Dear Ibrahim:

You may try to check P solubilization on NBRIP liquid medium and detect the amount P release. I remember once I was working with Bacillus megaterium checking its P solubilization capacity and in solid agar plate NBRIP medium I did not detect any clear halo around the colony, but when I did it in liquid medium the P released was really high.

Dear Tahir,

Dr. Vidal is right. Sometimes it is difficult to see the halo zone around growth but you can estimate the P solubilization in broth using several methods (available on internet). 

Had you purchased the Bacillus culture or isolated it from somewhere? 

If you isolated it then may it not solubilizing the P.

NBRIP-BPB medium is the indicator medium and it gives you good results for P solubilizing bacterial isolates.

Dear Vidal and Kunal

Thanks a lot for your answers that really are helpful for me. One more thing I am confronting is when I adjust pH of NBRIP-BPB medium to 7 with NaOH, precipitates appear. What should I use to avoid precipitation.

Thanks once again

check the concentration of NaOH.... I think you can use 0.1N NaOH..... or some buffer

One experiment you can perform without altering the pH..... 

Thanks a lot Kunal. I am using 10N NaOH. I will try as per your suggestions.

Thanks

Try using ammonia solution to adjust the pH. 

it helps when you boil the pikovskaya media before autoclaving it but the best way is to do it in broth. You can use the Fiske and Subbarow method to quantify the P release in the supernatant of your culture

Do the bacterium used as positive control give a  negative response on NBRIP and Pikovskaya media?

You are using the very classical method and that too very rightly for primary screening. Keep doing so. You have not mentioned the water used ( I presume it should be 100ml, in that case you could have given the composition in %). I think you should increase the agar concentration to 2.5% (in case you are using Difco or Oxoid agar). This will help you to see the zones of clearance after atleast 96 hours of incubation.  

Yes, try in liquid medium (Quantitative Estimation).

Many bacillus species are well known as a phosphorus solubilizers. In eroded soils like in desert, remarkable activity on rizhoplane of rock weathering included minerals like phosphorus is by bacillus sp.

Check this papers. http://www.bashanfoundation.org/gmaweb/pdfs/rhizoplane2.pdf

http://link.springer.com/article/10.1007%2Fs003740050024

it may be due to calcium phosphate.

Used Tri-calcium phospahte of Sigma company.

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Thanks all.

I used tri-calcium phosphate from Sigma in NBRIP-BPB agar medium and it worked fine.

 Dear Muhammad,

I think your bacterium is not PSB or you can try the liquid medium!

You can use a reference strain solubilizing P in the same condition that your bacterium and see the results!!

Where did you purchase the NBRIP-BPB media? I am conducting similar research but am not satisfied with Pikovskaya's medium. I searched the NBRI website as well as major manufacturers and cannot find a supplier. Do you have any suggestions?

Hello, how you overcome tri-calcium phosphate precipitation in NBRIP-BPB?

Dear Raheleh

Tri calcium phosphate is always an water insoluble compound under normal pH conditions. That is why you should sterilize it separately as dry powder in the same autoclave when you sterilize the agar or liquid medium. Then just before pouring plates (in case of agar medium) or inoculation of the liquid medium add the powder tri-calcium phosphate in the well cooled medium, shake well to disperse as evenly as possible and proceed.

Dear Dr. Jai Ghosh

Thank you I really appreciate your help.

i use 0.5% tri-calcium phosphate to PVK medium

but Bacillus indicated low halo zone

Enterobacter clocae only excellent

The thickness of the medium in Petri Dishes should be very thin.

Please suggest alternative for tricalcium phosphate for P solubilization Actinobacteria.

Nizara Ahmeda you can use Iron-Phosphate or Aluminium-Phosphate instead of tricalcium phosphate. You can get some idea from the article- Article New isolation method for microorganisms solubilizing iron an...

Thank you @chandra Prakash Paul sir

Sorry sir @Chandra Shekhar Paul