SEPARATION OF FORM AND FUNCTION 



167 



PEOCEDUEE: 



EFFECT ON THE SPERM AND FERTILIZATION 



1. Prepare an ovulating female, Sana pipiena. 



2. Prepare two sperm suapenaiona (Eana pipiena), aa followa: 



a. Control - 2 paira of teates in 10 cc, of Spring water. 



b. Experimental - 2 paira of teatea in 10 cc. of Spring water containing 



1/3,000 MS 222. Examine a drop of this auapenaion Just iDefore atripping 

 the eggs into it to determine whether the sperm are motile. 

 5. Fertilize eggs from the same female in the two aperm suapensiona Flood in I5 



minutes with more of the aame solutions used to make the suapensiona. 

 k. After 1, 5, and 5 hours remove eggs from the Experimental mass and place them in 



pure Spring Water. Lahel the container, and make a further change in Spring Water 

 in 5 minutea. Allow these eggs to develop under the same conditions aa the con- 

 trola. Observe frequently. 

 5. Obaerve the development of the eggs remaining in the MS 222. 



RECORD OF FERTILIZATION DATA (SpeciesL 



EFFECT ON EARLY MOTILE STAGES 



Anura St. #17-#S5: Urodela St. #27-#35 



1. Select a large number of embryos of the same age, atage, and species. There ahould 

 be evidence of motility. Anura stagea #21-25, Urodela Stages #31-#55 are best. 



2. Prepare finger bowls or Petri dishes of MS 222 in 1/5,000 concentration. 



5. Place the embryos, 5 at a time, in a finger bowl of anesthetic and, with a stop 

 watch and 2-3econd Interval stimulations, determine the time of the first and the 

 last of the 5 embryos to lose their responsiveness to tactile stimvilation. It is 

 best to use a hair loop and stimulate the same region, i.e., the side of the body. 

 Mark each finger bowl with the exact time of anesthesia. 



k. At intervals of 1, 6, 2k, 56, and 1*8 hours (or approximately similar houra) transfer 

 the anesthetized embryos from one of the finger bowla to pure Spring Water. With a 

 atop walch and 2-second interval stimulations (as above) determine whether snort or 

 long anesthesia has any effect on the period of recovery. Note, Incidentally, 

 whether there haa been any injury to the experimental embryos as evidenced by ab- 

 normal movements . 



