39© Transactions of the Canadian Institute [vol. ix 



occur when more than one of those methods, specially adapted, are 

 skilfully applied to investigate the distribution of salts in the cells of 

 the renal tubules in the various stages of acti\ity. Of the methods nou 

 available, that for the microchemical demonstration of potassium, first 

 employed by Professor A. B. Macallum,^ has been already used by him 

 in studying the exci^etion of the salts of that element in the renal cells 

 of frogs kept in the laboratory tanks througn the winter. The results 

 thus obtained were such as to indicate that the method might profitably 

 be applied in a more extended investigation along the same line, and the 

 author, acting on the suggestion of Professor Macallum, undertook the 

 research of which the present contribution is the outcome. 



II. Methods of Investigation. 



The method used is that fully described by Professor Macallum for 

 the microchemical demonstration of potassium in animal and vegetable 

 cells. It will, therefore, be necessary only to review here very briefly 

 the reaction and preparation of the reagent. 



The reagent is prepared by dissolving 20 grams of cobalt nitrite and 

 35 grams of sodium nitrite in 75 c.c. of dilute acetic acid (i.e., 10 c.c. 

 glacial acetic diluted to 75 c.r.). A vigorous evolution of nitrogen 

 peroxide results. It is allowed to stand for some hours, when, if any 

 trace of potassium has been present in the sodium nitrite used, a pre- 

 cipitate forms which may be removed by filtration. The filtrate is 

 diluted with water to 100 c.c, and is then ready for use. The precipi- 

 tate produced by the reagent, when added to a solution of potassium 

 salt, is a triple compound, the hexanitrite of cobalt, potassium and 

 sodium. Its composition is given by Gilbert^ as Co(N02)3 3(K/Na) 

 NO2, nHaO, the value of n being i 1/2, 2, or 2 1/2. Its composition 

 varies, however, with the potassium salt content of the original solution. 

 It consists of chrome yellow crystals of dodecahedra of varying micro- 

 scopic size. 



The tissue examined was removed from the body as soon as possible 

 after the animal was killed, so as to insure a perfectly fresh, normal 

 condition. It was at once placed on the plate of a C02-freezing micro- 

 tome, where it was immediately frozen and sections made from it, and 

 placed immediately in the reagent. Here lay perhaps the crucial point 

 of the technique. First, the tissue must be frozen quickly and while 

 perfectly fresh. In the second place, it is absolutely essential that the 

 knife and the atmosphere in which the sectioning is done should be 

 thoroughly chilled to a distinctly freezing temperature in order that the 

 section should not thaw before the entrance into its reagent. If this 



