72 CASWELL GRAVE 



METHODS 



Tadpole larvae of Amaroucium may be secured in abundance 

 at Woods Hole during the months of July, August, and Sep- 

 tember by placing large colonies of the ascidiozooids in glass 

 jars before a window in the laboratory. The best results have 

 been obtained when the colonies were collected the day before 

 tadpoles were desired and kept in running sea-water during 

 the night. Tadpoles escape from the colonies in swarms at and 

 just after sunrise, but they continue to be liberated in small 

 numbers at any hour of the day. When liberated the tadpoles 

 swim immediately to the surface of the water and collect at one 

 side of the jar where they may be easily captured with a pipette. 



Immature tadpoles and various earlier developmental stages 

 may be secured by squeezing a colony in the hand over a culture 

 dish of sea-water. With the mass of ascidiozooids, tadpoles, 

 embryos, and eggs thus forced from the colony, a considerable 

 quantity of gelatinous debris is included, which settles very 

 slowly, and may therefore be removed by decantation, two or 

 more changes of water being required. Mature tadpoles may 

 be had in small numbers in this way also. For a few seconds 

 after having been squeezed from the colony they lie motionless 

 upon the bottom of the dish, but soon begin to move Rightly, 

 then to swim about, apparently stimulated by light or by con- 

 tact with pure sea-water. 



For the study of the general and histological structure of the 

 tadpole, Bouin's and Flemming's mixtures have been found to 

 give the best fixation. Sections have been made by the paraffin 

 method and stained with Delafield's or iron haematoxylin. 

 For total mounts an excellent transparent stain for tadpoles 

 fixed with Bouin's solution is made by adding to 70 per cent 

 alcohol, slightly acidulated with HCl, an amount of borax carmiri 

 sufficient to give a delicate pink color to the solution. Specunens 

 should be allowed to remain in the stain twelve hours or more. 



