77 



substances or conditions that become changed after death. In the 

 reaction reduction of the methylene blue to its leucobase (colorless) 

 by the nervous tissues appears to play an important part. In general, 

 the technique involves: (1) Bringing a methylene blue solution of 

 sufficient strength in contact with the (essentially) living nervous 

 elements, (2) permitting it to remain a sufficient length of time for 

 the staining reaction, (3) exposing the tissue to the action of the 

 oxygen of the air until the stain is fully developed, re-oxidation 

 of any leuco-methylene blue and satisfying the reducing reaction of 

 the tissue; then (4) either examining at once or fixing the stain 

 in situ by its precipitation in an insoluble form for its preservation 

 (imbedding and sectioning) . 



1. The methylene blue may be brought in contact with the 

 neurones by injection, (a) through the vascular system, aorta if 

 the animal is small, artery supplying the part, if large; (b) into 

 the body cavities ; (c) subcutaneously : or, by immersion of the organ 

 or part, or the entire animal if small (many invertebrates). Keep- 

 ing in mind the end result desired, the best method will suggest itself 

 in a particular case. Cajal cut parallel slits in the cerebral cortex 

 and inserted the methylene blue in powdered form or as a saturated 

 solution. In general, the solutions should be as dilute as possible; 

 of 1/15 to 1/4% strength in physiological salt solution, the more 

 direct the application the weaker. It is well to have on hand a 1% 

 stock solution in physiological salt solution and dilute it (with salt 

 solution) as desired. Of the methylene blue preparations that are 

 available, Ehrlich's or "B. X." are more generally used. A combi- 

 nation of injection and immersion is often advisable. In introduc- 

 ing the stain by injection, first remove the blood by washing out with 

 physiological salt solution or by bleeding and let the injection be 

 full, i. e., through the capillaries into the veins. If a mammal is be- 

 ing dealt with, salt solution and staining solution should be warmed 

 to body temperature (35 to 38 C.). 



2. It is difficult to give any general rules as to the time the 

 methylene blue solution should remain in contact with the tissue 

 before exposure as it is best to determine it experimentally in each 

 case. The time should be shorter for warm blooded, longer for cold 

 blooded animals. If introduced by injection, 20 to 30 minutes for 

 a mammal, 2 to 12 hours for a cold blooded form, may be suggested; 

 the organ or part should then be removed, wet with the dilute stain 

 (perhaps 1 /15% strength) for another period of time, ^ to 1 hour, 



