I've found that it is possible to visualize this activity by culturing the bacterium on glucose free mineral mediun to which bromothymol blue was Added, appearance of blue green color change from green color indicated that the isolate had nitrogen fixing activity (Khin Mya Lwin and al., 2012). is any one has done this experiment?

We also did the isolation of Azotobacter spp from soil. We used Jensen's Nitrogen free medium to isolate. But, for screening on nitrogen fixing activity of isolates, we used glucose nitrogen free mineral medium and BTB as color indicator. First, we adjusted medium as green color with BTB and we inoculated strains on this medium. After 3-7 days, the medium color will change to blue color, if your isolates have Nitrogen fixing activity. Here is the composition of GNFMM;

Composition (gram per liter)

Glucose 20g

K2HPO4 1g

CaCl2 0.1 g

NaCl 0.5 g

MgSO4.7H2O 0.25 g

FeSO4.7H2O 0.01 g

Na2MoO4.2H2O 0.01 g

MnSO4.5H2O 0.01g

Agar 20 g

BTB trace

I hope this information is useful for you.

Good luck!

lynn

If you have access to a gas chromatograph and acetylen, I would suggest just a standard nitrogenase activity essay of the strains, growing on a nitrogen-free media. Other possibility is just a nitrogen-free media followed by nitrogen content determination, if you have access to a sensitive one

Please suggest that in  GNFMM Composition (gram per liter)., wthat is the pH adjusted to the medium secondly please suggest the exact amount of BTB added.

I used jensens media and added BTB as indicator. But I got colour change from green to yellow after 5 days but was expecting green to blue. Can anyone give any clue??

When did you add the color indicator, before or after autoclaving? 

Hi Rufus

I added indicator to the media before autoclaving. Could this be the reason??

Dear Anupama Rani,

We also add BTB before autoclaving. I would like to know your medium color after autoclaving. It should be green color and pH should be around 7. 

If your medium color is green after autoclaving and changed to yellow color after 5 days incubation means that your bacteria cannot fix excess ammonia to medium. I mean that your bacteria can fix nitrogen for their survival but cannot fix extra ammonia to change the color of medium. That's my assumption.

But, if possible,  a standard nitrogenase activity essay of the strains by using a gas chromatograph and acetylen will  give you more accurate results as Mr. Mario suggestion.

If not, You can also measure nitrogen fixing activity by ammonia test kit or indophenol blue method. 

Bergersen, F. J. Methods for Evaluating Biological Nitrogen Fixation. John Wiley & Sons, Ltd., London, 1980.

I hope this answer will help you.

lynn

 If your target is Azotobacter spp you should use Jensen N2 free medium or N2 free Ashbey  Mannitol agar medium for isolation. In this case you should observe a brown/black/yellowish colony development with  in your plate after spreading from your sample  within 2-3 days. If you do not get any then wait until 5-7 days for any brown/black/yellowish colony development.

Then watch them after gram staining or simple staining. If you get clump of cell, kind of oval shaped together (cyst), there is a huge probability (combining two factor; brown/black/yellowish colony and cyst) they will be azotobacter species.

We have successfully isolated few azotobacter strain by using this method.

Thanks.

Dear Amin,

I am testing for Azotobacter Chroococum and found that my culture is able to grow on N free Jensen media, but colonies not turned to brown/black/yellowish even after a week. Then I found that the color change properties are same as mentioned by Anupama Rani Mam.

Then I found there is background contamination in it as, one set turned to brown colored colonies and other remained white slimy colonies. In gram staining- the brown colonies are gram -ve rods but they are not changing color on the N free BTB media plates, but the white slimy colonies are Gram -ve cocci and are able to change the BTB color.

So what are the actual characteristics of Azotobacter chroococum- starting from its colony morphology on YEM and Jensen plates to Gram characters along with other properties?

Also share views on Azospirillum also.

I am following this conversation and I am in the same position right now. I am trying to screen for Nitrogen fixing rhizobacteria and using the Glucose nitrogen free medium with Bromothymol Blue. But the colonies are producing yellow colour instead of green colour. Somebody help me explain this.

Dear, Becky Aloo.

I work with nitrogen-fixing bacteria and use a medium with bromotomol blue. This is an indicator that can change the color according to the pH of the medium. My advice to you is to try to grow bacteria in a liquid medium without agar, if the medium changes again in yellow, then you will get a confirmation that this strain produces a organic acids and non fixing atmospheric nitrogen. Because nitrogen-fixing bacteria can form ammonia from atmospheric nitrogen, this led to change pH to alkaline and indicator indicator turns blue.

Tin Mar Lynn Hello, if I have BTB in liquid form, how can I prepare the medium?

Itzeel Rosalina Ramirez Hernández

Hi, sorry for late reply.

If you have BTB in liquid form, you can add it drop by drop to media until the color of the media becomes green color when it is stirring.

hope it will help.

lynn

Following

You can use semi solid mediums, reed about that in Article Isolation and Screening of Bacteria for Their Diazotrophic P...

Acela

By using Malic acid instead of Glucose in BTB nitrogen-free medium, I was able to detect color change ( Green to Blue) after 18 hrs, while glucose turned the medium into yellow.

You can use Winogradsky agar medium (Glucose 10 g. L-1, Agar 20 g. L-1, traces of CaCO3, and concentrated saline solution (KH2PO4 g. L-1, MgSO4 g. L-1, NaCl g. L-1, FeSO4 g. L-1, MnSO4 g. L-1, Na2MoO4 g. L-1))