434 BONE, TEETH 



staining embryos for a week in a solution of 0-25 grni. methylen 

 blue in 100 c.c. of 70 per cent, alcohol with 1 per cent, of hydro- 

 chloric acid. Wash out in alcohol with 1 per cent, of hydro- 

 chloric acid until no more colour comes away (about a week) 

 and mount in balsam. The cartilage remains blue, all the other 

 tissues being colourless. 



Similarly, Lundvall (Anat. Anz., xxv, 1904, p. 219, and xl, 1912, 

 p. 639), using toluidin blue. Thionin blue also may be used. 



Similarly also Bakay (Verh. Anat. Ges., 1902, p. 248), with Bismarck 

 brown (the embryos having been previously treated with nitric acid of 

 3 per cent.). 



For fish embryos. Professor E. S. Goodrich, of Oxford, informs us that 

 thionin (Griibler) is excellent. 



For the Spalteholz method of clearing such preparations see § 809. 



923. Demonstration of Centres of Osteoblastic Activity by 

 Trypan Blue (P. G. Shipley and C. C. Macklin, Anat. Record, 

 X, 1915 — 16). If an azo dye like trypan blue be administered 

 to a very young animal, the bones are stained quickly and very 

 intensely with vital colour. The dye is injected in a 1 per cent, 

 solution into the peritoneal cavity (less preferably subcutane- 

 ously). The animal is killed forty-eight hours after staining, 

 and the tissues are fixed by 10 per cent, neutral formalin injected 

 through blood-vessels, followed by immersion in 10 per cent, 

 formalin for twenty-four to forty-eight hours. Bones are washed 

 thoroughly, hardened in ascending grades of alcohol, after which 

 the soft parts are dissected away. Clear in benzol and then in 

 oil of wintergreen. Study with dissecting microscope. 



924. Potash Method for Osteoblastic Centres (Schultze, 

 Grundriss d. Entwickl. d. Menschens, 1897, and F. P. IVIall, Amer. 

 Journ. Anat., v, No, 4, 1905-06), 



Embryos of mammals after fixation in alcohol may be cleared, 

 for the study of the ossification centres, by means of weak potash. 

 For alcohol specimens Mall considers that Schultze's solution is 

 too strong, and uses instead a 1 per cent. KOH solution for a 

 few hours. With weak solutions the tissues of the smaller embryos 

 remain firm, and, in the end, the specimen is transparent, with the 

 bones held in place. After treatment with the potash, the 

 embryo is placed in the following solution for days, or even 

 months : — 



Water . . . . . .79 c.c. 



Glycerin . . . . . .20 c,c. 



Potash ...... 1 gm. 



From time to time the embryo may be returned to a 3 per 

 cent, solution of potash for a number of hours to hasten the 

 clearing process ; then returned to the glycerin solution, which 

 helps to hold the parts together. When properly cleared, up- 

 grade gradually to pure glycerin, in which they may remain. 



