514 H. H. New?nan 



5 Methods Proper 



The methods of experiment and study were naturally the direct 

 outcome of the experience outlined under the three previous heads. 

 By taking the precaution to equalize, as far as possible, the physio- 

 logical condition of the parents and the external conditions of the 

 developing embryos, and, at the same time, allowing for the factor of 

 variability, it was possible to get results of a somewhat regular 

 and invariable nature. 



The method of procedure that gave the best results was as 

 follows: Fresh, egg-laden females of good size of both species 

 were selected. The eggs of a F. heteroclitus female were then 

 stripped into one finger-bowl, those of a F. majalis female into 

 another. The eggs were then stirred up with the finger so that 

 those first extruded and those last extruded might be evenly 

 distributed. Then about half of the eggs in the two bowls were 

 transferred to two other bowls. Two males, one of each species, 

 at the height of their sexual tone, as indicated by their dark colors, 

 the presence of contact organs, etc., were then chosen. The milt 

 of the F. majalis male was stripped into a very little sea-water, 

 stirred and poured partly on one lot of F. heteroclitus eggs and 

 partly upon one lot of F. majalis eggs, being stirred up with the 

 eggs in both cases. After washing the hands in fresh water, 

 which certainly killed all adhering sperm from the F. majalis 

 male, the F. heteroclitus male was used to fertilize the remaining 

 two lots of eggs. After allowing the eggs in all four bowls to 

 stand for about fifteen minutes with the small amount of water 

 used in the fertilization process, the excess sperm was washed out 

 with fresh sea-water and the eggs were transfered to large, cov- 

 ered bacteria dishes, containing about a liter of fresh sea-water. 

 Usually from one to two hundred eggs were allowed to develop in 

 each dish. The water in these dishes was partially drawn off 

 and renewed nearly every day. Eggs were dissected apart when- 

 ever a tendency to clump up manifested itself. All dead eggs or 

 embryos were removed as soon as noticed. The water always 

 smelled sweet and fresh in cultures treated as described, and a 

 very large percentages of embryos developed and hatched in the 

 two pure strains, especially in those of F. heteroclitus. 



