IO8 THE FROG 



stain on the slide with Delafield's hsematoxylin and counter- 

 stain with orange G. 



14. Sections of the integument. It is desirable to se- 

 cure a frog with mucus glands in the resting condition, but 

 the circumstances that govern this have not been definitely 

 determined. Probably it is better to kill the frog by pithing, 

 not with anaesthetics. A frog whose skin is bright-colored 

 is probably better to show the golden cells. From the dorsal 

 surface cut out a strip of convenient size including both 

 lightly-pigmented and deeply-pigmented portions as well as 

 some poison glands; the latter are especially numerous in 

 the dorso-lateral line. Fix for a few hours in Zenker's 

 fluid. Cut sections 8 n thick. Stain on the slide with Dela- 

 field's hsematoxylin and counterstain with orange G or 

 Congo red. 



15. Cross-sections of decalcified femur. Cut out a 

 piece about I centimeter long from the middle of the femur 

 of a young frog, being careful not to injure the investing 

 membrane, the periosteum. Fix for a week or ten days in 

 a mixture consisting of Muller's fluid, 9 parts, and concen- 

 trated formalin, i part; the formalin must be added at the 

 time of using the mixture, which should be changed daily. 

 Wash for a few days in 5 per cent, formalin, in the dark, 

 changing the solution as often as it becomes discolored. De- 

 calcify for four weeks in 70 per cent, alcohol containing 5 

 per cent, nitric acid, changing the solution every two or 

 three days. Stain deeply with borax-carmine and destain 

 slightly with acid alcohol. Infiltrate in a mixture of xylol 

 and paraffin at about 40 C. for a day or two, and in several 

 changes of pure paraffin melting at 52 C. for 24 to 36 

 hours. Cut sections about 10^ thick. A counterstain is not 

 required. 



