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H. MOSER, I. HADJI-AZIMI AND S. SLATKINE 



température of 25° C yields satisfactory results with cells derived from advanced 

 embryos, tadpoles and adult Xenopus. 



Although successful cultivation of Amphibian cells in vitro has been reported 

 previously (Wolf, K., Quimby, M. C, Pyle, E. A. and Dexter, R. P., 1960; 

 Freed, J. J., 1962; Shah, V. C, 1962; Wolf, K., and Quimby, M. C, 1964; 

 Rafferty, K. A., 1965; Balls, M. and Ruben, L. N., 1966) we shall take the 

 opportunity of this conférence to make you familiar with the culture procédures 

 which have been developed in or adapted by our laboratory in Geneva, and to 

 report to you some of our preliminary observations with cultured Xenopus cells. 



The sources of primary cell and tissue cultures 



As source materials foi primary Xenopus laevis cell and tissue cultures we 

 have chosen so far (A) whole embryos and whole young swimming tadpoles, 

 (B) internai organs (for example kidney) of normally reared frogs and tadpoles. 

 Since contamination with micro-organisms and other parasites is quite a problem 

 with aquatic animais such as is Xenopus laevis, the source materials must be 

 decontaminated prior to explantation procédures. 



A. Decontamination of eggs and production of aseptic embryos 

 and young swimming tadpoles 



For the production of aseptic embryos and aseptic young swimming tadpoles 

 healthy fertilized Xenopus eggs (2-8 cell stages) are selected and decontaminated 

 by the following steps: 1. A 24-hour bath at room température in our basic 

 antibiotics rearing mediumi ABIOT-RS-A1, a médium which contains penicillin G 

 (300 units per ml), streptomycin sulfate (300 fjig per ml) and tetracycline-HCl 

 (10 (jig per ml) in 1/10-strenght NIU and TWITTY electrolyte solution (see 

 Table I); 2. treatment of normally developed eggs with 1% solutions of Lysol 

 or Desogen (GEIGY) for 10" to 15", and with 70% ethanol for 10" to 20"; 

 3. aseptic removal of the jelly and the vitelline membranes, with steiile watch- 

 maker's forceps in antibiotics rearing médium ABIOT-RS-A1. The liberated 

 embryos are subsequently transfered, with antibiotics-rearing médium ABIOT- 

 RS-A2 (see Table I) which contains in addition to the above mentioned anti- 

 biotica and electrolytes sulfathiazole at a concentration of 0.05% (see Table I) 

 into stérile covered 60 mm-diameter Petri-dishes or into small loosely capped 

 Erlenmeyers. The liberated embryos are reared by daily transfer into fresh ABIOT- 

 RS-A2 until they are selected for explantation. Individuals which have been let 

 to develop into ready-to-feed young tadpoles in ABIOT-RS-A2 are transfered, 

 prior to their use for explantation purposes, into our antibiotics-rearing médium 

 ABIOT-RS-A3 (see Table I) where they are exposed for not more than 2-3 hours 



