Effect of isolation method of Contracaecum rudolphii Hartwich, 1964 (Ascaridida: Anisakidae) eggs on embryonic development and the number of larvae hatched.

This study tested the isolation of C. rudolphii eggs using various methods and evaluated their course of embryogenesis and the number of larvae hatched. The pressing of eggs from the earlier-isolated uteruses was conducted in Petri dishes using bent preparatory needles to obtain eggs for a control culture. The experimental cultures contained incubated mature females in a culture medium which had been homogenized and digested by proteases. In the first experimental cultures, the females were incubated for three days at 40 degrees C in 10-ml plastic flasks. Eage's medium with the addition of 1% pepsin (pH 2) and 20% heat-inactivated foetal bovine serum was used as an incubation fluid. In the second method, the suspension of females homogenized with a blender was centrifuged for 3 minutes at 1000 rpm, the supernatant was removed and the sediment was then rinsed with a PS. In the third method, an attempt was undertaken to collect eggs by digestion of mature females with 0.1% and 1% aqueous solutions of pepsin adjusted to pH 2.0 with 1N HCl, as well as in 0.1% and 1% solutions of trypsin prepared in a Sörensen buffer (pH 7.6). The suspension obtained after complete digestion and still containing eggs was purified from proteases by washing several times with PS. In turn, no eggs were isolated by using the method of incubation of females in a culture medium nor by digestion with pepsin. The method of homogenization of whole nematodes resulted in egg damage. The best method of egg isolation was digestion with a 0.1% solution of trypsin. When the digestion was conducted with a 1% trypsin solution, the arrestment of embryogenesis was observed in a considerable percentage of the eggs, whereas eggs thecae were left by 31% of the larvae.