228 EDITH PINNEY 



MATERIAL AND METHODS 



The material for this study comprises many series of eggs 

 obtained from heterogenic crosses and fixed at successive stages 

 in development. Each cross was repeated as often as possible 

 in order to insure appropriate stages. Series of straight fertilized 

 eggs of all of the species used were also fixed for comparison with 

 the hybrids. The familiar methods of making the straight and 

 cross fertilizations were used and the usual precautions to pre- 

 vent undesired chance fertilizations were strictly followed. In 

 connection with every experiment unfertilized controls were kept. 

 In no case did these controls show dividing eggs. It is to be 

 regretted that in all cases the development was not followed 

 to the end, but, owing to the abundance of available data in regard 

 to development in fish hybrids, this was not thought necessary. 



Bouin's, Perenyi's, Gilson's, and Kleinenberg's fixatives were 

 used. The Bouin fixative gave uniformly good results. The 

 fixation with Perenyi proved to be very capricious. Eggs, 

 fixed in Perenyi's fluid in July, 1914, and kept in alcohol for over 

 a year and a half, afforded cytological material which from the 

 standpoint of fixation left nothing to be desired. In view of the 

 splendid results of its first trial, I used it freely and with great 

 confidence in preserving material in the summer of 1916. None 

 of the later material killed in Perenyi has been well fixed. By 

 its action the entire system of astral fibers which extends through- 

 out the cell during the later mitotic phases is often entirely ob- 

 literated. More frequently the spindle remains, but its fibers 

 are twisted and distorted and the chromosomes are hopelessly 

 massed together. The best results obtained were when the eggs 

 were fixed during anaphase stages. Such material affords good 

 evidence on some points. The original plan was to fix parallel 

 series, using at least two fixatives, but it was soon found that the 

 amount of available material did not permit this, and the plan 

 was abandoned. The best that could be done was to use one 

 fixative for an entire series and supplement this with occasional 

 stages fixed in some other fluid. In cases where Perenyi's fluid 

 was used for the main series the number of well-fixed stages is 

 small. 



