) 
ARTIFICIAL PARTHEXOGEXESIS IX ECHINUS ESCULEXTUS. 529 
have used almost exclusively ‘‘outside water ” foe the early 
stages of all our experiments. 
AVhen the egg.^, after treatment with hypertonic sea-water, 
have been returned to normal sea-water and have segmented 
and developed to the actively swimming blastula stage, they 
have been pipetted off, and placed in breffets^ of Bei kefeld 
water. In this way any contamination of our cultiu-es from 
the outside water has been avoided as far as possible, any 
contamination also being watched by proper controls made 
Avith every experiment. 
The stock solutions of our reagents used have been made 
from doubly re-crystallised salts dissolved in water re- 
distilled from glass. 
The hypertonic sodium-chloride solution was tested by 
precipitation with silver nitrate. 
The solutions used in the experiments were made by adding 
quantities of the stock solutions to sterile sea-water as they 
were required. 
In all our operations proper attention has been paid to 
sterilisation of instruments, pipettes, glassware, etc. Each 
sea-urchin, before being opened, was placed under a tap of 
fresh water to kill any sperm on its surface, and after each 
one opened the hands and instruments have been re-sterilised. 
Of every experiment made a proper control has been kept, 
as well as ev'ery batch of eggs being tested by normal 
fertilisation. In any case where the control has gone Avrong, 
or the eggs failed to fertilise in the normal way when sperm 
Avas added to the fertilisation control, the entire batch of 
eggs Avas thrown away, and the experiment discontinued. 
The meiliod Ave ha\'e followed in rearing our larvm is 
that elaborated by Allen and Xelson (1), and which has beeu 
already extensively applied by one of us (20) to the rearing 
of Echinoid hybrids. It is therefore unnecessary to describe 
this method here, as a detailed description has already been 
published by Allen and Xelson (1). We have made use of 
pure cultures of diatoms to feed the parthenogenetic plutei, 
' Glass jars of •2500 c.c. capacity. 
