Hello,

I have been trying to perform the acetylene reduction assay using pure bacterial cultures. Despite numerous attempts, including trials with positive control bacteria, I have not been able to detect any ethylene gas formation.

Here is the procedure I have been following:

• Pre-cultured the bacteria for 2 days in medium containing a nitrogen source
• Harvested the cells (8000 rpm, 5 min)
• Washed the cell pellet twice with 500 µL Burk’s N-free medium
• Resuspended in 1000 µL Burk’s N-free medium
• Inoculated 200 µL of the cell suspension into Burk’s N-free semi-solid medium (20 ml of medium in 50 ml serum bottle)
• Incubated the semi-solid medium for at least 2 days
• Sealed serum bottles with rubber stoppers and aluminum crimp caps
• Flushed the headspace with N₂ gas for 20 minutes at 1 cmHg to reduce oxygen
• Replaced 10% of the headspace (3 ml) with acetylene
• Collected 100 µL of headspace gas for 0 h GC measurement
• Incubated at 30°C
• Collected 50 µL of headspace gas every 12 h for GC analysis

I would greatly appreciate any insights or suggestions you might have regarding this issue.

Thank you very much for your time and support.