290 TEXT-BOOK OF EMBRYOLOGY. 



mation of the primitive blood vessels is seen in the visceral mesoderm. The vessels often 

 contain masses of nucleated cells (erythroblasts) derived from the blood islands 



The study of the developing vessels within the embryo is much more difficult, requiring 

 complete serial sets of sections at different stages, and involving tedious methods of recon- 

 struction. Any particular vessel at one stage must be traced from section to section through- 

 out its entire length, and its relations to other vessels and to surrounding structures must be 

 observed. Furthermore, such observations must be made at many different stages. It is 

 obviously impossible to retain a clear mental picture of all such vessels throughout a series 

 of observations; consequently it is necessary to reconstruct graphically or otherwise the 

 vessels in the succeeding stages in order to make comparisons and observe the progress of 

 development. 



For the successful study of developing blood vessels (and lymphatic vessels) two things 

 are essential: 



1. In the first place the embryo at any particular stage must be treated in such a manner 

 as to differentiate the vessels from the surrounding tissues, and so enable one to reestablish 

 their continuity throughout a long series of sections. The differentiation of the vessels is 

 usually obtained by the use of certain stains which have strong affinities for blood cells. 

 A chick embryo or mammalian embryo is fixed in Zenker's fluid. As much blood as possi- 

 ble should be left in the vessels. The embryo is embedded in paraffin and cut into trans- 

 verse serial sections, care being taken to have the series complete. The technic for serial 

 sections will be found in the Appendix. 



The method of staining is as follows: 



1. Xylol, graded alcohols, water. 



2. Weigert's haematoxylin, several minutes. 



3. Rinse in water. 



4. Decolorize in water acidulated with HC1 (six drops to 50 c.c. of water) until 

 tissues appear gray. 



5. Rinse in water. 



6. Dip in water containing a few drops of ammonia (three drops to 50 c.c. of 

 water) until tissues are blue. 



7. Rinse thoroughly in distilled water. 



8. One-half to i per cent, solution of Orange G. in distilled water until tissues 

 acquire a brownish tinge. 



9. Rinse in distilled water. 



10. Graded alcohols, xylol, xylol-damar. 



This method gives the blood cells a bright orange color, thus differentiating the vessels 

 from the surrounding tissues. In case the blood does not take the stain readily, a drop of 

 acid (acetic is good) in the Orange G. solution (one drop to 100 c.c.) will usually remove 

 the difficulty. 



Similar results may be obtained by using picric acid instead of Orange G. 



2. Since the sections are arranged serially, it is possible to trace the course of a vessel 

 from section to section; but to obtain a complete picture of the vessels it is necessary to 

 reconstruct (a) diagrams or (b) models, (a) Where conditions are not too complicated the 

 graphic reconstructions are sufficient. It is possible to make a map, as it were, of the vessels 

 on paper so as to get a comprehensive view of a whole system of vessels (see Appendix), 

 (b) Where there are many vessels it is best to make plastic reconstructions. By this method 

 any number of vessels may be reproduced in wax, and a model of an entire system obtained 

 (see Appendix). 



