Dear all,
I encounter problems of precipitation when I am trying to reproduce a known culture media with buffers like HEPES ; PIPES or MOPS
I am preparing a minimal synthetic medium for the growth of halophilic bacteria. To do so, I prepared separately the following solutions :
- NaCl (156g/L)
- MgCl2.6H2O (13g/L)
- MgSO4.7H2O (20g/L)
- CaCl2.6H2O (1g/L)
- KCl (4g/L)
- NaHCO3 (0,2g/L)
- NaBr (0,5g/L)
- Adjust to pH 7,3
2. Trace element solution SL6 composed of :
- ZnSO4.7H2O (0,1g/L)
- MnCl2.4H2O (0,03g/L)
- H3BO3 (0,3g/L)
- CoCl2.6H2O (0,2g/L)
- CuCl2.2H2O (0,01g/L)
- NiCl2.6H2O (0,02g/L)
- Na2MoO4.2H2O (0,03g/L)
- Adjust to pH 7,3
3. NH4Cl concentrated solution
- Adjust to pH 6,8
4. KH2PO4 concentrated solution
- Adjust to pH 6,8
5. Glucose concentrated solution
- Adjust to pH 6,8
6. FeCl3 concentrated solution
7. Buffer (HEPES or PIPES or MOPS) concentrated solution
- Adjust to pH 7,3
To prepare my flask for inoculating, I mix all these solutions in the following order : saline media > Glucose > NH4Cl > KH2PO4 > SL6 > FeCl3 > Buffer.
5min after stirring at 37°C, the mixture started to form a precipitate, eventhough I didn't inoculate yet. Control experiments with the same media without buffer did not show any precipitation.
Did you face the same problems ? Articles using the same media did not mention any precipitations problems. I am wondering if there could be any importance in the order of addition ?
Thanks for your help,
Dr. Antoine Brège
Has this medium been reported before? I think part of he problem may be that the high NaCl concentration is salting out one or more of the components. Also, several of the metals may be precipitating as the phosphate salts.
I agree that the concentrations look very high in salt concentrations. Are you sure that there is not a final dilution after the additions of the trace elements etc to bring the final concentrations closer to isotonicity which would reduce the salt concentrations and the precipitation. You do not give the volumes of each added to make the final medium
Dear Paul and Adam,
Thanks for your answers. This medium is indeed reported in a few publications, such as doi : 10.1007/s00792-017-0964-9. It's a minimal synthetic medium that has been adapted from ATCC 1176 medium with substitution of yeast extracts
This medium is suitable for the growth of halobacterium, thus requires a high concentration of salts, especially NaCl. These bacteria are known to accumulate salts in their membrane so they can resist osmotic pressure of highly saline media.
It wasn't clear that the trace element solution is rediluted at 1%v in the final culture medium. The final concentrations of the different salts are then :
- NaCl (156g/L)
- MgCl2.6H2O (13g/L)
- MgSO4.7H2O (20g/L)
- CaCl2.6H2O (1g/L)
- KCl (4g/L)
- NaHCO3 (0,2g/L)
- NaBr (0,5g/L)
- ZnSO4.7H2O (1mg/L)
- MnCl2.4H2O (0,3mg/L)
- H3BO3 (3mg/L)
- CoCl2.6H2O (2mg/L)
- CuCl2.2H2O (0,1mg/L)
- NiCl2.6H2O (0,2mg/L)
- Na2MoO4.2H2O (0,3mg/L)
- NH4Cl (2g/L)
- KH2PO4 (0,5g/L)
- Glucose (10g/L)
- FeCl3 (5mg/L)
- Buffer (50mmol/L)
To achieve such concentrations, I prepared concentrated solutions of SL6, NH4Cl, KH2PO4, Glucose, FeCl3 and buffer and just dilute them to the saline medium. For instance, when I prepare 1L of medium, I weight the salts of saline medium (i.e NaCl ; MgCl2 ; MgSO4 ; CaCl2 ; NaHCO3 and NaBr) in a working volume of 800mL. Then I add the calculated volume of concentrated solutions to my working volume to achieve the desired final concentration (e.g 2g/L NH4Cl), and I complete with sterile H2O to obtain 1L of culture medium.
I must add that all of my separated concentrated solution did not show any precipitations
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