the radiolabel procedure or the conventional single filter, leading-front method. Using either method, a highly pathogenic cloned strain of N. fowleri amoebae moved in a directional manner (chemotactically) in vitro to B103 rat neuroblastoma cell extract. In contrast, a weakly pathogenic strain of amoebae responded in a nondirectional manner (chemokinetically) to nerve cell extract. While both the leading-front assay and the radiolabel assay give accurate results, the measurement of radiolabeled cells allows one to test a greater number of attractants in one assay and the procedure eliminates observer bias.