Two successive extractions of the sap were made from the vacuole of each living cell of Nitella flexilis. The first extraction, called for convenience “sap”, was made without pressing on the cell wall, while the second extraction, called “sap mixture”, was made by squeezing the cell wall vigorously. The pH value of the sap was found to be 0.6 pH lower than that of the sap mixture. This difference may be entirely due to the presence of the protoplasm in the latter, or partly due to the presence of fluid from the space between the cell wall and the protoplasm.
The pH values of the sap and the sap mixture remained unchanged during the period of extraction and of the subsequent measurement by the glass electrode.
The sap. (1) 0.07% cresyl blue, azure B, and methylene blue salt do not alter the pH value when dissolved in the sap in vitro, but 0.07% free base of cresyl blue and azure B cause it to increase about 0.5 pH with the former and 1 pH with the latter.
(2) a. When 0.07% dye has penetrated from the solution at pH 9.2 there is an increase in the pH value of the sap of 0.5 pH with cresyl blue, and 1 pH with the azure B or with the dye penetrating from methylene blue solution.
b. Spectrophotometric measurements show that cresyl blue and azure B penetrate as such while it is chiefly azure B that penetrates from methylene blue solution which always contains azure B as impurity.
(3) a. 0.07% cresyl blue penetrating in 15 minutes from the dye solution made up in buffer at pH 9.2 or at pH 6.8, or in tap water, raises the pH value of the sap equally (0.5 pH).
