1. In the plant cell vacuoles, preliminarily stained in vivo with thiazine dyes (but excepting methylene blue), there appear numerous crystals of the dyestuffs, when they are treated with the solutions of thiocyanide, iodide, bromide, chloride or nitrate of alkali metals (Na, K, or Ca). These demixed crystals show various types of spiral shape. 2. This demixing reaction is accelerated with the increase of the concentration of the dye-solution used, but also related to the sort of the anion of the salt employed, giving the following anion-series, concerning the acceleration of the reaction: SCN->I->Br->NO3->Cl- 3. The same demixing reaction is proved to take place in the mere water solutions of the dyestuffs, when they are treated with solutions of the same salts through a semipermeable membrane of collodion or pig bladder. The anion-series obtained from these model experiments perfectly coincide with the one obtained from the vital stainings of cell vacuoles. 4. It may be concluded, therefore, that the demixing of the spiral formed thiazine dyes in plant vacuoles has been aroused by a simple reaction of salting-out of dyecrystals in solutions. Although their shapes were various and often curious, no parts would be played by the constituents of cell saps or cytoplasms.
A critical study of the occurence of silver nitrate reduction in plant cells was carried out with the following results. 1) Minimum amount of ascorbic acid (reduced form) clearly detectable with acidified silver nitrate solution after GIROUD is 300mg% at 25°C and 150mg% at 40°C (Table 1). At 25°C, 150mg% ascorbic acid is clearly detectable with a si1ver nitrate solution at pH6. 3 (Table 2). 2) As a rule, the more contains the tissue ascorbic acid (reduced form), the more markedly occurs the reduction of silver nitrate. But this relation does not hold good in some cases (Table 4). 3) In most cases, chloroplasts remain colourless when a tissue is treated with acidified silver nitrate solution after GIROUD in darkness. 4) When the tissue treated with the acidified silver nitrate solution is immersed in ordinary alcohol, the alcohol causes a secondary blackening of chloroplasts.