IMPROVED METHOD OF ARTIFICIAL PARTHENOGENESIS 69 
Hence membrane formation is essential for development, and 
it is immaterial how the membrane formation has been pro- 
duced; except that the membrane formation induced by a 
fatty acid injures the egg less than that provoked by most of 
the other substances. 
4. In the experiments thus far discussed the treatment with 
the hypertonic solution preceded the artificial membrane forma- 
tion with the fatty acid. Fertilization with sperm, however, 
begins with membrane formation; and hence it was natural to 
find out, whether in artificial parthenogenesis also, membrane 
formation by means of a fatty acid might not be applied as the 
first step. 
The unfertilized eggs were therefore subjected to treatment 
with a fatty acid until they all formed membranes. Ten to 
twenty minutes later they were placed in hypertonic sea-water. 
It turned out that in this order of events the eggs of S. purpur- 
atus need remain in the hypertonic solution only a relatively 
short time, some thirty to sixty minutes at about 15262 
The development of the eggs is nearly identical with that evoked 
by sperm, and practically all the eggs develop; provided that 
the details of the method are correctly carried out. 
. The procedure for the chemical activation of the unferti- 
lized egg of S. purpuratus is therefore as follows. The eggs 
are placed in 50 c.c. of sea-water+2.8 c.c. of N/10 butyric acid 
(which must be thoroughly mixed beforehand). At 15°C. 
a portion of the eggs is transferred after one and one-half, 
two and one-half, and three or four minutes to 200 c.c. of sea- 
water which had previously been made ready for this purpose. 
In one or all of the dishes all the eggs form a normal fertilization 
membrane. 
It may be observed, by the way, that too many eggs must not 
be put into the acid sea-water, since otherwise the mass of acid 
1 The eggs of Arbacia need remain in the hypertonic solution only about 20 
minutes at a temperature of 22° C. 
