144 s - J- HUNTER. 



for chemicals. For this reason it was found advisable to use 

 only new glassware. 



Much time was consumed in the work of sterilization to pre- 

 vent contamination from spermatozoa. This, however, is essen- 

 tial. In the eighty-three experiments referred to sterilization was 

 performed in accordance with the plan mentioned in the paper 

 cited. If, after this treatment, there were eggs that developed 

 through normal fertilization, such escaped notice. In five other 

 experiments made to determine the necessity of this sterilization, 

 the sea-water was not sterilized nor was the sea-urchin carefully 

 washed in hydrant water. In these five experiments a few nor- 

 mally developing forms were noted. 1 



The relative proportion of eggs to concentrated sea-water is an 

 important factor in determining the percentage of development. 

 In the paper referred to mention was made of the necessity of 

 placing comparatively few eggs in the solution. Tabulated re- 

 sults of some experiments on this point make the relative value 

 of this condition more apparent. In two bowls containing equal 

 amounts of condensed sea-water there were placed in the first 

 bowl a greater number of eggs and in the second bowl very few. 

 Out of the first bowl 14 per cent, reached the swimming gastrula 

 stage ; out of the second bowl 87.5 per cent, reached the same 

 stage. Notes taken on early stages of the culture showed that 

 in the bowl containing the less number, segmentation began in a 

 greater number of eggs. 



In other experiments an endeavor was made to measure ap- 

 proximately the number of eggs placed in a given amount of sea- 

 water. One of these experiments is placed in tabulated form. 



No. i, i pipette full of eggs in 50 c.c. concentrated sea-water. 



No. 2, 5 pipettes full of eggs in 50 c.c. concentrated sea-water. 



No. 3, 10 pipettes full of eggs in looc.c. concentrated sea-water. 



After two hours, transferred to sterilized sea-water, frequently 

 changed at first --examined twenty -seven hours later with re- 

 sults as follows : 



!The test of purity of culture was based on the facts, (i) that cleavage in parthe- 

 nogenetically developing eggs of Arbacia at no time prior to blastula stage, resemble 

 normal processes ; (2) the shortest time in which any culture under constant observa- 

 tion reached the active swimming stage was nine hours and seven minutes, under 

 like conditions normally fertilized embryos become active in about six hours; (31 

 the absence of t.he perivitelline membrane. 



