A B C

Figure A-C: Test tubes having a brim-filled of 0.001% Methylene blue; Fig. A with and without Mung
Bean seedlings before incubation; Fig. B- after incubation within 24 hours and Fig. C-after aeration

Test tube with 0.001%
Methylene blue
Color
Original After 24 hours After Aeration
With Germinated Mung bean
seedlings


Blue

Colorless

Light Blue
Without germinated Mung bean
seedling


Blue

Blue

Blue


The test for dehydrogenases has a set-up of 2 test tubes brim-filled with 0.001% methylene blue. The
first test tube contains 10 grams of germinated Vigna radiata seedling while the second one doesnt
contain Vigna radiata and serves as the control. Both test tubes were covered by a stopper and
incubated overnight. After 24 hours there was a change of color in test tube 1. From blue it becomes
colorless and the test. tube 2 remains unchanged (see figure B). After aeration, the solution in test tube
1 becomes blue in color again (see figure C).


During respiration, hydrogen atoms are removed from glucose molecules by enzymes called
dehydrogenases and passed to various chemicals called hydrogen acceptors. As the hydrogen atoms
pass from one hydrogen acceptor to another, energy is made available for chemical reactions in the cell.
In this way, substances such as glucose provide energy for vital reactions in living organisms. In this
experiment, a dye called methylene blue acts as an artificial hydrogen acceptor. When this dye is
reduced by accepting hydrogen atoms it goes colourless.

O2 + methylene blue ---> methylene blue
(dissolved) (colorless) (color blue)
(reduced form) (oxidized form)



Although a great deal of information has been amassed concerning dehydrogenases in animal tissues,
there was for a long time little evidence that certain of these important enzymes even existed in plants.
Malic and citric dehydrogenases were reported in 1929 in cucumber seeds (Thunberg 1929), but it was
not until 1939 that succinic dehydrogenase was found, first in pollen. (Okunuki 1939) and then in certain
other tissues (Damoran1941). Nevertheless, the apparent absence or near-absence of succinic
dehydrogenase in some tissues (Bartlett 1943) as well as the occasional reports of the presence of
individual enzymes (Thunberg 1938) seemed to indicate that the dehydrogenases, at least those of the
4carbon and 6 carbon acids, were distributed only sporadically. It was during this period that the
tricarboxylic acid cycle of Krebs (Krebs 1943), embodying many of these dehydrogenases, was becoming
accepted as the main pathway of respiration in animal tissues. Respiration studies in plants (Bonner
1948) pointed in the same direction.

Reference:
THE ESTIMATION OF DEHYDROGENASES IN PLANT TISSUE1 CARL A. PRICE AND
KENNETH V. THIMANN BIOLOGICAL LABORATORIES, HARVARD UNIVERSITY,
CAMBRIDGE, MASS, March , 1954
DAMODARAN, M. and VENKATESAN, T. R. Amide synthesis in plants. I. The succinic oxidase
system in plants. Proc. Ind. Acad. Sci. (B) 13: 345- 359. 1941.