226 StncUes in the Physioloou of Fertilization 



of even a minute quantity of spermatozoa of any other individual com- 

 pletely vitiates the results. This applies to cross-fertilizations as much 

 as to experiments on self-fertilization, but in the latter the effects of 

 such contamination are more obvious. It is with comparative difficulty 

 that self-fertilization can be brought about at all in Ciona, and when it 

 is found that this difficulty is lessened by the changing of a certain 

 factor, the elementary precaution of ensuring the CDinplete absence of 

 spermatozoa from any other individual must naturally be taken before 

 certainty can be attained that the alteration is really due tu the con- 

 dition which has been changed in the e.xperiment. 



The precautions which were taken to ensure the absence of sperm- 

 contamination can be divided under three heads : 



(1) Sterilization of instruments and luater. 



All glass-ware was cleaned with hot water before being used. 

 Similarly scissors, forceps, etc. were dipped into hot water after every 

 operation. The sea-water used in the experiments was taken from the 

 circulation in the laboratory, and passed through a Berkefeld filter. The 

 complete absence of spermatozoa from such water was shown by the fact 

 that Ascidian or Echinoid eggs never segmented wh(,'n left in it without 

 the addition of sperm. 



(2) Method of removing the eggs and sperm from the animals. 

 The first source of contamination of the genital products of Ciona 



with the spermatozoa of other individuals is the possible jiresence in 

 the pharynx, branchial basket and atrium of the animals of spermatozoa 

 taken in with the water. To guard against this, the animals (which 

 were in almost every case brought in from the sea on the day they 

 were used) were treated as follows. The atrial cavity of each was slit 

 up by inserting one point of a pair of scissors into the exhalent aperture. 

 The animal was then pinned down and wa.shed with a copious stream 

 of fresh-water. 



The second pos.sible source of contamination is that in removing the 

 eggs from the oviduct some sperm might accidentally be taken with 

 them from the adjacent vas deferens. It is extremely easy to puncture 

 the oviduct and then suck out the contained eggs with a pipette with- 

 out injuring the adjacent vas deferens at all. Sperm, however, will 

 always exude from the terminal aperture of the vas deferens during 

 this operation. In order to prevent this, the tops of the oviduct and 

 vas deferens were always tightl}' closed by means of a "bull-dog" clip 

 before the animal was washed under the stream of fresh-watei'. 



