I am currently working on isolating and screening rhizobacteria. One of the properties am concerned about is nitrogen fixation ability and so I am growing the bacteria in nitrogen free media. Literature suggests that the colour of the medium should change from green to blue. However, am getting colour change from green to yellow and this is quite confusing.
Due to the fact that nitrogen fixation by diazotroph bacteria is a H+ consumer process and also ammonia is subsequently produced, the color of the NFB medium should be changed from green to blue (due to the presence of Bromothymol blue pH indicator). The production of yellow color by bacteria in this medium indicates a decrease in pH that can confuse you. As regards that the bacteria could grow in this medium, it can be said that it also has the ability to nitrogen fixation.
The color change is a qualitative hypothetical test for the N2 fixer in N free media (either Jensen's media/Nfb media) with BTB indicator. But, this is not the confirmatory test for N2 fixation estimation. (even the color may be changed for pH unstability or suspicious culture contamination!!)
You need a GC (gas chromatography) analysis for its quantitative assessment. If you have no GC access, simply you may estimate amount of Nitrogen by Micro-Kjeldahl analysis.
The thumb role is the colony of N2 fixer (diazotrophs) are more largers with watery ring in the jensen/nfb medium.
The production of acid was indicated by a change in color from green to yellow.
Colour change of media does not indicate much about the nitrogen fixing ability of the bacteria.The nitrogen fixation can be confirmed by gas chromatography or by microKjeldhal method for in vitro grown bacteria
Thank you all for your wonderful insights and advice. Can I also perform nesslerization of broth cultures to quantify ammonia produced in nitrogen free medium?
@Surendra Gopal sorry I meant use of the Nessler's reagent to quantify ammonia with the aid of standard curve prepared from known ammonia concentrations
@ Becky Aloo, I think you can do it. Please look at the reference of Jackson (1971).
Thank you everyone. Both my student had got colonies growing on Jensen agar, but one got green colonies while the other obtained yellow colonies after staining with Bromothymol blue.
I wonder how one can use Kjeldahl method for estimation of nitrogen fixation in bacterial cells. Researchers generally used Kjeldahl methods for find the T-N form soil and plant digest samples.
@ Seema Dharni
The total nitrogen fixed in the bacterial cell and released into the media can be estimated by Kjeldhal method
the color change is due to the color indicator in your media. do you know which indicator you used? you can look up the pH scale for it to interpret your results. I find luck with using Nfb Semi-solid media for N fixation screening, color change to blue is a positive result. (PDF) The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semi-solid media: a practical guide for microbiologists (researchgate.net)
I can assume that bromophenol blue is added to the medium, as a result of which the color of the indicator changes from blue (alkaline reaction) or green (neutral reaction) to yellow (acid reaction). Many nitrogen-fixing bacteria produce acids as a result of the utilization of sugars. As a result, the color changes to yellow.
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