18 Mr. J. S. Macdonald. [July 31, 



Staining Due to Salt Concentration. 



We may now turn to another striking fact which has made its appearance 

 in the course of these investigations. Basic dyes, toluidine blue, and neutral 

 red have been made use of to map out the distribution of a material, 

 which, upon further examination, turns out to be potassium chloride. Set 

 forth in these direct terms, there is nothing surprising in this statement, 

 since both dyes can be " salted out " from their solutions by the addition of 

 potassium chloride. Neutral red is a most insoluble body when tested in 

 this manner, and it was for this reason that I selected its staining effects as 

 a guide to the distribution of the potassium chloride solutions in nerve-fibres. 

 Toluidine blue is more soluble, nevertheless it is easily capable of being 

 " salted out " by solutions of the concentration that my electrical experiments 

 have led me to anticipate. I have also since found that it is more easily 

 " salted out " by potassium chloride in the presence of small traces of alkali, 

 0"03 sodium carbonate. This latter point has an obvious bearing upon the 

 salting out of toluidine blue in alkaline cell-structures, and more especially 

 at kathodal points. 



This definite coincidence between toluidine blue staining and results 

 obtained by the use of reagents precipitating potassium and " chloride "" 

 renders it possible at once to advance a general proposition of some interest : 

 All the appearances made evident in the nerve-fibre by the use of toluidine blue 

 are due to the manner in which this dye is salted out from its solutions by 

 potassium chloride. 



There is also a further point. The appearances which Bethe developed in 

 nerves fixed in alcohol, or in alcohol and ether, 1 have obtained in nerves 

 freshly teased in " normal saline." Bethe's results coincide with mine, and 

 must be equally accepted as providing a picture of solutions of different con- 

 centrations of potassium chloride. Nor is this to be wondered at. Potassium 

 chloride is only slightly soluble in alcohol, although extremely soluble in 

 water. The addition of alcohol, the rapid dehydration which ensues, and the 

 replacement of the water by alcohol, must be definitely expected to leave the 

 salts present as precipitates. Wherever it exists the potassium chloride is 

 precipitated practically in the same quantity as it was originally present in 

 solution. Alcohol fixation, therefore, is precisely calculated to leave in situ 

 precipitates of salt for subsequent micro-chemical investigation with toluidine 

 blue and similar dyes. 



It is impossible to leave this point without remarking upon the general 

 bearing of this conclusion. The nerve-fibre cannot be the only case in which 

 considerations of this kind are worthy of examination. Granules in the 



