Abstract
Since some confusion has arisen lately concerning the identity of the dye which penetrates into the vacuole of living plants of Nitella and Valonia, the writer has undertaken to analyze the causes of this apparent discrepancy.
Irwin 1 , 2 has attempted to show by spectrophotometric determinations that the dye from solutions of methylene blue which penetrates the sap of these plants, is not methylene blue as originally stated by the writer 3 , 4 but is rather some lower oxidation product, trimethyl thionine. This forms readily in alkaline solutions of methylene blue and penetrates as a free base, whereas methylene blue is completely dissociated and according to Irwin does not penetrate living cells except during injury.
The writer, on the other hand, also using the spectrophotometer to determine the identity of the dye found in the sap of these plants, found only methylene blue present.
An analysis of the differences in methods used by Irwin and the writer has been made. The following main points of divergence have been tested.
1. pH value. The writer used pH 8.83 while Irwin used 9.5 and 10.9. The use of the higher pH values in the external solution introduces 2 errors: first, precipitation of Mg (at pH 10.9) from sea water which produces a solution unbalanced with respect to cations. Such unbalanced solutions affect the permeability of all plant cells for which we have any data. The second effect of high pH value is the relatively rapid conversion of methylene blue into trimethyl thionine and related dyestuffs.
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