58 THE AMERICAN ARBACIA 



There is of course a great advantage in procuring large quantities of 

 eggs by the KCl method, without destroying the animals. Forced 

 shedding by the electrical method has already been described in 

 Section c of this chapter. 



h. Removal of Gonads 



The old method of obtaining eggs and sperm, practiced for many 

 years, was to remove the ovaries and testes intact from the animal. If 

 animals are not scarce, or if for some reason shedding with KCl or 

 electricity is contra-indicated, the following procedure is advocated 

 (E.B.Harvey, 1939 b). 



Wash the animal with running cold fresh water for a moment or two to kill any 

 sperm possibly adhering to the shell. Take the animal, oral (teeth) side up, and cut 

 around the shell with scissors, at about its widest circumference ; then remove the 

 upper (oral) part of the shell. The five gonads are now in view in the lower part, the 

 ovaries red, the testes white. 



To prepare the eggs for experimental work, run a pair of curved forceps gently 

 under an ovary, to snip the duct, and then remove it intact to a finger bowl about 

 a quarter full of fresh sea water. Remove the other four ovaries in the same way. Con- 

 trary to the prevalent opinion, the fluid in the body cavity (in a small amount), is 

 not toxic to the eggs. Let the bowl stand for five or ten minutes so that the ripe eggs 

 may flow out of the ovaries. Then put a piece of cheese cloth whose holes are five to 

 ten times the diameter of the egg (i.e. about 0.5 mm.) and which has been wet with 

 sea water, over another finger bowl and pour the egg suspension through. The debris 

 and pieces of tissue will be be held back and you will have eggs free and clean in the 

 dish, ready for use. To keep the eggs for several hours, there should not be too many 

 eggs in the dish, just enough to form a thin layer on the bottom. The bowls of eggs 

 are best kept on the floor of the cement aquarium tables where cool water will flow 

 around them, and they should be kept covered to prevent evaporation. The eggs 

 treated in this way are suitable for use throughout the day though they change 

 slightly on standing. Individual batches of eggs vary greatly in shape, percentage 

 of fertilization, reaction to centrifugal force, etc. Any batch which does not give 

 98 % fertilization membranes or which shows abnormalities in cleavage should be 

 discarded. 



The testes should be removed with curved forceps in the same way as the ovaries, 

 but put into a small salt-cellar or a very small Stender dish (3 cm. diameter) without 

 water. This dish should be covered and kept cool. The sperm are inactive when kept 

 concentrated, but become active in sea water, soon wearing themselves out. If the 

 dish of sperm is placed immediately in the refrigerator and kept at about 8° C, the 

 sperm will keep perfectly for 4-5 days. After dilution, the sperm are in optimum 

 condition for only an hour or so. 



After opening a male, care should be taken to prevent contamination of females 

 opened subsequently. Scissors and forceps should be put into a bowl of tap water 

 and one's hands should be thoroughly washed, immediately after opening an animal. 



