Dr. Murai Éva szerk.: Parasitologia Hungarica 23. (Budapest, 1990)

MATERIALS AND METHODS T. borelli was isolated from its leech vector Piscicola geometra, which was collected from carp (Cyprinus carpio) obtained from pond farm. The isolate was cultivated in SNB-9 diphasic blood agar medium at 21 °C. The cultivated strain was passaged at 12- to 14-day intervals and has been propagated in vitro up to the present time. Material for TEM was taken from the 45th passage, while that for SEM investigations from the 12th passage. Flagellates were harvested by gentle centrifugation in the log pha e. They were washed in PBS three times with centrifugation at 2,500 RPM/min for 10 minutes. Preparation of samples for scanning electron microscopy fSEM^) The pellet was digested with 0.005 % trypsin in PBS (pH 7.4) with 0.05 % EDTA for 30 minutes to remove the protein-peptid content of the medium. After digestion the specimens were washed in ice-cold PBS. The rinsed trypanoplasma were fixed for 6 hours in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2 - 7.4) and washed three times in ice-cold 0.1 M cacodylate buffer. They were post-fixed in 0.2 % OsO^ for 4 hours and washed twice in cacodylate buffer. Dehydration was done through ascending concentration of ethanol. Specimens were allowed to settle onto cover-glasses dried and coated with gold. The cover-glasses were mounted on stubs. Electron micrographs were taken with a JEOL 25S2 electron microscope. Preparation of samples for transmission electron microscopy (TEM) The rinsed and concentrated flagellates were fixed for 3 hours in 1 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2 - 7.4) and washed three times in o.l M cacodylate buffer. They were post-fixed in 2 % (Durcupan ). Ultrathin sections were stained with 1 % solution of uranyl acetate. Electron micrographs were taken using a JEOL 100B electron microscope. The cultivated forms vary considerably in morphology. In the culture three types can be distinguished: (1) small, slender forms typical of the leech phase (Figs 2 and 3); (2) broader, contorted forms (Fig. 1); and (3) large, stubby or rounded forms. Specimens reproducing by longitudinal binary fission are "V"-like in the last phase of fission (Fig. 4), while those dividing by multiple division are rosette-like. The two flagella emerge from flagellar pocket with the preoral ridge. The recurrent flagellum is attached to the cell membrane, passing the posterior end of the body showing an enlarged tip. The recurrent flagellum of the rounded forms is attached to the body surface only in a very short distance. Flagella of the dividing rounded forms, especially the short new ones, do not attach to the body surface, or they attach only in the last stage of division. The undulating membrane especially that of the broader forms is well developed. The preoral ridge arises from the flagellar pocket, extends posteriorly along the cell surface and ends in the cytostome. Scanning electron micrographs of in vitro cultivated T. borelli reveal Os0

4 in Qjl M cacodylate buffer ethanol and embedded in Araldite RESULTS SEM