To rapidly isolate Leishmania donovani promastigotes in samples from Novy-MacNeal-Nicolle (NNN) cultures, a method of staining with acridine orange was developed. Such vital staining combines the advantages of direct microscopic examination (e.g., observation of motility) with more accurate cytological and structural imaging of the stained parasites (usually obtained by Giemsa staining). Progressive immobilization of Leishmania promastigotes associated with a change in fluorescence color was also studied. Our findings may be useful for the early confirmation of a positive culture inoculated with a clinical sample.