M9 medium prepared from M9 salts mixture from sigma. Medium is usually filter sterilized, followed by addition of glucose, CaCl2 and MgSO4. Forms colourless crystalline ppt after 2-3 of storage at room temperature or at 4 degrees. Can this medium be stored over long periods of time or does the medium have to be freshly prepared?
Hello Maria, Yes Surely you can store M9 medium for long periods (2-3 months). I think to avoid the problem of precipitation you should take care of few things like always use new chemicals and while preparing the medium add the ingredients one by one. Also after complete dissolution of one chemical add the next one. The storage container should be sterilised as well.
M9 medium can be stored at 4 degrees, but make sure the salts are completely dissolved! Especially when you are adding CaCl2 and MgSO4. You can also fiter sterilize all the solutions and store seperately, and add just before you want to use the medium.
Thank you for your replies.
We use a M9 salts mix so our recipe is much less complex. At first the complete medium was made (w/ antibiotics) and stored at 4degrees thats when I first saw the ppt after a few days of storage. Next I tried only adding Glu, CaCl2 and MgSO4 to the autoclaved M9, which was stored RT (around 22-25 degrees). All salts remain dissolved for the first few days, and then there was the same crystalline ppt (maybe MgCl2? - not sure). Im not sure what the problem is but for now I am only adding glucose to the autoclaved M9 medium (so far no ppt). This is stored at RT and I add the CaCl2, MgSO4 and antibiotics to aliquots as I need them.
Hi Maria,
Though I didn't use M9 before, but I think the precipitate is due to the formation of hydrated calcium sulphate (CaSO4, 2H2O) crystals.
You could do the following to avoid this:
1) use deionized (demineralized) water for preparing medium
2) at first dissolve CaCl2 (completely) and then dissolve other ingredients one by one and then filter sterilize or autoclave (whatever works better)
Hope this could help you to avoid crystal formation.
Good luck!
Hi Maria,
I would assume that Calcium Phosphate precipitates from your medium since it is only poorly soluble (0.1 g/L at 25°C: http://gestis-en.itrust.de/nxt/gateway.dll?f=templates$fn=default.htm$vid=gestiseng:sdbeng). I use M9 as well and I always add the filter sterilized CaCl2 right before use without having any precipitation problems.
best wishes and good luck,
Christoph
We're having the same problem in our lab. We bought 2x M9 salts from Life Technologies, it came as a sterile clear liquid, but upon addition of CaCl2 and MgSO4, both previously prepared as 5x solutions and filter-sterilized, a white precipitate appears immediately. We're adding also glucose, antibiotics and a carbonate buffer to keep the pH between 7.2 and 7.4. The media seems to work ok, but this precipitate might be interfering with some measurements.
What should we do? Prepare M9 from scratch, from the solid salts, filter the precipitate once the media is prepared?
Thanks!
Hi Aleixandre, I now use water purchased from Fisher to make up my solutions. I get no precipitate, even when stored at 4C.
Maybe try making fresh CaCl2 and MgSO4 in purchased water free of unwanted ions.
Hi Maria, we finally figured out the problem... we were adding concentrated CaCl2 and MgSO4 at the beginning, and there was a precipitate of CaSO4. Now the solutions are more diluted and the medium is clear. It looks like those salts must be added at the end to avoid the formation of precipitates, after the M9 salts, glucose and everything else.
We use ultrapure water from a machine we have, so there's no interfering ions in the liquid.
Thanks!
I've been having this problem lately, myself. Here's what I've found.
On what the precipitate is: Adding 25 uL 1M CaCl2 to 1 mL 1M MgSO4 resulted in some formation of precipitate after several minutes. Adding the same amount of CaCl2 to 1 mL 1x M9 salts resulted in large amounts of precipitate immediately, so in my case at least it seems calcium phosphate is the larger problem (no precipitate is formed upon mixing MgSO4 and M9 salts).
The main issue: The pH of the salt solution I was using turned out to be about 6.8; upon bumping it up to 7.25 the CaCl2 went in fine after everything else was diluted properly (specifically, adding 25 uL 1 M CaCl2 to 250 mL 1x M9 salts and MgSO4 worked fine).
Bonus: Calcium phosphate actually becomes less soluble at higher temperatures, so do not autoclave after mixing together the CaCl2 and the M9 salts (yes, I can verify this from experience).
Summary: Make sure the pH is at least 7.0, add the CaCl2 last, and do not autoclave after doing so.
https://en.wikipedia.org/wiki/Calcium_phosphate
Hi Maria. The precipitation is from calcium sulfate formed when calcium chloride reacts with magnesium sulfate when the calcium is added to the solution containing sulfate ions. To avoid this, calcium chloride should be added to demineralized water before adding the other salts. This allow the calcium to be dissolved homogeneously at a very low concentration; thus when sulfate ions are added later on the formed calcium sulfate concentration is still below its solubility at the room temperature.
- Badges
- Science method