190 METHYLEN BLUE. 



has directly experimented on this point, and sums up the necessary 

 conditions as follows : The tissue need not be living, but must be 

 fresh ; nothing must have been extracted from it chemically, and 

 its natural state must not have been essentially changed by physical 

 means. For example, the tissue must not have been treated with 

 even dilute glycerin, nor with alcohol, though a treatment for a 

 short time with physiological salt solution is not very hurtful ; it 

 must not have been coagulated by heat. MICHAILOW (ibid., xxvii, 

 1910, p. 7) prefers tissues that have lain from one and a half to two 

 hours after the death of the subject in Ringer's salt solution. 



As above explained, the primary stain obtained by injecting 

 methylen blue, or immersing tissues in it, only lasts a very short 

 time. In order to get it to last long enough for study, it must be 

 re-blued by oxidation (see last ). It is therefore the usual practice 

 to dissect out the tissues to be examined, and leave them for some 

 time exposed to the air. This is done in order that they may take 

 up from the air the necessary oxygen. 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. EHRLICH 

 also (op. tit.) holds that an alkaline reaction of the tissues is a neces- 

 sary condition to the stain. APATHY further holds that the stain is 

 a regressive one, easily washed out by the surrounding liquid ; and 

 in order to prevent this washing-out being excessively rapid, it is 

 desirable to have it go on in presence of as little liquid as possible. 



341. The Modes of Staining. The practice of the earlier workers 

 at this subject was (following EHRLICH) to inject methylen blue into 

 the vascular system or body-cavity of a living animal, wait a suffi- 

 cient time, then remove the organ for further preparation and study. 

 And there appears to have been a belief with some workers that it 

 was essential that the stain should have been brought about by 

 injection of the colouring matter into the entire animal. It is now 

 known that the reaction can often be equally well obtained by re- 

 moving an organ and subjecting it to a bath of the colouring matter 

 in the usual way. But in some cases it seems that injection is 

 preferable, if not necessary. 



342. The Solutions employed. The solutions used for injection 

 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. The earlier workers generally 

 took concentrated solutions. Thus ARNSTEIN (Anat. Anz., 1887, 



