574 



SCIENCE 



[N. S. Vol. XXXIV. No. 878 



less lecithin would be required unless one as- 

 sumes that this substance functions in more 

 than one way in the cell, which is, of course, 

 quite possible. With these several possibili- 

 ties open it seemed that some method of ex- 

 perimental attack could be used; that if the 

 idea of nuclein synthesis during cleavage were 

 true it should be possible to show as cell-di- 

 vision progresses a diminution of alcohol-sol- 

 uble phosphorus with a corresponding increase 

 of phosphorized material not digestible by 

 pepsin. 



Using the eggs of Arhacia punctulata, the 

 phosphorus partition in the 2-4 cell stage was 

 compared with that in early blastula. Two 

 separate experiments are reported. The meth- 

 ods used were the same in each; so the only 

 difference between them was that in Experi- 

 ment II. a smaller mass of eggs was available 

 — though the latter fertilized and developed a 

 trifle better — than in Experiment I. The de- 

 tails follow. 



After removal from the ovaries the eggs 

 were washed several times with large quanti- 

 ties of filtered sea-water until they settled 

 rapidly and left no material suspended in the 

 supernatant liquid. They were then sus- 

 pended in 2,000 c.c. of sea-water and fertilized 

 with a small amount of sperm. The eggs 

 were stirred and oxygen allowed to bubble 

 through the suspension at short intervals. 

 When the first cleavage occurred 1,000 c.c. 

 were measured off and allowed to settle. The 

 remaining eggs were allowed to divide for five 

 hours from the time of fertilization, reaching 

 then the early blastula stage. These eggs 

 were not allowed to develop to the swimming 

 stage because of the probable difficulty of 

 separating them from the surrounding sea- 

 water without causing cytolysis. 1,000 c.c. 

 were taken and from this point were treated 

 in precisely the same manner as the previous 

 mass of eggs at first cleavage. 



In each case — first cleavage and early 

 blastulae — after settling in sea-water, the lat- 

 ter was drawn off, using a ceRtrifuge to con- 

 centrate the eggs as much as possible. It may 

 be added here that previous trials had shown 

 the practical impossibility of separating whole 



or cytolyzed eggs from the surrounding 

 medium by filtration. Even if by special meth- 

 ods clear filtrates were obtained, the process 

 of filtration was so prolonged that the mate- 

 rial decomposed though thymol was added as a 

 preservative. Eesort was had, therefore, to a 

 small hand centrifuge, which proved very 

 satisfactory in separating the solid materials 

 from the various liquids. Remarkably clear 

 liquids were obtained in a very short time. 

 During the time that undesirable chemical 

 changes might take place the materials were 

 kept on ice. 



The eggs, freed now from sea-water as far 

 as possible, were cytolyzed with successive 

 small portions of distilled water (25-50 c.c.) 

 until the combined filtrates in each case 

 equaled 700 c.c. By this time the pigment 

 was practically entirely removed and it was 

 assumed that all water-soluble material had 

 been taken out. The residual material was 

 then extracted with successive 25 c.c. portions 

 of boiling neutral ethyl alcohol (95 per cent, 

 redistilled over NaOH) until the volume, in- 

 cluding two final extractions with boiling ab- 

 solute alcohol, equalled in each case 400 c.c. 



Each of the residues after alcoholic extrac- 

 tion was dried at 60° C. and then digested for 

 four days with 150 c.c. of pepsin-hydrochloric 

 (2 grams of pepsin in 1,000 c.c. of .4 per cent. 

 HCl). The peptic digests were then filtered 

 and washed with cold water. 



Phosphorus determinations were now made 

 on (a) water-soluble material ; (b) alcohol-sol- 

 uble material; (c) the filtrates and (d) the 

 residues from peptic digestion. Controls 

 were also run on the pepsin solution and on the 

 sperm used in each experiment. The material 

 in each case was digested with 10 c.c. of con- 

 centrated sulphuric acid. The digestion was 

 finished by the addition of a few drops of 

 fuming nitric acid. The phosphorus was first 

 precipitated by molybdic acid and weighed 

 finally as magnesium pyrophosphate.' The 

 data obtained are tabulated below. The fig- 

 ures represent milligrams of magnesium pyro- 

 phosphate. 



' ' ' Methods of Analysis, ' ' Bureau of Ghemistry 

 Bulletin 107 (revised), p. 2. 1907. 



