Hi.

You can use Ashby's nitrogen-free medium to identify the ability of strains to fix N (positive or negative). In the attachment the paper where we used the method.

Good luck.

i would be very careful in interpreting any growth on any N-free medium because bacteria that grow may have AcdS (1-aminocyclopropane-1-carboxylic acid deaminase) activity, which results in growth by breaking down the precursor to ethylene, ACC, into 2-oxobutanoate and ammonia. If you can obtain 15N, that is a good way, but in the past some samples of 15N gas were contaminated with ammonia. Another way is to check to see whether or not the nitrogenase fixation genes are present. See Schwartz et al. 2013 (Agronomy 2013, 3, 1-x manuscripts; doi:10.3390/agronomy30x0000x ) to see how we addressed this issue.

Thanks and I hope everything goes well in your research.

Dear Ayaz Farzand

Dra Johanna Döbereiner developed in 1960´s and interesting approach to isolate putative diazotrophic bacteria from non-legumes. In this approach, you can inoculate the bacteria (or plant tissues) in semi-solid N-free media and evaluate the presence a thin pellicule (or biofilm) in the subsurface of the medium. This is an strong indicator of diazotrophic activity. The pellicule is formed in place in the medium column where the O2 pressure allows both the bacterial growth (aerobically) and N2-fixation (anaerobically).

The amplification of N2ase/N2ase reductase genes (as recommended Dr Ann M. Hirsch), along with the pellicule formation in semi-solid media, are strong indicators of N2-fixation. To assure this activity, you can carry on the acetylene reduction assay in an GC.

Please take a look in the reference below, this is a complete illustrated guide.

Baldani, J.I. et al. Plant Soil (2014) 384: 413. https://doi.org/10.1007/s11104-014-2186-6

Yes, I am sorry that I did not mention Dra. Döbereiner's work. Thanks for doing so. Her work is so important for the nitrogen fixation field.

I think using NFB medium and distinguish based on color changing is an easy way, if bacteria have N fixation ability the color of NFB goes from green to blue.

Please take a look at Handbook for Azospirillum

Good luck

Hi Ayaz

for evaluation of nitrogen fixation or the efficiency of the nitrogen fixation you can use acetylene reduction assay (ARA). I have used it in my thesis frequently, here down I will paste the text

''

Nitrogenase activity was measured by using acetylene reduction assay (ARA) (Hardy et al., 1973, Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biology and Biochemistry 5, 47–81 ). Individual roots with attached nodules were immediately placed in 35 ml bottle and sealed. 3.5ml volume of air was withdrawn with a syringe and replaced by 3.5 ml of acetylene. After 1h incubation in room temperature, 1ml of gas were taken from the bottle and injected into the gas chromatograph system. Ethylene production was determined by gas chromatograph (Chrompack Packard 438A equipped with Porapak N column (110 cm x 1.6 mm ID ) using a N2 as a carrier gas at a flow rate of 30 ml/min at 60 °C.

Good luck

Yes, this works very well but the most reliable assay especially when dealing with difficult organisms or field sites is 15N.