We have a dire need to use Complete S medium (over supplemented M9) but have always had problems with precipitation.
Anyone that can let us know how to avoid ppt when making Complete S medium, write me at chammell@cshl.edu.
Here is the recipe we are using: S basal (before use, add what is described in the recepie below) 1 Liter
5.9 g NaCl
50 ml of 1M potassium phosphate pH 6.0
1 ml of 5 mg/ml cholesterol (in EtOH) Complete S Basal
To each 500 ml bottle add:
5 ml 1M Potassium citrate, pH 6.0
5 ml Trace Metals solution
1.5 ml 1M MgCl2
1.5 ml 1M CaCl2 Trace Metals Solution
Disodium EDTA 1.86 g (5 mM)
FeSO4 7H2O 0.69 g (2.5 mM)
MnCl2 4H2O 0.20 g (1 mM)
ZnSO4 7H2O 0.29 g (1 mM)
CuSO4 5H2O 0.025g (0.1 mM)
Dissolve in 1L water; aliquot into 50 ml conicals and store in dark.
Yes we autoclave everything and we see precipitation even in the trace metals solution. I’m going back to make sure that the ZnSO4 and CuSO4 contain the correct dH20 content but it is really annoying.
some possible causes spring to mind, but then you say that all of the component Solutions also Show signs of precipitation (or is it just S-complete and trace metals)?
Local precipitation will be seen when shooting cholesterol into Phosphate-containing Solutions as the EtOH precipitates the phosphate out. You see this with NGM agar, but then it’s difficult to assess whether that seeds further precipitation.
Alkaline pH could also be a factor…worth checking.
The thing that makes me consider something else is the fact that more than one solution is hit. We have had a similar Problem with the S-basal accumulating a precipitate over a couple of days after being prepared…the crystals formed looked suspiciously like KH2PO4. We are trying to exclude that our water prep system is not compromised, but it’s clear that it starts off relatively small scale and increases with time.
Adding the Mg/Ca too hot gives you a nice precipitate in the S-complete. But then, that doesn’t explain your Trace soln. unless the pH is wrong.
Has there been a change in personnel responsible for the buffer preps?
You wrote:
3. The thing that makes me consider something else is the fact that more than one solution is hit. We have had a similar Problem with the S-basal accumulating a precipitate over a couple of days after being prepared…the crystals formed looked suspiciously like KH2PO4. We are trying to exclude that our water prep system is not compromised, but it’s clear that it starts off relatively small scale and increases with time.
I think that this is exactly what is happening and we have gone through the idea that it is contamination or operator error and think it must be something like pH that we have not accounted for though everything seems correct.
After looking again at the crystals I found after several days littering the bottom of my S buffer, I think they may be calcium phosphate rather than dihydrogen potassium phosphate (which makes more sense).
So, two observations.
in S basal / trace metals, the source of calcium (e.g. calcium chloride) is not there (and in the trace solution you have a chelator anyway)…I think (in our S basal solutions at least) that residual calcium on the bottles or flasks might be the culprit as they were not acid washed. Another source of Ca2+ is of course when the deioniser is not pulling it’s weight. For the trace solution, there must be another reagent precipitating out, but it’s not clear what would…
In the 'complete s basal the concentration of CaCl2 is way over what’s needed to precipitate out CaPO4. Additionally, the hotter the solution the more you get. Therefore, adding CaCl2 to cold S basal should produce less precipitate…something worth trying?
We’ve observed the same phenomenon on occasion, and identified residual detergent from poorly rinsed glassware as the culprit. Thorough rinsing with ddH2O solved the problem.