When living cells of Nitella are placed in methylene blue solution the rate of penetration of dye into the vacuole is more rapid at pH 9.2 than at pH 5.5. The penetration at pH 5.5 is too slow for satisfactory spectrophotometric analysis but at pH 9.2 this is possible. The dye in the vacuole gives an absorption curve characteristic of a dye consisting chiefly of azure B and a trace of methylene blue with an absorption maximum at 655mμ, while the external solution gives a curve characteristic of methylene blue with an absorption maximum at 664mμ. The presence of methylene blue in the sap is not due to contamination because the sap is extracted by cutting the end of the cell which is wrapped in dampened absorbent cotton and kept outside the solution. The same result is obtained whether the analysis is made immediately or several hours after extraction of the sap from the vacuole.
This result shows that the dye in the sap of Nitella has more methylene blue than that of Valonia (the primary absorption maximum of the latter being at 650mμ). This difference may be due either to the difference in the conditions of cells brought about by experiments or due to the difference normally existing. As soon as cells are injured more methylene blue penetrates.
The sap of Nitella is incapable of changing methylene blue to azure B even in 20 hours, when a sample of methylene blue is dissolved in the sap.
The rate of penetration of dye from pure azure B solution at pH 9.2 is much more rapid than from methylene blue solution at pH 9.2. The dye which has penetrated is found to be azure B with an absorption maximum of 650 mμ, which is identical with that of the external solution.
