FERTILIZATION OF THE FROG'S EGG 



117 



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 con- 

 centrations. Each succeeding dish should contain a sperm suspension representing a 

 10% concentration of the previous dish. Using paired Petri dishes, the number of eggs 

 per dilution can be doubled. 



Exactly 30 minutes after the testes are first cut into pieces, begin stripping eggs 

 from an ovulating female into bottom Petri dish marked "N". Estimate about 50 eggs 

 per dish, and then strip into the bottom Petri dish containing the next concentration. 

 A single ovulating female should provide enough eggs for the 5 bottom Petri dishes. 

 A second female will be used for the remaining 5 top Petri dish series. Gently rotate 

 the dishes so that each egg is exposed to the sperm suspension. 



After 5 minutes, flood each dish with Spring Water so that the eggs are entirely cov- 

 ered. Do not disturb them further until three (3) hours after the time of insemination. 

 Series "A" and series "B" below represents the top and bottom Petri dish series, or 

 eggs from two different females. 



SERIES "A" 



SERIES "B" 



CONCENTRATION 



COMPOSITE GRAPH OF SPERM CONCENTRATIONS AND FERTILIZATION 

 100% 



20% 



0% 



N 

 (Control) 



0. IN 



0. OIN 



0. OOIN 



0. OOOIN 



CONCENTRATIONS OF SPERMATOZOA 



These observations need not be carried beyond the 4-cell stage, or about 4 hours after 

 insemination. 



