42 BASIC METHODS FOR EXPERIMENTS 



That the presence of the egg-jelly is not necessary for mem- 

 brane-separation has been abundantly shown by several workers, 

 notablv Harvey, Lillie, Just and Hobson. The fact that shed 

 eggs, which in some instances are devoid of jelly, on insemination 

 separate perfectly normal membranes argues against any neces- 

 sary role of the jelly in the process of membrane-separation. 

 Moreover, frequently through shaking, the eggs lose their jelly 

 but retain their capacity to separate membranes. Centrifuging 

 removes the jelly without impairing the normal response of the 

 eggs to insemination. What is true, therefore, is not that 

 the egg-jelly is the sine qua non for this response, but that 

 the methods for its removal may be deleterious to the eggs 

 themselves. This is the case with HCl in sea-water. 



HCl in sea-water, employed to remove the jelly from echinid 

 eggs, may be harmful to the eggs either because the concentra- 

 tion of the acid is too great or the washings in sea-water subse- 

 quent to the acid treatment are not sufficrently thorough. If 

 the concentration of the acid is too great, it blocks fertilization. 

 According to Clowes and Smith (1923), the acid limit (of COo- 

 free sea-water) for a ten minute exposure for -eggs of Arhacia 

 and Asterias is about pH 4.4. That is, at this pH and below, 

 the eggs are irreversibly injured. 



If the worker insists on using acid sea-water to remove the 

 egg-jelly he may find the following directions useful: 



Treat each of four to six lots of eggs from the same female 

 with 50 cc. of HCl sea-water of pH ranging from 3.5 (eggs 

 exposed for five minutes) to 5.0 (eggs exposed for eight minutes). 

 Wash the eggs thoroughly by carrying them over, with as little 

 of the acid sea-water as possible, to 1000 cc. of normal sea-water 

 in a large flat bottom dish of 3000 cc. capacity. Gently stir 

 the water with a circular motion when the eggs have settled, in 

 order to bring them to the centre of the dish. Pipette them oflF 

 and place in 1000 cc. of sea-water. Take samples of each lot 

 and inseminate. At least one lot should show as many mem- 

 branes as the untreated inseminated eggs; if no lot gives this 

 result, the eggs have suffered from the acid treatment. If, how- 

 ever, one or more of the samples show normal membranes fol- 

 lowing insemination, take additional samples from the original 

 lots and without insemination examine each in turn under the 



