. 2 3 APPENDIX. 



in distilled water to sunlight in a porcelain dish until it becomes dark 

 brown ; the reduction is arrested, when sufficiently advanced, by 

 thorough washing in water to which a few grains of sodium chloride 

 have been added. The stained tissue may be mounted either in 

 glycerin or in balsam, soaking in dilute and later strong glycerin, 

 or dehydration and clearing, being the subsequent respective manipu- 

 lations. 



Staining chromatin filaments for the display of karyokinetic 

 figures and other studies of cell-structure can be successfully carried 

 out only after accurate fixation of the cells, for which purpose the 

 stronger Flemming's solution will be found most trustworthy. 



The tissue after such treatment is embedded in paraffin and cut, 

 the fixed sections on the slide being subsequently stained by saffranin 

 or by Delafield's haematoxylin. When karyomitosis is the especial 

 object of study, preparations made by stripping off the epidermis of 

 suitable animals (very young larval newts being excellent) are more 

 favorable than sections, as the cells are preserved intact and contain 

 the entire chromatin figures, and not merely the parts included within 

 the planes of the section. Place small pieces of such tissues in 



a. Saffranin 2 gm. 



Alcohol, 50 per cent 60 c.c. 



24-48 hours. 



b. Wash off in water for a few moments. 



c. Transfer to acidulated absolute alcohol (10 drops of pure hydro- 

 chloric acid to loo c.c. of absolute alcohol) for a few moments (^-i 

 minute) until the clouds of color cease to be copiously given off; 

 then 



d. Transfer to fresh absolute alcohol for i to 2 minutes. 



e. Clear in clove oil and mount. 



Care must be taken not to remove too much color by prolonged 

 action of either the acidulated or the plain absolute alcohol, since 

 the preparation can be almost entirely bleached by inattention to 

 this point. In a successful preparation the chromatin figures are 

 brilliantly stained of a bright red, while the other parts of the cells 

 are almost uncolored. 



Injection of capillary blood-vessels requires considerable ex- 

 perience, and at best an element of uncertainty enters into every 

 attempt, since the condition of the tissues, particularly of the vessels, 

 largely influences the manner in which the fluid runs. While car- 

 mine-gelatin injections make very attractive pictures, a successful 

 blue mass possesses many advantages when used in connection with 



