Glass electrode measurements of the pH value of the sap of Nitella show that cresyl blue in form of free base penetrates the vacuoles and raises pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, while the dye salt dissolved in the sap does not alter its pH value. It is proved conclusively that the increase in the pH value of the sap is due only to the presence of the dye and not to some other alkaline substance.
Spectrophotometric measurements show that the dye which penetrates the vacuole is chiefly cresyl blue.
When the protoplasm is squeezed into the sap, the pH value of the sap is higher than that of the pure sap. Such a mixture behaves very much like the sap in respect to the dye.