Dr. Murai Éva - Gubányi András szerk.: Parasitologia Hungarica 28. (Budapest, 1995)

incubated for 30 min at 0°C. After centrifugádon (12,000 g for 10 min at 4 °C), the supernatant fluid was transferred to a fresh Eppendorf tube and an equal volume of phenol/chloroform (1:1) solution was added. The supernatant fluid was collected following centrifugádon at 12,000 g for 10 min at 4 °C. The DNA fraction was sedimented by adding 1.0 ml of cold absolute ethanol and 35 pi of 3 M sodium acetate solution at -70 °C. The supernatant fluid was removed following centrifugádon at 18,000 g for 15 min and the sample was dried with a centrifugal evaporator (Tomy, Tokyo) at 230 g for 2-3 min at 42 °C. The sediment was dissolved in 30-50 pi of TE buffer (5 mM Tris HCl pH 8.0, 1 mM EDTA) and stored at -20 °C until used. Approximately, 0.15 pg of the mtDNA sample was incubated overnight with 5-10 units of restriction enzymes Bgl II and EcoK I at 37 °C in a total volume of 50 pi using buffers specified by the enzyme supplier. The enzymes were purchased from Takara Shuzo (Kyoto, Japan). The digests were electrophoresed in 0.7% agarose gel at a constant voltage of 3 V/cm for 6 h in TBE buffer (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA). The gel was stained with ethidium bromide and observed with a short wave ultraviolet transilluminator. The Hae III digests of O XI

74 phage and the Sal I and Hind III digests of Lambda phage were included in each gel run as size standards. Table 2 Estimates of mtDNA restriction fragment sizes for 2 endonucleases Endonuclease Fragments CI Dun Mos Bgl II 1 20,100 9,200 11,300 2 6,300 6,300 10,200 3 6,000 5,700 6,600 4 5,400 5,500 4,500 5 2,000 4,500 3,600 6 800 3,100 2,300 7 2,500 1,400 EcoK I 1 10,500 13,200 11,700 2 8,500 12,700 10,700 3 7,500 8,600 7,600 4 6,600 5,000 6,900 5 4,300 1,800 3,200 6 3,300 1,400 2,500 RESULTS Morphologically, all the three isolated strains were identified as Acanthamoeba spp mainly on the basis of cyst structure (Fig. 1. A, B, C). The endocysts are usually stellated and the ectocysts (the outer wall) are wrinkled. The vegetative form is larger, nuclei and granular cytoplasm can be seen. Comparing A, B and C, no morphologic distinction can be made. For the RFLP analysis, the amoebae were cultured on agar plates covered with