precipitate coming out of media! - (May/10/2006 )
hello,
i am having trouble with some media i have been using for quite a while. it consists of:
5g/L yeast extract
10g/L d-mannitol
1.0g/L (NH4)2HPO4
2.0g/L MgSO4 X 7H20
I have been making and using it for a while, but now i am having a substance falling out of solution. all the chemicals are the same. the people at the university were doing something with the milli pore system last week, and this is the first time i have made media since.
the precipitate is very unsoluble. i've heated to boiling and still nothing. i have narrowed it down to the addition of the di-ammonium phosphate to the magnesium sulfate. both will go into solutions of their own, but in each, when the complement is added, the precip forms. when i pour out the solution, the stuff sticks to the inside of the glass bottles and won't come off with water.. took some scrubbing with sparkleen. any ideas?
thanks
cheers
dez
is it possible that someone mislabeled the bottle of mgso4 and it is caso4? could your mg have been contaminated with ca?
sounds like a caphos precipitate.
the mgso4 is pretty old and i am the only one that uses it in a lab. the amount of precipitate formed is fairly consistent each time. if it were contaminated with Ca, wouldn't it vary in relationship to different amounts of Ca in the mgso4 bottle?
what are the odds that i am getting calcium from the water supply? the millipore is reading around 18.3 consistently
Check the pH of the water? Of the media? Do you have any old aliquots of the media you can check the pH of? Could you have used monobasic ammonium phosphate (NH4H2PO4) by mistake?
the pH of the water is unstable... first test puts it about 7.6 ish, after sitting it hits about 5.6, reaction with CO2, from what i've been told.
the media is supposed to be at pH 5.5, so i've been adding HCl to drop the pH down. it usually is around 6.8 before i add the acid, but that's not always constant. and we only have di-ammonium phosphate in the lab...
can the mgso4 or the di-ammonium phosphate go bad? the mgso4 is old, but the ammonium is fairly new. i'm stopping by another university across town in the morning and getting a carboy of millipore from them to test the h2o
just an update,
i've used 2 different sets of each chemical and each produces the same precipitate. i've added the ammonium phosphate to dissolved mgs04 and vice versa and still getting the stuff falling out. i tried using the water from the carboys here in the lab to see if the pH in those were a bit more stable, but no luck.
The precipitate that is coming out looks really fine.. almost like talcum powder.
I guess at this point, it must be the water. Can you get a water hardness test kit? You could confirm that it's the water by buying some sterile water for injection USP (from a pharmacy or a vendor) and try making your media with that, as a source of water completely independent of your Millipore system.
What kind of system do you have? Can the vendor of your water system offer any advice?
Maybe what you're seeing is magnesium phosphate or magnesium ammonium phosphate?
yeah.. i'm at a loss... i tried a whole different water system today at a different university, 20 miles away and still had the precipitate form. while there, i chatted with my old organic professor and we looked int the Merck and figured we were making magnesium diphosphate noting the 'slight solubility' at a normal pH and soluble at an acidic pH. so we dropped the solution to 5.5 (where the media is supposed to be at) and the silky sediments disappeared. there were still some larger complexes left, so i dropped the pH down to around 2ish and everything went in. i then brought the pH back up to 5.5 and everything stayed in.
so tomorrow, i am going to try making molar solutions out of the 2 salts and pH them, and play around with the addition of the salts in different circumstances and see what i can find out. i also bought 2 new stocks of the salts. last weekend my lab went up to 95 degrees so i just want to make sure that my salts are still ok.
any ideas?
I've always been told to autoclave the phosphates and the rest of the media components separately, then mix them afterwards. Terrific broth, e.g. is made this way. I think there are components (maybe the magnesium) which will precipitate in the presence of phosphates during autoclaving.
I also faced the same problem with precipitation of media after autoclave. My media consisted of calciumnitrate and potassiumdihydrogenphosphate, and they precipitated during autoclave. So I prepared solution of calcium and phosphate seperatedly and then mixed them after autoclave at room temperature. there was no precipitation after all.
However, i am now preparing my media by preparing each chem. reagents and autoclave them seperately. Then, i mix every thing at room temperature. It should be better
Hopefully u can fix this problem soon. Good luck