FERTILIZATION OF THE FROG'S EGG 



PURPOSE: To determine the Ideal conditions of concentration, time, and temperature for 

 maximum fertilization of normal eggs of the frog, Eana pipiene. 



MATEBIAI3: (This experiment can be done hest with groups of h students) 

 Biological : Mature male frogs 



Ovulating female frogs 



Technical : Finger howls, paired Petri dishes 



Controlled temperatures at I+OC. , 10°C., 200C., and ?.6°C. 

 (If these exact temperatures are not available, the range 

 should be covered and the temperatures should be stable.) 



METHOD : 



Precautions : 



a. All glassware must be biologically clean. 



b. Glassware and solutions must be brought to the various temperatures prior to the 

 introduction of spermatozoa, unless otherwise directed. 



c. Separate pipettes (medicine droppers) must be used for the transfer of sperm 

 suspensions from each container! They should be biologically clean and marked 

 for identification. Mark the pipettes at the 1 cc. level. 



d. l^gs from the ovulating female should be tested against normal sperm suspensions 

 to determine whether they are fertllizable. 



Control : The control consists of "d" above, and those eggs which are exposed to the 

 "normal" concentration. This is considered as one (1) pair of adult testes per 

 10 cc. of Spring Water. 



Procedure : Three distinct sets of observations are to be made, and the data is to be 

 plotted on graphs. 



A. DILUTION AND FERTILIZATION 



In this first experiment, the temperature shall be that of the laboratory and eggs 

 are to be stripped Into the sperm suspensions within 50 minutes of the maceration of the 

 testes. The only variable will therefore be the dilution, or the concentration of the 

 spermatozoa. 



a. Prepare 10 Petri dishes, (5 covers and 5 bases). Label the pairs N, 0.1 N. , 

 0.01 N. , 0.001 N., and 0,0001 N. 



b. Quickly remove and cut into small pieces (with fine scissors) two (2) pairs of 

 testes from adult frogs. These should be further mashed with a flattened end of 

 a glass rod, all in 20 cc. of Spring Water. Note the time of testes excision. 



0. If possible centrifuge by hand the sperm suspension to throw down the larger 

 pieces of testes, or allow them to settle In a tapered test tube. See that the 

 total volume is 20 cc. Decant off the homogeneous suspension (do not remove any 

 pieces of tissue) into the two Petri dishes marked "N", 10 cc. in each. 



d. With a clean and dry pipette (medicine dropper), previously marked at the 1 cc. 

 level, remove 1 cc5. of the spenn suspension from the bottom Petri dish marked "N" 

 and mix thoroughly with 9 cc. of Spring Water in bottom Petri dish marked 0.1 N. 

 In a similar manner transfer 1 cc. of "N" from the top Petri dish to the top Petri 

 dish marked 0.1 N, adding 9 cc. of Spring Water. In a similar manner transfer 

 1 cc. of each thoroughly mixed sperm suspension to 9 cc. of Spring Water in the 

 next dish, so that there will be progressive dilution as indicated above. It is 

 very important that clean and dry pipettes be used for each transfer, in order to 

 insure proper concentrations. Each succeeding dish should contain a sperm suspen- 

 sion representing a 10^ concentration of the previous dish. Using paired Petri 

 dishes, the number of eggs per dilution ceui be doubled. 



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