192 METHYLEN BLUE 



simple immersion of intact animals in a dilute staining solution in the 

 manner we are considering. Chief amongst these are nerve-fibres and 

 ganglion-cells, ivhich remain unstained in the intact organism. To get 

 these stained, it is necessary to isolate them sufficiently, as explained 

 in the following sections. 



379, Staining Nervous Tissue during Life. Ehrlich (op. cit., 

 last §) found that by injecting a solution of methylen blue into 

 the vessels or tissues of living animals and shortly after- 

 wards cutting out and examining small pieces of their tissues, 

 these will be found to be intensely stained in some of their ele- 

 ments (chiefly nervous). If the tissues are mounted under a 

 cover-glass, the stain will fade in a short time ; but if the cover- 

 glass be removed, so that oxygen can have access to the tissues, 

 the stain will be restored, as explained last §, The chief elements 

 stained in this way are peripheral nerves, and amongst these 

 more especially axis-cylinders of sensory nerves. 



Ehrlich held that the stain so obtained is a product of a vital 

 reaction of the tissues, and that it cannot be obtained with dead 

 material. Dogiel, however (Arch. mik. Anat., xxxv, 1890, 

 pp. 305 et seq.), found that muscle nerves of limbs of the frog 

 could be stained as much as from three to eight days after the 

 limbs had been removed from the animal. He concludes, indeed, 

 that the reaction shows that the nerves were still living at that 

 time. But it seems more natural to conclude with ApIthy 

 {Zeit. wiss. Mik., ix, 1892, pp. 15 et seq.) that nerve-tissue can be 

 stained after life has ceased. Apathy 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. Ehrlich also {op. cit.) 

 holds that an alkaline reaction of the tissues is a necessary 

 condition to the stain. Apathy further holds that the stain is 

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



