Rapid Detection of Plastmodium by a New ‘Thick Smear’ Method using Transmission Fluorescence Microscopy : Direct Staining with Acridine Orange

A new staining technique of \u27thick smears\u27 was developed tor diagnosis of malaria using transmission fluorescence microscopy. Plasmodium falciparum cultured in vitro was used as a model. The infected blood was mixed 1 : 1 -1 :2 with acridine orange (AO) solution (final concentrations of 50-lOOu.g/ml in 0.01 M Tris-HCI buffer or in PBS, pH 7.0-7.5).Immediately or several minutes later, haemolyzed or non-haemolyzed \u27thick smears\u27 were observed directly by fluorescence microscopy using an interference filter specially designed for excitation of AO or commercially available, three (interference-, glass- and triacetyl cellulose film-) types of B-excitation filters with halogen- or daylight-illuminated microscopes. All of these filters were capable of detecting the parasites rapidly in these wet mounts at a magnification of x200 using standard lenses. Among them, the interferencetype filters having higher transmission efficiencies gave the best results. Film filter system, which was the cheapest, could also be used, especially in combination with daylightilluminated microscopes, although it was necessary to be exposed accurately to the direct rays of the sun. These results strongly suggest that transmission fluorescence microscopy using any B-excitation filter in light microscopes may be a useful economic system for rapid diagnosis of malaria