Dear Dr. Pedro B. Costa,

Did you try to change the order of the reagents to dissolve them?

In my laboratory work to prepare a specific media, when we change the order of the reagents it precipitated.

Did you try change the order of reagents before autoclaving the medium?

I believe there is not the water or pH, but the order of mixing the reactants.

Best regards.

As Hani said, your P-sources are not soluble.

This kind of solubilisation tests are usually performed on agar plates and evaluated based on the changes on the transparency of the medium over time

Dear researchers, Thank you for your colaborations. It is good to know that we were not actually making something obviously wrong with our solutions. Still, let me elaborate our situation here a little bit further.

We used tricalcum phospahte solubilization assays in agar plates for years, but based on Bashan et al. 2013* we decided to try other phopahte sourcers and liquid media for spectrophometric quantification. The P solubilization assays we found on literature, however, use large volues of media for each strain tested, and we wanted to make a microtube-sized aproach to test hundreds of strains simultaneously. The difficulty in measuring water-soluble P, and substract it from total added P, is that the autoclaved media gets fractioned in the microtubes. Since the P salts in autoclaved media are precipitated, fractioning in many tubes is not homogeneous even when stirred constantly, and P added to each tube is not as constant as required for a propper assay. weighting and preparing media for several hundreds microtubes indepedently is too larborous and prone to error. Thus we wanted to dissolve these P sources to make sure all tubes get the same amount of P

Biomass quantification is another problem. NaOH, used to cell lysis, reacts with FeO4P.2H2O, oxidating and even changes the color of the media. Rust is not something we want to have in our assays! We made cell lysis by heat shock, solving that issue. Still, centrifugation to collect cell pellets often comes with the P precipitates, making spectrophometric quantification of bacterial biomass quite misleading.

Considering i cannot dissolve these P salts at all, how could I fractionate a media with a precipitated P source into several small tubes, making sure all tubes get the same amount of P? (stirring the media while fractioning it did not function well, specially by the last ml of the media)

furthermore, how could we separate the precipirated P salts form the cells to measure abcteria biomass? (if we just let the P salts fully precipitate to the bottom of the tubes, so might the bacterial cells!)

Thanking you,

Pedro Costa

*Yoav Bashan et al.,, "A proposal for isolating and testing phosphate-solubilizing bacteria that enhance plant growth" Biol Fertil Soils (2013) 49:1–2

Hi pedro, how much volume did you use in the microtube? I did the same essay in 40 bacterial strains using 50ml of media. The only way I could secure all flask had the same P amount was weigthed the P individually for each one.  Also, I doubt you could have a mass quantification by spectophotmetric method, you could instead use plate counting. This paper helped me a lot, Chen et al 2006 Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. 

Having other solids on suspension, biomass quantification is a no-go...

You shouldn't prepare the tubes independently, but aliquot from the same stock. Otherwise you are introducing a considerable source of error, specially considering the small amounts.

In general, you mustn't autoclave phosphates with divalent cations, e.g. Ca and Mg, even if the phosphates are soluble, as they are going to form insoluble phosphates. Saying that, you should autoclave the solid phosphates as a suspension of fine powder separately from the rest of the medium and mix them together after cooling. This medium will be a suspension and you should aliquot it while stirring.

By the way, are your bacteria P-solubilisers or you are still screening? Because the medium you describe is not really selective. Almost all bacteria can grow in culture media without added P, likely a bit slower but still... many bacteria are actually inhibited by the high-P of most usual culture media. It is one of the reasons of the 'unculturability' of many bugs.

Have you considered to use qPCR for the quantification? Or direct counting the bacteria under a microscope with e.g. DAPI?