378 CONNECTIVE TISSUES. 



780. 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 peri- 

 toneal cavity (less preferably subcutaneously). 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. Refer also to J. Thornton Carter, 775A. 



780A. Potash Method for Osteoblastic Centres (SCHULTZE, 

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

 Journ. Anat., v, No. 4, 1905-6). 



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 



Glycerin ....... 20 



Potash ........ 1 



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, upgrade gradually 

 to pure glycerin, in which they may remain. 



MALL (op. cit.) clears formalin embryos in 10 per cent, potash for 

 about a month or longer. Formalin renders the connective tissues 

 very tough, and this strong KOH solution is necessary. Refer also 

 to 592, 



