CHAPTER 16 



Methods 



a. Whole Eggs 



When Arbacia punctulata eggs are centrifuged in sea water, they are 

 thrown to the bottom of the tube and crushed, because they are heavier 

 than the sea water. They must be centrifuged in a medium of the same 

 density as themselves, in which they will be suspended. The solution 

 must also be of the same osmotic pressure as the eggs, so that they will 

 not swell or shrink. Since individual eggs vary in density, it is best to 

 centrifuge the eggs in a medium of graded density, made by partially 

 mixing sea water and 0.85 M cane sugar. The sugar solution' is 

 osmotic with but of slightly greater density than the eggs. Two parts 

 of the cane sugar solution are placed in the bottom of a small slender 

 centrifuge tube and one part of sea water containing the eggs on top. 

 By a slight rotation of the tube the solution can be partly mixed and 

 a density gradient established, so that when centrifuged, the eggs come 

 to lie in a region of their own density. As they break apart during 

 centrifuging, the half-eggs move to a new region equal to their density. 

 They are consequently separated into layers, the heavy (red) halves 

 at the bottom and the hght (white) halves at the top (the unbroken 

 eggs are in the middle), as shown in Plate VII, Photograph 10. With 

 a fine pipette the eggs of each layer can be removed without conta- 

 mination with the eggs of other layers, and large numbers can be 

 collected for experimental work. 



A small electric centrifuge has been used with two (or four) glass tubes 

 6.5 cm. long and 0.4 cm. inside diameter, narrow in order to prevent 

 mixing by convection currents. The centrifugal force, F, in terms of 

 force of gravity (g), is given by the equation F = 0.04 X radius (in 

 cm.) X (r.p.s.)'. A centrifugal force of 10,000 X g for four minutes 



' The 0.85 M sugar (molecular weight 342.24) solution is prepared by adding 29 gm. 

 sugar (commercial samples are as good as "chemically pure") to sufficient tap water to 

 make 100 cc. Tap water is used rather than distilled water as the slight alkalinity prevents 

 stickiness of the eggs. I have found it best to weigh out several lots of 5.8 gm. sugar and to 

 add tap water to make 20 cc. when needed; the sugar solution will keep for several days in 

 the refrigerator (8 '^C), but becomes acid on standing at room temperature. 



