Studies on Larval Fascioloides magna. II. In vitro Survival of Axenic Rediae in Amino Acids and Sugars

Abstract

Preparation of salt solutions and test media. A salt solution termed Ringer C was earlier found satisfactory for use (Friedl, 1961). It was prepared as a stock solution in concentrated form, i.e., in 600 ml of distilled water rather than a liter, and had the following saline composition: NaCl, 6.5 g; KC1, 0.14 g; CaCl2, 2H2O, 0.16 g; NaHCO:, 0.10 g; NaH2PO4, 0.012 g. In these experiments, the solution was autoclaved at 10 lbs for 20 min, the precipitate forming in the presence of bicarbonate being allowed to settle out. Because it obscured subsequent observation, it was generally not resuspended when the supernate was used. Upon combining 3 ml of the above stock with 2 ml of water, or an equivalent volume of water plus appropriate amounts of stock test substance (giving the desired 2, 4, and 6 mM concentrations of the latter) the proper Ringer C concentration resulted. Stock amino acids and sugars were prepared in 100 mM aqueous solutions and autoclaved (except tryptophan which was Seitz filtered). Ringer C tended to equilibrate in air to about pH 7.8 and in combination with test substances generally gave solutions approaching this value from neutrality. Fraction V Bovine Serum Albumin (Armour) in a 2.5 percent aqueous solution was Seitz-filtered. A final concentration of 0.12 percent in Ringer C, used for washing rediae, is referred to as wash solution. Glassware. Numerous capillary pipettes were indispensable. They were conveniently made by drawing the center of a 23 cm length of 6 mm diameter soft glass tubing to about 30 cm over a Bunsen flame without a wing-top. When broken apart, this gave two pipettes of the desired 0.8 mm outside diameter; tips were not fire-polished. They were used with plastic mouth tubes. Silicone was used to coat the interiors of capillary pipettes and 16 x 150 mm test tubes, and the concavities of 75 x 45 x 6 mm tissue culture slides.