166 



SEPARATION OF FORM AND FUNCTION 



Controls : The controls consist of untreated embryos of the same age and stage, kept 

 under identical conditions except for the drug being tested on the experimentals. 



PROCEDURE: 



EFFECT ON THE SPERM AND FERTILIZATION 



1. Prepare an ovulating female, Rana pipiens. 



2. Prepare two sperm suspensions (Rana pipiens), as follows: 



a. Control - 2 pairs of testes in 10 cc. of Spring water. 



b. Experimental - 2 pairs of testes in 10 cc. of Spring water containing 1/3, 000 



MS 222. Examine a drop of this suspension just before stripping the eggs 

 into it to determine whether the sperm are motile. 



3. Fertilize eggs from the same female in the two sperm suspensions. Flood in 15. 

 minutes with more of the same solutions used to make the suspensions. 



4. After 1, 3, and 5 hours remove eggs from the Experimental mass and place them 

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

 Water in 5 minutes. Allow these eggs to develop under the same conditions as 

 the controls. Observe frequently. 



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



RECORD OF FERTILIZATION DATA (Species ) 



EFFECT ON EARLY MOTILE STAGES 

 Anura St. #17-#25: Urodela St. #27-#35 



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

 should be evidence of motility. Anura stages #21-25, Urodela Stages #31-#35 are 

 best. 



2. Prepare finger bowls or Petri dishes of MS 222 in 1/3, 000 concentration. 



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

 watch and 2-second interval stimulations, determine the time of the first and the 

 last of the 5 embryos to lose their responsiveness to tactile stimulation. 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. 



4. At intervals of 1 , 6, 24, 36, and 48 hours (or approximately similar hours) trans- 

 fer the anesthetized embryos from one of the finger bowls to pure Spring Water. 

 With a stop watch and 2-second interval stimulations (as above) determine whether 

 short or long anesthesia has any effect on the period of recovery. Note, inci- 

 dentally, whether there has been any injury to the experimental embryos as evi- 

 denced by abnormal movements. 



