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Studies on penetration of dyes with glass electrode. IV. Penetration of brilliant cresyl blue into Nitella flexilis. V. Why does azure B penetrate more readily than methylene blue or crystal violet?

Studies on penetration of dyes with glass electrode. IV. Penetration of brilliant cresyl blue into Nitella flexilis. V. Why does azure B penetrate more readily than methylene blue or crystal violet?

Jour Gen Physiol 14(1): 1-29

IV. It was previously shown that penetration of basic dyes into living cells increased with increase of pH of the solution, indicating that the free base rather than the salt penetrated to the vacuoles. The present work establishes this presumption with regard to cresyl blue. Free base was obtained by shaking a solution of cresyl blue with chloroform, separating from the aqueous phase, and evaporating the chloroform. Sap of Nitella added to this free base in vitro raised the pH about 0.55, whereas the salt of cresyl blue had a negligible effect. Immersing living cells in a dye solution at pH 9.2 for about 15 min., then determining the pH of the sap with a glass electrode, showed an increase in pH of 0.52 in 1 test, and 0.37 in a 2nd test. Similar results were obtained at pH 6.8, proving that the penetration of the dye rather than the alkaline buffer salts was responsible for the change in pH. Spectrophotometric measurements showed that the dye which penetrated had the characteristic absorption curve of cresyl blue.[long dash]V. Results with azure B were the same as with cresyl blue except that pH was raised about 1 and results were not so decisive, due to greater injury to the living cells by the dye. Methylene blue does not give free base at the pH 9.2 used and did not penetrate until the cells were injured. Methylene blue contained azure B as an impurity, which penetrated. Crystal violet, also undisso-ciated, was more toxic and penetrated slowly, probably not until some injury was produced. It is concluded that basic dyes penetrate to the vacuole of Nitella as free base and are there partly converted to the dye salt, the extent of conversion depending primarily upon the pH of the sap. At equilibrium the concentration of free base in the sap is proportional to the free base in the external solution, which explains why the concentration of total dye (free base and salt) in the sap is greater when the pH of the external solution is raised. The results are in accord with the "multiple partition coefficient theory" of penetration previously advanced, but represent a simple type in that only 1 non-aqueous layer need be assumed to exist between the aqueous external solution and the sap.

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