hi
I want to measure N fixation as a H2 evolution with a specific device and I need to know if my strain has an active hydrogenase enzyme.
Methylene blue reduction assay
The method described by Lambert et al. (1985) and
Zhang (2006) was used for the methylene blue reduction
assay. All nodules were thoroughly washed in water to
remove soil particles and excess plant matter. The number
and distribution of root nodules varied from plant to
plant, but were generally no less than ten per legume.
Washed nodules were then placed in small labeled seed
germination plates with filter paper that had been soaked
in a methylene blue reduction solution of 200 mmol/L
iodoacetic acid, 200 mmol/L malonic acid, 2.5 mmol/L
MgCl2, 50 mmol/L K2HPO4, 10 mmol/L Methylene Blue
dye, and adjusted to pH 5.6 with KOH. The nodules were
distributed evenly on the plates, crushed and left to incubate in air for 15 min. The plates were then placed under
a 100% H2 atmosphere in a sealed gas chamber overnight
at room temperature. Plates were removed and photographed using a Canon PowerShot S21S digital camera.
The presence of colorless zones around each nodule represented uptake hydrogenase activity (Hup+) while the
absence of colorless zones represented Hup microsymbionts.
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