232 CHAPTER XVII. 



The larger the volume of liquid with which the tissue is sur- 

 rounded, the faster will this washing-out process go on ; and 

 in order that it may not go on with excessive rapidity, wash- 

 ing out the stain from the nerve-fibres as well as from the 

 earlier stained elements (which alone it is desired to wash 

 out, so as to leave a differentiated specific stain of nervous 

 elements), it is in many cases desirable to have the process 

 go on in presence of as little liquid as possible. Another 

 consideration that justifies the practice is that by exposure to 

 air the preparations take up a trace of ammonia, and APATHY 

 has experimentally established that this is an important factor 

 in the sharpness of the stain. Oxygen has, according to 

 him, nothing to do with it. But the point does not seem to 

 be definitively settled. RUBASCHKJN (see Zeit. f. wiss. Milt., 

 xvi, 3,, 1899, p. 372) has lately concluded that the staining 

 of nerve-fibres (not of all tissues) is furthered by oxygen, and 

 also by CO 2 . 



325. Staining Nerve Tissue by Injection or Immersion The 



practice of the earlier workers at this subject was (following 

 EHKLICH) to inject methylen blue into the vascular system or 

 body-cavity of a living animal, wait a sufficient time for it to 

 take effect on the organ to be stained, then remove the organ 

 for further preparation and study. And there appears to 

 have been a belief with some workers that it was an essential 

 or at least a desirable condition to the production of the stain 

 that it should have been brought about by injection of the 

 colouring matter into the entire animal. It is now known 

 that this is generally immaterial, and that the reaction can 

 equally well be obtained by removing the organ and subject- 

 ing it to a bath of the colouring matter in the usual way. 

 But it would also appear that in some cases the procedure 

 by injection is preferable, if not necessary. 



326. The Solutions employed. The solutions used for in- 

 jection are generally made in salt solution (physiological, or a 

 little weaker) ; those for staining by immersion either in salt 

 solution or other " indifferent " liquid, or in pure water. 

 Very various strengths have been employed. The earlier 

 workers generally took concentrated solutions. Thug 

 ARNSTEIN (Anat. Anz. } 1887, p. 125) injected 1 c.c. of satu- 



