538 CKESSWELL SHEARER AND DOROTHY .IORDAN EEOYD. 
As the result o£ many experiments we have found that with 
Loeh’s improved method the figures and procedure given 
below are generally the most satisfactory for E. escu- 
lent u s . 
(1) 3 c.c. N/10 butyric acid + 50 c.c. sea-water for 3 
minutes. 
(2) Wash in two or three changes of sea-water and 
transfer to : 
(3) ‘2 c.c. N/10 NaOH + 50 c.c. sea-water for 6 minutes. 
Transfer to : 
(4) 8 c.c. 2‘5 M NaCl + 50 c.c. sea-water for ‘ 75-1 hour. 
Transfer to — 
(5) Nornml sea-water. 
IV. Dei.age’s Method. 
Delage (5) used tannic acid followed by ammonia to induce 
parthenogenesis in Strougylocentrotus lividus at Ros- 
colf. Various media were used as bases for these reagents, 
but the one finally adopted was a mixture of 30 c.c. sea-water 
and 70 c.c. of a solution of cane-sugar containing 388 grin, 
per litre. Delage fixed on this strength of sugar solution, 
which is about D13 molecular, under the impression that it is 
isotonic with sea-water. Mr. D. J. Matthews, who has kindly 
determined the salinity of the “ outside water ” from one of 
the jars used in our experiments, gives us the figure S = 
.34'60. From Krummel’s tables this gives an osmotic 
pressure of about 23 atmospheres, whereas the experimental 
number obtained by Lord Berkeley and Mr. Hartley (2) for 
the osmotic pressure of a solution of sucrose of the strength 
used by Delage is somewhere about forty atmospheres. 
The tannic acid solution used by Delage contained 5'4 
N 
grm. of the acid per litre. In his paper he calls this 
assuming that the carboxyl and five hydroxyl groups of 
tannic acid are all dissociated in solution. The dissociation 
constant for tannic acid has never been taken experimentally. 
