218 TEXT-BOOK OF EMBRYOLOGY. 



dermal cells, the formation of the primitive segments, and the further differentiation of the 

 primitive segments into sclerotomes and myotomes may be studied in successive stages. 



The embryos are removed from the egg, fixed in Flemming's fluid or Zenker's 

 fluid sectioned transversely in paraffin, stained with Heidenhain's hematoxylin and 

 mounted in xylol damar (see Appendix). A counterstain with a weak solution of 

 fuchsin (0.5 per cent, in distilled water) is of value in studying the fibers. This stain is 

 used after the hematoxylin, and the sections are then rinsed in distilled water before passing 

 them into alcohol. 



Sections of the umbilical cord of any mammalian embryo, prepared by the above technic, 

 are also very instructive. 



Primitive Segments. — The primitive segments, from which the vertebra, etc., are derived, 

 can be studied in transverse sections of chick embryos during the second and third days of 

 incubation. The material is prepared as described above under the head of embryonic 

 connective tissue. For a comprehensive picture of the series of primitive segments, sagittal 

 sections should also be prepared. 



Blastemal Stage of 'the Skeletal System— -Pig embryos of 12 to 14 mm. are fixed in Zenker's 

 fluid or in Bouin's fluid, cut transversely in celloidin or paraffin, stained with Weigert's 

 hematoxylin and eosin, and mounted in xylol-damar (see Appendix). The anlagen of the 

 vertebrae appear as condensations in the embryonic connective tissue. Similar condensa- 

 tions in the extremities indicate the anlagen of the appendicular skeleton, and in the region 

 of the base of the skull the anlage of the chondrocranium. 



By the use of serial sections, and the method of plastic reconstruction, models of the 

 anlagen of the bones may be made. (For method of reconstruction, see p. 638.) 



Cartilaginous Stage of the Skeletal System. — Pig embryos of about 35 mm., prepared as de- 

 scribed above under the head of the Blastemal Stage, show the cartilaginous elements which 

 precede true bone in the vertebra, ribs, base of the skull, and certain portions of the appen- 

 dicular skeleton. In order to get a comprehensive idea of the relation of the cartilaginous 

 elements to one another, it is often necessary to make models of parts or of the whole of the 

 cartilaginous skeleton by the method of plastic reconstruction (p. 638). Serial sections are 

 of course necessary for this. 



Stage of Ossification, (a) Intramembranous Bone. — Small pieces, including the skin and 

 dura mater, are removed from the skull cap of a four-month human foetus or of a four -inch 

 pig embryo, fixed in Orth's fluid, hardened for several days in alcohol, decalcified in 1 per 

 cent, hydrochloric acid (several days), washed in running water to remove the acid, sectioned 

 at right angles to the surface in celloidin or paraffin, stained with Weigert's hematoxylin 

 and picro-acid-fuchsin, and mounted in xylol-damar. 



(b) Intracartilaginous and Subperiosteal Bone. — Remove the forearms and legs of fcetal pigs 

 of five to six inches, and prepare by the technic given in the preceding paragraph, cutting the 

 sections parallel to the long axes of the developing bones. 



In preparing specimens for the study of either intramembranous or intracartilaginous de- 

 velopment, both fixation and decalcification can be done at the same time. Put the fresh 

 material in Bouin's picro-formalin-acetic mixture and let it remain for a week, with 

 one or two changes of the fluid; then wash in several changes of alcohol (40 to 50 per cent.). 

 A few drops of ammonia added to the alcohol facilitates the removal of the picric acid. 

 Further treatment is the same as after any ordinary fixation. The results are good. 



The earlier stages in the ossification of the vertebre and the base of the skull can be studied 

 in pig embryos of 40 to 50 mm. The embryos are fixed, and at the same time decalci- 

 fied (see preceding paragraph), in Bouin's fluid. They are sectioned transversely in 



