Greetings all,

Im trying to estimate the phosphate solubilization by soil bateria using NBRIP broth as suggested by C. Shekhar Nautiyal.

Broth composition:

Glucose, 10 g: Ca3(PO4)2, 5 g; MgCl2W6H2O, 5 g; MgSO4W7H2O, 0.25 g; KCl, 0.2 g and (NH4)2SO4, 0.1 g, agar 15g, pH 7.

After inoculation of the broth (10ml) with the bacteria (100 μl ), it was placed in incubator shaker for 3 days/30°C at 160 rpm.

The inoculated broth was centrifugated at 6000 rpm/14min, 750μl of supernatant was mixed with 750μl of ammonium molybdate reagent (ammonium molybdate ((NH4)6Mo7O24·4H2O) 1.5% (w/v), sulfuric acid (H2SO4) 5.5% (v/v) and Iron sulfate(FeSO4) 2.7% (w/v) ).

After 30 min in the dark, the OD of the mixture was mesured at 700 nm.

All bacterial samples gave the same dark blue color, and a maximum optical density equal to 3. what could possibly be the problem? does ammonium molybdate interacte with the insoluble phosphate Ca3(PO4)2 to give the blue color?