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 norma! 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. 24 m NaCi.! From 
this they are transferred to normal sea-water after from thirty 
to sixty minutes at 15° C., 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. Usualiy 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 
_jarvae 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.” ann 
‘A mixture of 2} m NaCl+KCl+CaClh 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. 
