180 Animal Microlo<j/j 



Ordinary commercial methylen blue usually contains, in addi- 

 tion to the blue dye, a small quantity of a reddish-violet dye. Such 

 methylen blue is termed polychromatic and is especially service- 

 able in staining certain cell granules. Only the pure methylen 

 blue, however, should be used for nerve staining and other infra 

 vitam work. 



a) Intra Vitam Stain for Small, Comparatively Transparent 

 Aquatic Organisms. Add sufficient methylen blue to the water 

 containing the organisms to tinge it a light blue. Different tis- 

 sues will take up the color after different intervals of time, and a 

 given tissue after having attained a maximum degree of colora- 

 tion will rapidly lose its color again. It is necessary, therefore, 

 to watch the organisms closely for the maximum of color in the 

 tissue desired. If the observer wishes, the stain may be fixed 

 for more prolonged study by following the processes indicated 

 under 6). The order in which various tissues take the stain 

 seems to vary in different organisms. Usually gland cells stain 

 first, then with more or less deviation, other epithelial cells, fat 

 cells, blood and lymph cells, elastic fibers, smooth muscle, and 

 striated muscle. Nerve cells and nerve fibers do not ordinarily 

 take the stain when the entire animal is immersed. 



b] Ehrlich's Method for Nerve-Terminations and the Relations 

 of Nerve Cells and Fibers to the Central Nervous System. 



The stain should be Grrtibler's methylen blue (rectificiert 

 nach Ehrlich). A 1 per cent, solution in normal saline is used 

 Warm the solution till it steams, stir it thoroughly and when 

 cool, filter. The tissue must be perfectly fresh. Chloroform 

 the animal and immediately inject the stain into the main 

 artery of the part to be investigated. If the animal is small, 

 the entire body may be injected. The vessels should be filled 

 full but care must be taken not to rupture them. The part 

 should become decidedly blue in color. It is well after 10 or 

 15 minutes to inject more stain. At the expiration of half an 

 hour after the second injection remove small pieces of tissue 

 containing the nerve elements desired, and e.rpoxc them freely 

 fo the air on a slide wet with normal saline. Examine every 

 two minutes under the microscope (without cover-glass) until 



