332 ORILLA STOTLER WERNER. 



the egg with warm instruments and placed in Allen's modification 

 of Bouin's fluid for two hours. The temperature of the fixative 

 was kept at 37. The amnion was punctured at the end of an 

 hour to allow the fixative free access to the embryo. 



The tissues of the first three embryos were rendered practically 

 useless by increasing the strength of the alcohol too rapidly. The 

 chromosomes were clumped and massed so that it was almost im- 

 possible to make a count. For this reason the following procedure 

 was worked out. When the tissues were removed from the 

 fixative they were rinsed in several changes of distilled water at a 

 temperature of 37, then passed successively through the following 

 grades of alcohol: ^, 1,2, 3, 4, 5, 6, 7, 8, 9 and 10 per cent. 

 During this time the alcohols were maintained at the same tem- 

 perature and the tissues were allowed to remain approximately 

 twenty minutes in each fluid. While the material was in the 10 

 per cent, alcohol the membranes were removed from the embryos, 

 and placed in a 2 per cent, solution of iron alum ; one and one half 

 hours. They were then rinsed in several changes of distilled 

 water and placed in an aqueous solution of Heidenhain's hema- 

 toxylin, two hours; rinsed in tap water and destained in iron 

 alum. They were then passed successively through the following 

 grades of alcohol, remaining about ten minutes in each grade: 

 12, 14, 16, 18, 20, 22, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 and 

 100 per cent. They were then passed through xylol, fifteen 

 minutes, cedar oil three hours, back to xylol ten minutes, 100 per 

 cent, alcohol one hour, forward to cedar oil two hours, then xylol 

 fifteen minutes, and finally cut into small pieces for mounting. 

 The double clearing makes the tissues beautifully transparent. 



As suggested by Painter in his study of mammalian material, 

 the large mesodermal cells were found to be the best for study. 

 For this reason the tissue was placed on the slide with the meso- 

 dermal surface upward. They were mounted in gum damar, a 

 small leaden weight being placed on the cover slip while the slides 

 were drying. 



Technique for Embryos. The embryos were taken from the ten 

 per cent, alcohol and passed through the same grades as were the 

 membranes but because of their greater bulk were left thirty 

 minutes in each grade. They were then passed through half 



