Hello.

I am attempting to perform the acetylene reduction assay (ARA) to detect nitrogenase activity in bacteria known to fix nitrogen. However, the assay is not producing any detectable ethylene, even when using a positive control strain (Ciceribacter azotifigens).

Several articles and protocols mention flushing the headspace with nitrogen (N₂), argon (Ar), or helium (He) to remove oxygen, but they generally do not specify how long or at what flow rate the flushing should be done. I tried flushing with nitrogen gas 0.5 cmHg for 5 minutes, but no ethylene peak was observed.

I would like to ask the following:

1. To what extent should oxygen be removed for nitrogenase activity to occur?

2. Is there a way to measure or monitor oxygen removal, such as using resazurin dye?

3. Would using a vinyl anaerobic chamber to prepare and seal the bottles be more effective than flushing?

Here is what I have in mind for using the anaerobic chamber:

a. Transfer the bacterial culture (in N-free medium) to serum bottle

b. Let the bottle sit open in the chamber for at least 30 minutes

c. Seal the bottle with rubber stopper and aluminium crimp, all inside the anaerobic chamber.

Any insight would be helpful. Thank you very much.