70 ARTIFICIAL PARTHENOGENESIS AND FERTILIZATION 



is not sufficient. It is also necessary to collect the eggs into 

 a heap by gentle rotation of the dish before transferring them 

 to normal sea-water, so that they can be transferred in a 

 pipette with as little acid as possible. This gentle agitation 

 also prevents the eggs from sticking to the glass. 



After the eggs are transferred from the acid to normal sea- 

 water, they should not be placed at once in the hypertonic 

 sea-water, but only after from fifteen or twenty minutes, or 

 even later still. The hypertonic sea-water used in this case 

 is a mixture of 50 c.c. of sea-water +8 c.c. 2| m NaCl. 1 From 

 this they are transferred to normal sea-water after from thirty 

 to sixty minutes at 15 G., at intervals of from two and one- 

 half to five minutes. After transference to normal sea-water, 

 those eggs which have been just long enough in the hypertonic 

 solution begin to segment and develop. Usually only two 

 astrospheres or centrosomes are formed by this method. 

 When the length of exposure is correct, the first division is a 

 cleavage of the egg into two cells as in normal fertilization, 

 although the cleavage in the first division is often somewhat 

 asymmetrical. That is probably a result of treating the egg 

 with hypertonic sea-water. This anomaly, however, only 

 presents itself in the first division and has no further influence 

 on the development. All the eggs that divide into two cells 

 develop into apparently normal larvae, while eggs which go 

 into more than two cells at one division develop into crippled 

 larvae and most of them die in or before the gastrula stage. 

 This latter abnormal kind of development is obtained regularly 

 if the eggs remain too long in the hypertonic solution and an 

 over-exposure of only a few minutes may produce this fatal 

 effect. This shows how necessary it is that the eggs be trans- 

 ferred from the hypertonic solution to normal sea-water at the 

 right time. 2 



1 A mixture of 2 m NaCl +KC1 +CaCL in the proportions in which these 

 salts exist in the sea-water is still better than 2 m NaCl, since it is less injurious. 



2 This time varies somewhat for the different eggs even of the same female. 



