When the indicator is initially added to the solution, the solution turns blue. The color gradually fades and becomes colorless. The color reappears when the flask is vigorously shaken. The color then begins to fade again.
The color change results from the reversible oxidation-reduction reaction of the methylene blue indicator. In alkaline solutions, glucose is oxidized to D-gluconic acid or alpha-D-gluconolactone
HOCH2(CHOH)4CHO + 3 OH - ---> HOCH2(CHOH)4CO2 + 2 H2O + 2 e -
In the course of this reaction, methylene blue is reduced from the blue (oxidized) form to the colorless (reduced) form.
Shaking the flask dissolves O2 in the solution, which oxidizes the indicator back to the blue (oxidized) form.
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