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

970 ml, autoclaved at 121 °C for 15 min. The following reagents are added aseptically: 50% glucose in water 10 ml (Millipore filtered), Penicillin K. 200,000 U/l, Streptomycin Sulfate 200,000 ug/1. Inoculation of plates and observation: A small volume of water (preferably the sediment of the centrifuged water) or medical samples are placed on the center of two agar plates coated with bacteria and incubated parallel at 25-30 °C and 42-45 °C, respectively. Observations are made daily with the unaided eye and under a microscope (preferably an inverted microscope) for up to 7 days. If amoebae are present, they will feed on bacteria and multiply, then move away centripetally from the area of inoculation. After several days of incubation, the amoebae will begin to encyst. A small piece of agar is excised with a sterile lancet from an area positive for amoebae, and transferred upside down onto a fresh agar plate precoated with bacteria. Table 1 Sources of the isolated strains Strain Source CI Contact lens container and corneal scrapings of a patient with severe eye soreness. Her symptoms appeared when using daily-wear soft contact lenses during her vacation at Lake Balaton Mos The root of a moss Dun The River Danube Cloning and axenization - micromanipulation method: A single cell (preferably cyst) is collected using a sterile microcapillary tube and transferred to a fresh agar plate precoated with bacteria. The dish is then placed into an incubator. For axenization, the cysts from a clonal culture are kept in 0.1 N HCl at room temperature overnight. They are washed several times with an excess amount of sterile water to neutralize the pH of the suspension, and are inoculated into an axenic culture medium (PYG medium) containing antibiotics. The cloned isolates were adapted and cultured axenically at 26 °C in PYG medium for large-scale sampling of the organisms. All the procedures should be carried out in a laminar flow cabinet or similar clean environment to eliminate all risk of airborne contaminants. Restriction fragment length polymorphism (RFLP) analysis Mitochondrial DNA from the isolates of Acanthamoeba was obtained by the method of alkaline lysis. Aliquots of midlog phase amoebae (5x10 cells) grown in PYG medium were collected and washed three times in phosphate buffered saline (pH 7.2) by centrifuging at 300 g for 5 min. The pelleted amoebae were washed again three times in a 1.5 ml Eppendorf centrifuge tube in TES buffer (50 mM Tris HCl, 10 mM ethylenediamine tetraacetic acid (EDTA), 50 mM sucrose, pH 8.0). The pellet was resuspended in 100 pi of chilled TES and added to 200 pi of freshly prepared 1% sodium dodecyl sulfate solution in 0.2 N NaOH. The suspension was gently mixed by inverting the tubes and incubated at 0 °C for 5 min. Then, 150 pi of chilled 3 M potassium acetate buffer was added to the suspension. The suspension was mixed and