294 ADVENTURES IN RADIOISOTOPE RESEARCH 



labelled. If the yolk piiosphatide remains unlabelled while that of 

 embryo becomes radioactive, we can conclude that the phosphatide 

 molecules present in the embryo have not come from the yolk but 

 have been built up in the embryo with the participation of labelled 

 inorganic P. Similar considerations apply to certain other compounds 

 occurring in the embryo. 



METHODS 



The phosphorus content of a series of solutions is usually determined 

 colorimetrically. For example, the inorganic P present in one sample 

 of an acid-soluble fraction can be determined in this way, and then in 

 another sample the phosphagen-P present can be converted into inor- 

 ganic P, so that colorimetric determination now supplies the value for 

 the inorganic P + phosphagen-P. In our experiments this was inade- 

 quate. We had to measure not only the P content but also the activity 

 of the various fractions, so we had to obtain precipitates in each case. 

 To obtain sufficient precipitate when dealing with eggs only incubated 

 for a few days, it was necessary to work with several eggs simultaneously. 



We precipitated the phosphorus, after bringing it into the inorganic 

 state, as ammonium magnesium phosphate. The precipitate was then 

 dissolved in 0.1 N HCl and an aliquot part was sucked into a glass 

 cuvette. This was placed below the Geiger counter used to determine 

 the activity of the preparations, while another aliquot part was utilized 

 for the colorimetric determination of the phosphorus content. The glass 

 cuvettes were covered with a thin mica window (5—6 mgm per cm'^) 

 which only absorbed to a negligible extent the B-rays emitted by the 

 radioactive phosphorus; the area of the mica window was I.l cm- and 

 the liquid content of the cuvette amounted to about 0.5 ml. 



We were interested in the determination of the activity of 1 mgm P 

 prepared from different phosphorus compounds present in the embryo 

 or in the remains. Accordingly we were not concerned with quantitative 

 determination of the P compounds present but concentrated our 

 efforts on obtaining the various fractions in a pure state — to avoid, 

 for example, traces of inorganic phosphate remaining in the phospha- 

 tides extracted from the yolk. As the phosphatides of the yolk were 

 found to be but slightly active, while the inorganic P was strongly active, 

 even a small contamination of the former by the latter was to be avoided. 

 The white, the yolk, the embryo and, in some cases, the amniotic and 

 allantoic liquids were worked up simultaneously. 



As regards the white we were only interested in the total activity 

 present after incubation. The white was ignited (reduced to ash) and its 

 phosphorus precipitated as ammonium magnesium phosphate. 



