AETIFICIAL PAETHENOGEXESIS IX ECHIXUS ESCULEXTUS. 547 
Loeb’s inetlioJ consists in ti-eating the unfertilised eggs 
witli butyric acid to cause inetnbrane-forination^ and sub- 
sequently with hypertonic sea-water. 
Owing to the condition of the sea-water at Plymouth it 
is necessary, after membrane formation, to place the eggs in 
water of raised OH ion concentration before treating them 
with hypertonic sea-water. The modification of Loeb’s 
method which we employed is as follows. The unfertilised 
■eggs were placed in — 
(1) 3 c.c.X/10 butyric acid -|- 50 c.c. sea- water for 1 '5 minutes. 
(2) 0'2 c.c. X/10 XaOH -f- 50 c.c. sea-water for 6 minutes. 
(3) 8 c.c. 2’5 M/NaCl -f 5 c.c. sea-water for •75-1 hour. 
The best experiments with this method gave 60 per cent. 
blastulse. The larvae were healthy and grew well. All 
those plutei that underwent metamorphosis were obtained by 
this method. 
Deluge’s method consists in treating" the unfertilised eggs 
with tannic acid and ammonia in a mixture of sea-water 
and cane-sugar. The proportions we found most favourable 
for E. esculentusat Plymouth were 10 c.c. sea-water -f 40 
<3.c. cane-sugar solution (strength, 388 grms. to a litre) + P4 
c.c. lM/60 tannic acid. The unfertilised eggs were placed in 
this for six minutes, and then P5 c.c. M/10 ammonia was 
added for one hour. AVe have obtained 80 percent, blastulte 
by this method, but they were not healthy, and died off 
rapidly during the first week. There is no membrane forma- 
tion by this method. 
The method which we have found most successful consisted 
in treating the unfertilised eggs first with butyric acid to 
cause membrane formation, as in Loeb’s method, and then by 
Delage’s method, as described above. In this way we have 
obtained as many as 90 per cent, blastulte. The larvae are 
vigorous, and grow for the first three weeks more rapidly 
than larvae from fertilised eggs. AVe were, however, unable 
to get any of these larvae to successfully metamorphose. 
Given in detail, this method consists in treating the eggs 
as follows : 
