AXENIC CULTIVATION OF THE BRINE SHRIMP ARTEMIA SALINA LUIGI PROVASOLI AND KAGEHIDE SHIRAISHI Haskins Laboratories, Nciv York 17, N. Y., and Dcpt. of Fisheries, I'aculty of Tohokn University, Sendai, Japan In a previous paper (Provasoli, Shiraishi and Lance, 1959) we have added to Gibor's (1956) observations that related species of algal flagellates may be either good or bad food for Artcmia. This idiosyncrasy may depend upon nutritional deficiencies in the algal food, on toxic metabolic products, or even upon some nutrient in excess. One way to attack this ecological problem is to grow Artcmia on a non-living medium as a step toward a chemically defined medium and, finally, identification of all its nutritional requirements. The present paper concerns the first stage, i.e., the growth of Artcinia on a IK in-living complex medium. MATERIALS AND METHODS The amphigonic American race of Artcmia salina was employed. Utah brine shrimp eggs (Aquarium Stock Co. Inc., 31 Warren Street, New York 7, New York) proved more satisfactory than other samples tried in respect to percentage of hatching and speed of development. Sterilization of the eggs. Durable eggs of Artcmia obtained commercially always contain many dead dried eggs whose chorion is cracked. These eggs are lighter and cannot be disinfected as rapidly as the intact viable ones and should be eliminated at the onset to avoid infections from the inoculum. The technique of disinfection is a modification of the one employed by Gibor (1956). The dead eggs, being lighter than the viable ones, are eliminated by the flotations in sea water. The eggs are disinfected in screw cap tubes for 10 minutes in Merthiolate solution (1 : 1000 in H,O)+ 0.2 ml.^ of a lO^c solution of Aerosol OT. to improve wettability. The disinfectant is decanted and the eggs washed in three baths of sterile sea water. The egg slurry is distributed into several tubes of a sterility-test medium (STP, Table I) and allowed to hatch 2-3 days at 22-26 C.). Contamination generally shows in 2-4 days. The new-born nauplii de-velop to second metanauplii at the expense of the reserves of yolk within 4-7 days ; the third metanauplii. if not fed, die. The metanauplii are transferred into a nutrient medium 1-2 days after the first nauplii have hatched, to secure a more uniform inoculum in respect to age; hatching is spread over several days. The growing larvae consume the participate food rapidly and must be transferred approximately every 6-8 days, especially after the fourth stage. Transfers are made with Pasteur pipettes connected to a mouthpiece by rubber tubing, to allow a clear view while fishing the larval forms. The later larval stages and the young adults defy suction unless sucked head first, while swimming toward the tip of the pipette. To avoid air-borne infection we used a transfer hood (top and back glass. 347
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